This thcsis consists of a collection of 5 papers dealing with ultraviolet inactivation and excision-repair in the transformable bacterium 'Bacillus subtilis' specially with respect to transforming DNA. One of the main objectives was to study UV inacyivation of a number of transforming markers. To that purpose, a multiple auxotrophic mutant, strain 8G-5 was constructed, which served as as the recipient strain. ...

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Bron, Sierd,

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Ultraviolet inactivation and excision-repair in bacillus subtilus

Bron, Sierd

Proceedings - University of Groningen

   University of GroningenUltraviolet inactivation and excision-repair in bacillus subtilusBron, SierdIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1972Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bron, S. (1972). Ultraviolet inactivation and excision-repair in bacillus subtilus. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 16-07-2023SUMMARYThis  thcs is  cons is ts  o f  a  co l l cc t ion  o f  5  papcrs  dca l ing  w i th  u l t rav io lc r  inac t iva t ionat rd  cxc is ion- rcpa i r  in  the  t r : rns fo r rnab le  bac ter iunr  Bac i l lus  subt i l i s ,  spcc ia l l y  w i thresPcc t  to  t ra t ts fo rn ' r ing  DNA.  Onc o f  thc  m: r in  ob lcc t rvcs  was to  s t r - rdy  UVi r r a c i i v a t i o r r  o f  a  n u t n b e r  o f  t r a r r s f o r r n i n g  n r : r r k c r s .  T o  t h a t  p u r p ( ) s c . : l  n r u l t i p l caL lxo i rc - 'ph i t  t - ' ru tá r t r t .  s r : . r i r  BG-5 ,  w i l s  cons t ru lc tcd .  wh ich  scrvcd  as  thc  rcc ip icn ts t ra i r r .  Thc  procedurc  o f  i so la t ion  and the  ch : r rac tc r iza t ion  o f  s t ra in  BG-5 arcdescr ibc ,d  in  papcr  l .  Th is  s t ra in  t rans forms c lu i te  sz r t i s fac to ry :  s ing le  n rarkcrt ra t ts fo r tna t io t r  f i ' cquenc ics  o f  : i  few pcrccn t  a rc  usua l l t ,  ob ta incc l .  A l thouchthc v:rr ious mark"r ,  " . "  s l ight ly di f fercnt  wi th rcspccr to " f f i . i . , r .1, . r f  r r , , , i r -fo r - tna t io t r ,  t l i cy  p robab ly  bc long to  on ly  onc  c lass .  a l l  bc ing  o f  thc  h ish-c f f i c ic t rcy  typc .  Thc  markers  were  loca tcd  re la t i vc  to  cach o thcr  by  , r r " r , . , ,o f  rnarkcr  f rcc luency  ar - ra lys is .  Of  the  B nrarkers  s tud icd .  t rp , , ,  a r rc i  t y r í  Arcc losc ly  l inkcd  in  t rans format ion .  Thc  r ib  rnarkcr  wá ls  shown-  to  be  wcak lyl inked to  thc  t rp  r - ty r r  pa i r  o f  n rarkers .  No fu r ther  l inkagcs  c -x is t  bcrwccnthc  r la rkcrs  in  s t r : r in  BG-5 .UV inac t iv : r t io t r  o f  thc  B  rnarkcrs  i s  dcscr ibcd  in  papcrs  l l  anc l  I l t .  DNA iso la tcc lf ro tn  p ro to t roph ic  cc l l s  w; rs  i r rad ia ted  w i t l i  var ious  doscs  o f  UV: rnd  subscc lucn t lyuscd to  t rans for tn  8G-5  rec ip icn t  cc l l s .  Thc  rcsu l ts  dcnrons t ra tc  tha t  thc  vur iousnt : r rkc rs  a rc  inac t iva tcd  a t  d i f f c rcn t  ra tcs .  Or - r r  work ing  hypothcs is  was th r r td i f fe rcn t ia l  markcr  inac t iva t ion  is  duc  to  d i f f c rcnccs  in  t l - rc  f rcc lucncv , - , ipo tcn t ia l  photochcrn ica l  les ions  in  the  rnarkcd  segmcnts  o f  ONA.  Howcvcr .in tc rpre ta t ion  o f  the  resu l ts  o f  our  marker  inac t iva t ion  cxper in ren ts  accord i r rgto  th is  hypothes is  w: rs  compl ica ted  fo r  a t  l cas t  two rcasons .  F i rs t l y .  thc  rcsu l tsshow tha t  marker  scns i t i v i t i cs  to  UV depend on  thc  DNA conccnt ra r lo l l  c iu r ingt rans for tna t ion .  A t  sa tura t ing  amounts  o f  DNA,  thc  rnarkcrs  a rc  : rpparcn t lyn lo re  scns i t i vc  to  UV than á i t  non-sa tura t ing  ár ïnounts .  Th is  i s  p robab ly  duct o  t h e  f a c t  t h a t  a t  s a t u r a t i n g  a m o u n t s  o f  D N A ,  i n  a c i d i t i o n  t o  t h c  m o l c c u l c scar ry ing  se lec tcd  nrarkcrs .  morc  non-sc lec tcd  danragcc l  rno lccu lcs  : r rc  in tcgra tcdin to  thê  rec ip icn t  genomc th : rn  a t  non-s i l tu r l r t ing  iu r louuts .  Thcrc fo rc .  r r - lo rcpoten t ia l  t rans fornran ts  a rc  k i l l cd  undcr  thc  fo rnrc r  cond i t ions .  wh ich  rcsu l tsin  overes t in la t ion  o f  n rarkcr  scns i t i v i t i cs .  Th is  compl icu t io r . r  was  avo idcd  bydeter rn in ing  the  markcr  scns i t i v i t i cs  to  UV a t  DNA conccnt r r r t io r " rs  fa r  bc lowsatura t ion .  Second ly ,  cornp l i ca t ions  w i l l  a lso  ar isc ,  i f  a f t " r  up takc  l . r r  rcc ip ic r r tce l l s ,  the  inac t iva tcd  mzt rkers  a rc  rcpa i rcd  to  d i f f c rcn t  dcgrccs .  To  uvo id  t l r i scompl icat ion,  an excis ion-repair-def ic ient  c ler ivat ivc w;rs iÀolatcd f rorn strainB G - 5 .  T h c  p r o p c r t i c s  o f  t h i s  s t r : r i n : r r c  a l s o  d c s c r i b e d  i n  p a p c r  l .  T h i s  s r n r i n .B G - 1  1 ,  i s  u n a b l c  t o  c x c i s e  p y r i m i c l i n c  d i n r c r s .  t o  r c a s s u r r r c  D N A  s v n t h c s i s  a f t c rU V  i r r a d i a t i o r - r ,  a n d  t o  r c a c t i v u t c  U V - i n a c t i v a t c d  b a c t c r i o p h a q c .  A s  c o r n p l r c c lt t t  s t r : t i t - t  I JG-5 ,  i t  t rans for r r rs  nornra l l v  r ,v i r l r  t rn i r r l c l i l t c r ]  I )NA.  bu t  1 ' , . . r , , r l vw i t h  U V - i r r a c l i : r t c c l  l ) N A .  B c c : r r - r s c  o f  t l r c s c  p r o p c r t i c s  s r r i r i n  S ( ;  l l  . 1 , , n  t r . ,1 8-classi f ied as being def ic ient  in excis ion-repair  (uvr-) .  When in v ivo photo-react ivat ion of  UV-irradiated donor DNA is prevented by working undcr diml ight ,  ancl  excis ion-repair  by using strain 8G-11 as recipient,  d i f ferent ia l  markerinact ivat ion by UV of 254 nm st i l l  exists (paper I I ) ,  which supports thc hy-pothesis that  the f requency of  potent ia l  photochemical  lesions determinesthe sensi t iv i ty to UV of  the marked segmenrs of  ONA.Comparison of the results on marker inactivation obtained with strain BG-5and strain BG-11 al lowed us to compute the f ract ion of  inact ivat ing lesionsrvhich is not repaired by the 8G-5 (uvr+) recipient.  The size of  th is f ract ionis marker-specific and varies from 13t/o to 26o/o, indicating that excision-repairis not equally efficient for different markers in irradiated DNA. On the averageabout 80' /o of  the induced lesions are succesful ly repaired.The involvement "f fr dimers in both differeniial rnarker inactivation anddifferential marker repair was studied in the following way. TransformingDNA was i r radiated with 313 nm l ight  in the presence of  acerophenone assensi t izer.  Under these condi t ions exclusively TT dimers should be induc "642,43 .H^owever,  our resul ts (presented in paper I I I )  demonstrate that ,  in addi t ion toTT dimers,  non repairable s ingle-stranded DNA breaks are : r lso produced. Invi t ro photoreact ivat ion of  the TT dimers in the i r radiated DNA al lowed usto colnPute the relat ive contr ibut ions of  the dirners and the single-strandedDNA breaks to total  marker sensi t iv i t ies.  Al though only one break occurs perapproximately 90 f f  d imers,  i t  appears that  the breaks account for  abàut20' /o of  the observed marker sensi t iv i t ies.Di f ferent ia l  marker inact ivat ion and di f ferent ia l  markcr repair  werc also ob-served in thc acetophenone systein of  i r radiat ion.  Thc resul ts dernonstrate thar,i r respect ive of  the uvr genotype of  the recipicnt ,  thc s inglc-strandcd DNA brcaLsinact ivate the var ious markers to the same extcnt.  From this observat ion i tcan be concluded that in the acetophenonc system di f ferent ia l  marker in-act ivat ion is exclusively duc to di f fercnces in t i re f requency " f  f f  d i rncrs inthe marked segments of  DNA. In addi t ion.  d i f fe rcnt ia i  markcr rcpair  is  exclusivelydue to di f ferenccs in thc ef f ic iency of  repair  of  r r  d imcrs.  Support  for  thcconclusion that di f ference s in the f requency " f  f r  d imers in thc rrr" . t "d scgmenrsof DNA causc di f fcrcnt ia l  rnarkcr inact ivat ion is that  photoreact ivat ion exper-imcnts  ind ica tc  tha t  exce l l cn t  cor re la t ion  ex is ts  be tween the  f requency  o fTT dirners in thc rnarked scgmcnts of  DNA arrd thc scnsi t iv i ty , r f  th.  '^^r-kc rs  to  i r rad ia t ion  in  thc  acc tophenonc sys tem.  Fur thcr  suppor t  fo r  th is  con-c lus ion  is  p rov idcc l  by  thc  obscrva t ion  tha t ,  as  judge d  f ro rn  heat  dcnarurar ions tud ics ,  markcrs  rc la t i vc ly  r i ch  in  thyrn inc  usua l ly  a rc  rc la r ivc ly  scns i t i ve  toi r rad ia t ion  and,  convcrsa ly ,  tha t  tnarkers  rc la t i ve ly  poor  in  thyrn ine  usua l lyare  rc la t i vc lv  rcs is tan t  to  i r rad ia t ion .Cot r ipar ison  c , f  thc  rcsu l ts  ob ta i r - rcd  in  thc  acc tophcnonc sys tc rn  w i th  thoseobta incd  in  thc  254 nrn  svs tc rn  o f  i r rad ia t ion ,  shows tha t  bo th  t l - rc  rc la t i vcordcrs , .> f  n ta rkcr  sc r rs i t i v i t i t : s  and o f  rnarkcr  rcpa i rab i l i t i cs  a rc  c lc - rse ly  s imi la rrn  thc  two svs tcnrs  o f  i r rad ia t ion .  Wc cons idcr  t i r i s  as  cv idcncc  t l ia t  a lso  in  thcl 92 5 4  tmarkare t lThethe  efirstllthe  kof  U'lo l lo rrecipiand sof d:abscnjuclue+uvrtranslis intgratelirradicxc luconcl iatnouT h c  Istr-rditstrai  nThc  Iandtnolc,rc iso liÀ int20-4 !for fdatnar e e x tS C C  O TThc 1was  Ihypot h c  ct igatrt h c  cl igascsigni lt y  c cvVhen in v ivo photo-y working under dimrt, differential markerhich supporrs rhe hy-r l  lesions determinesred with strain 8G-5f inact ivat ing lesions: size of this fractiong that excision-repairDNA. On the average'ker inact ivat ion andi way. Transformingof  ace tophenone asu ld  be  induceda 2  ,43  .:  thar ,  in  add i t ion  tore  a lso  produced.  Ined DNA al lowed usI  the s ingle-strandedrne break occurs pers account for  aboutrepair  werc also ob-I ts c le morlstrate that ,t randcd DNA breaLsr th is obscrvat ion i tí c rc r r t ia l  markcr  in -rcy of ff di.n"rs inr  rcpair  is  e xclusivclycrs.  Support  for  thehc nrarked scgmentsorcact ivat ion exper-r  the f requency ofs i t iv i ty of  the mar-rPporr  for  th is con-r  l icat  dcnaturat ionat ivc ly  scns i t i ve  ton  thyminc  usua l lysys tcm wi th  thoseboth  thc  rc la t i vearc c loscly s i rn i larrcc  tha t  a lso  in  thc254 nm system of irradiation TT dimers are the main cause for differentialmarker inactivation and that differences in the efficiency of repair "f fT dimersare the main cause for differential marker repair.The fate of UV lesions in transforming DNA was studied more detailed inthe exper iments descr ibed in paper IV.  The pr incipal  a ims were to invest igate:f i rst ly,  genet ic integrat ion of  UV-irradiated transforming DNA, and secondly,the k inet ics of  in v ivo repair  of  Uv- inact ivated transforming DNA. Tl-re ef fectof  UV lesions in t ransÍorming DNA on gener ic intcgrat ion was studied in theÍc. ' l lowing way. UV-irradiated DNA was used to t ransform a repair less uvr-recipient.  Af tcr  a l lowing t ime for intcgrat ion to occur,  DNA was reextractedand subsequent ly assayed on uvr-  and uvr-  second recipicnts for  the prcsenceof dark-repairable damage in rccombinant type moleculcs.  " ihe presence orabsence of  dark-repairable darnage in thc recombinant type molccules wasjudged by the number of  recornbinant typc t ransformants obt: i incd on theuvr*  re la t i ve  to  the  number  on  the  uvr -  second rcc ip ien t .  Thc  rcsu l ts  show tha ttransfortning DNA irradiatcd wi th modcrate doscs of  UV (2,000 erg/mrn2)is integrated almost equal ly ef f ic , icnt ly : rs non- i r radiarcd DNA, and that inrc-grated UV lesions account alrnost exclusivcly for  t l ie inact iv:r t ion.  Howcver,i r rad ia t ion  o f  thc  DNA wi th  20 .000 " .g / - -2  o f  uV rcsu l ts  in  a  s ign i f i can texclusion of  darnagcd DNA frorn th" p.oc"ss of  intcgrar ion.  Under thcsecond i t ions ,  the  cont r ibu t ion  o f  thc  exc lus ion  to  to ta l  markcr  inac t iva t ionamounts  to  approx i rna te ly  1 }o / , t  in  uvr *  and to  less  than 7oh in  uvr -  rcc ip icn ts .The kinet ics of  in v ivo cxcis ion-repair  of  Uv- i r radiated rransforming DNA we rcs tud icd  by  a  c losc ly  s imi la r  method.  ln  th is  case.  e r  rcpa i r -p ro f i c icn t  uvr *s t ra in  sc rvcd  as  thc  f i rs t  rcc ip ien t  in  t rans format ion  w i th  UV- i r rad ia rcd  DNA.The DNA was rccx t rac tcd  a t  var ious  in te rva ls  a f te r  n r i r ing  cc l l s  ; inc i  donor  DNA,and the rcsidual  amount of  dark-repairablc damage in recombin: int  ry pctno iccu les  in  the  rcex t rac ted  DNA was de tc rmincd as  a  func t ion  o f  t i rnc  o fre iso la t ion .  Thc  rcsu l ts  ind ica te  tha t  repa i r  on ly  s ta r ts  a f - tc r  the  donor  DNAi i  i r - r tcgra tcd  in to  the  rcc ip ien t  gcnomc.  Cornp le t ion  o f  repa i r  rcqLr i res  f ro r r r20-45  rn in  fo r  DNA i r rad ia tcd  w i th  2 ,000 c rg /n r rn l ,  and approx i r r ia tc ly  2  hfo r  DNA i r rad i : r ted  w i th  15 ,000 erg lmrn2.  T l i c  rcsu l ts  a lso  i r - rd ica tc  rha t  UV-danragcd cc l ipse  phase DNA (DNA tha t  i s  taken up  bu t  nor  in tcgra tcd)reex t rac tcd  f rom rec ip icn t  ce l l s ,  i s  no t  sub jcc t  to  cxc is ion- rcpa i r  in  uur*s c c o n d  r c c i p i e n t  s t r a i n s .The poss ib i l i t y  o f  in  v i t ro  cxc is ion- rcpa i r  o f  UV- inac t iva tcd  t rans for r r i ing  DNAwas s tud ied  in  thc  expcr i rnents  dcscr ibcd  in  thc  las t  papcr  o i  th is  t l r cs is .  Thchypothes is  pu t  fo rwardby  KELLY e t  a l .a0  th r t  DNA po lynrerasc  pcr fc , r r r rs  bo ththe  cxc is ion  and the  rep l i ca t ion  s tep  in  exc is ion- repa i r ,  p ron lp rcd  us  ro  invcs-t iga tc  whcthcr  UV- i r rad ia ted  t rans forming DNA can bc  rcpa i rcd  in  v i t ro  bythe  combined ac t ion  o f  UV-spcc i f i c  cndonuc lcasc ,  DNA po ly rncrasc  and DNAl igasc. Thc resul ts show th:r t  the biological  : rct iv i ty of  inact ivarcd DNA incrc: : rsess ign i f i can t ly  fo l low ing  incubat ion  w i th  the  th rec  cnzyrnes .  Thc  rcs to rcd  acr iv i -ty  can  no t  bc  fu r thcr  inc reascd by  photoreac t iva t ion  in  v i t ro .  ind ica t ing  r l ra t20-photorepairable dimcrs are no longer present.  I t  could be demonstrated thatái-"rr  àre speci f ical ly excised upon incubat ion of  the i r radiated DNA withthe thre e enzymes, and that under our condi t ions approximatciy 50' /o of  thcinduccdlesionsare succesful ly repaired. These rcsul ts s l - row that,  at  lcast  in v i t ro,KELLY's40 mode l  app l i cs  to  repa i r  o f  UV les ions  in  DNA.  Whether  th is  mode lalso appl ics io repair  in v ivo,  remains to be establ ished. I t  may also be envisagedthat al tcrnat ive pathwat 's of  excis ion-re pair  exist .Dit proefsclheid voorsubt i l is ,  deherstel vanEén van drvan inaktivwerd een nDe isolerinschreven inmerkers wckregen. Hcbehoren detransformervan merkerbij transforgekoppeldstam werdeDe inaktiveen I I I  bes ,bestraald nmee te t rarongelijke nàeze differwaalmee P(deze hyportweetal  korgevoelighedDNA, die rheden trandan met overzadigencongemerktegreerd danmeer PoteÍkeli jke gev<opgelost dctraties DNIkatie ortStrmerkers inkomen,  wet l- l

English

University of Groningen

   University of GroningenUltraviolet inactivation and excision-repair in bacillus subtilusBron, SierdIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1972Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bron, S. (1972). Ultraviolet inactivation and excision-repair in bacillus subtilus. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019SUMMARYThis  thcs is  cons is ts  o f  a  co l l cc t ion  o f  5  papcrs  dca l ing  w i th  u l t rav io lc r  inac t iva t ionat rd  cxc is ion- rcpa i r  in  the  t r : rns fo r rnab le  bac ter iunr  Bac i l lus  subt i l i s ,  spcc ia l l y  w i thresPcc t  to  t ra t ts fo rn ' r ing  DNA.  Onc o f  thc  m: r in  ob lcc t rvcs  was to  s t r - rdy  UVi r r a c i i v a t i o r r  o f  a  n u t n b e r  o f  t r a r r s f o r r n i n g  n r : r r k c r s .  T o  t h a t  p u r p ( ) s c . : l  n r u l t i p l caL lxo i rc - 'ph i t  t - ' ru tá r t r t .  s r : . r i r  BG-5 ,  w i l s  cons t ru lc tcd .  wh ich  scrvcd  as  thc  rcc ip icn ts t ra i r r .  Thc  procedurc  o f  i so la t ion  and the  ch : r rac tc r iza t ion  o f  s t ra in  BG-5 arcdescr ibc ,d  in  papcr  l .  Th is  s t ra in  t rans forms c lu i te  sz r t i s fac to ry :  s ing le  n rarkcrt ra t ts fo r tna t io t r  f i ' cquenc ics  o f  : i  few pcrccn t  a rc  usua l l t ,  ob ta incc l .  A l thouchthc v:rr ious mark"r ,  " . "  s l ight ly di f fercnt  wi th rcspccr to " f f i . i . , r .1, . r f  r r , , , i r -fo r - tna t io t r ,  t l i cy  p robab ly  bc long to  on ly  onc  c lass .  a l l  bc ing  o f  thc  h ish-c f f i c ic t rcy  typc .  Thc  markers  were  loca tcd  re la t i vc  to  cach o thcr  by  , r r " r , . , ,o f  rnarkcr  f rcc luency  ar - ra lys is .  Of  the  B nrarkers  s tud icd .  t rp , , ,  a r rc i  t y r í  Arcc losc ly  l inkcd  in  t rans format ion .  Thc  r ib  rnarkcr  wá ls  shown-  to  be  wcak lyl inked to  thc  t rp  r - ty r r  pa i r  o f  n rarkers .  No fu r ther  l inkagcs  c -x is t  bcrwccnthc  r la rkcrs  in  s t r : r in  BG-5 .UV inac t iv : r t io t r  o f  thc  B  rnarkcrs  i s  dcscr ibcd  in  papcrs  l l  anc l  I l t .  DNA iso la tcc lf ro tn  p ro to t roph ic  cc l l s  w; rs  i r rad ia ted  w i t l i  var ious  doscs  o f  UV: rnd  subscc lucn t lyuscd to  t rans for tn  8G-5  rec ip icn t  cc l l s .  Thc  rcsu l ts  dcnrons t ra tc  tha t  thc  vur iousnt : r rkc rs  a rc  inac t iva tcd  a t  d i f f c rcn t  ra tcs .  Or - r r  work ing  hypothcs is  was th r r td i f fe rcn t ia l  markcr  inac t iva t ion  is  duc  to  d i f f c rcnccs  in  t l - rc  f rcc lucncv , - , ipo tcn t ia l  photochcrn ica l  les ions  in  the  rnarkcd  segmcnts  o f  ONA.  Howcvcr .in tc rpre ta t ion  o f  the  resu l ts  o f  our  marker  inac t iva t ion  cxper in ren ts  accord i r rgto  th is  hypothes is  w: rs  compl ica ted  fo r  a t  l cas t  two rcasons .  F i rs t l y .  thc  rcsu l tsshow tha t  marker  scns i t i v i t i cs  to  UV depend on  thc  DNA conccnt ra r lo l l  c iu r ingt rans for tna t ion .  A t  sa tura t ing  amounts  o f  DNA,  thc  rnarkcrs  a rc  : rpparcn t lyn lo re  scns i t i vc  to  UV than á i t  non-sa tura t ing  ár ïnounts .  Th is  i s  p robab ly  duct o  t h e  f a c t  t h a t  a t  s a t u r a t i n g  a m o u n t s  o f  D N A ,  i n  a c i d i t i o n  t o  t h c  m o l c c u l c scar ry ing  se lec tcd  nrarkcrs .  morc  non-sc lec tcd  danragcc l  rno lccu lcs  : r rc  in tcgra tcdin to  thê  rec ip icn t  genomc th : rn  a t  non-s i l tu r l r t ing  iu r louuts .  Thcrc fo rc .  r r - lo rcpoten t ia l  t rans fornran ts  a rc  k i l l cd  undcr  thc  fo rnrc r  cond i t ions .  wh ich  rcsu l tsin  overes t in la t ion  o f  n rarkcr  scns i t i v i t i cs .  Th is  compl icu t io r . r  was  avo idcd  bydeter rn in ing  the  markcr  scns i t i v i t i cs  to  UV a t  DNA conccnt r r r t io r " rs  fa r  bc lowsatura t ion .  Second ly ,  cornp l i ca t ions  w i l l  a lso  ar isc ,  i f  a f t " r  up takc  l . r r  rcc ip ic r r tce l l s ,  the  inac t iva tcd  mzt rkers  a rc  rcpa i rcd  to  d i f f c rcn t  dcgrccs .  To  uvo id  t l r i scompl icat ion,  an excis ion-repair-def ic ient  c ler ivat ivc w;rs iÀolatcd f rorn strainB G - 5 .  T h c  p r o p c r t i c s  o f  t h i s  s t r : r i n : r r c  a l s o  d c s c r i b e d  i n  p a p c r  l .  T h i s  s r n r i n .B G - 1  1 ,  i s  u n a b l c  t o  c x c i s e  p y r i m i c l i n c  d i n r c r s .  t o  r c a s s u r r r c  D N A  s v n t h c s i s  a f t c rU V  i r r a d i a t i o r - r ,  a n d  t o  r c a c t i v u t c  U V - i n a c t i v a t c d  b a c t c r i o p h a q c .  A s  c o r n p l r c c lt t t  s t r : t i t - t  I JG-5 ,  i t  t rans for r r rs  nornra l l v  r ,v i r l r  t rn i r r l c l i l t c r ]  I )NA.  bu t  1 ' , . . r , , r l vw i t h  U V - i r r a c l i : r t c c l  l ) N A .  B c c : r r - r s c  o f  t l r c s c  p r o p c r t i c s  s r r i r i n  S ( ;  l l  . 1 , , n  t r . ,1 8-classi f ied as being def ic ient  in excis ion-repair  (uvr-) .  When in v ivo photo-react ivat ion of  UV-irradiated donor DNA is prevented by working undcr diml ight ,  ancl  excis ion-repair  by using strain 8G-11 as recipient,  d i f ferent ia l  markerinact ivat ion by UV of 254 nm st i l l  exists (paper I I ) ,  which supports thc hy-pothesis that  the f requency of  potent ia l  photochemical  lesions determinesthe sensi t iv i ty to UV of  the marked segmenrs of  ONA.Comparison of the results on marker inactivation obtained with strain BG-5and strain BG-11 al lowed us to compute the f ract ion of  inact ivat ing lesionsrvhich is not repaired by the 8G-5 (uvr+) recipient.  The size of  th is f ract ionis marker-specific and varies from 13t/o to 26o/o, indicating that excision-repairis not equally efficient for different markers in irradiated DNA. On the averageabout 80' /o of  the induced lesions are succesful ly repaired.The involvement "f fr dimers in both differeniial rnarker inactivation anddifferential marker repair was studied in the following way. TransformingDNA was i r radiated with 313 nm l ight  in the presence of  acerophenone assensi t izer.  Under these condi t ions exclusively TT dimers should be induc "642,43 .H^owever,  our resul ts (presented in paper I I I )  demonstrate that ,  in addi t ion toTT dimers,  non repairable s ingle-stranded DNA breaks are : r lso produced. Invi t ro photoreact ivat ion of  the TT dimers in the i r radiated DNA al lowed usto colnPute the relat ive contr ibut ions of  the dirners and the single-strandedDNA breaks to total  marker sensi t iv i t ies.  Al though only one break occurs perapproximately 90 f f  d imers,  i t  appears that  the breaks account for  abàut20' /o of  the observed marker sensi t iv i t ies.Di f ferent ia l  marker inact ivat ion and di f ferent ia l  markcr repair  werc also ob-served in thc acetophenone systein of  i r radiat ion.  Thc resul ts dernonstrate thar,i r respect ive of  the uvr genotype of  the recipicnt ,  thc s inglc-strandcd DNA brcaLsinact ivate the var ious markers to the same extcnt.  From this observat ion i tcan be concluded that in the acetophenonc system di f ferent ia l  marker in-act ivat ion is exclusively duc to di f fercnces in t i re f requency " f  f f  d i rncrs inthe marked segments of  DNA. In addi t ion.  d i f fe rcnt ia i  markcr rcpair  is  exclusivelydue to di f ferenccs in thc ef f ic iency of  repair  of  r r  d imcrs.  Support  for  thcconclusion that di f ference s in the f requency " f  f r  d imers in thc rrr" . t "d scgmenrsof DNA causc di f fcrcnt ia l  rnarkcr inact ivat ion is that  photoreact ivat ion exper-imcnts  ind ica tc  tha t  exce l l cn t  cor re la t ion  ex is ts  be tween the  f requency  o fTT dirners in thc rnarked scgmcnts of  DNA arrd thc scnsi t iv i ty , r f  th.  '^^r-kc rs  to  i r rad ia t ion  in  thc  acc tophenonc sys tem.  Fur thcr  suppor t  fo r  th is  con-c lus ion  is  p rov idcc l  by  thc  obscrva t ion  tha t ,  as  judge d  f ro rn  heat  dcnarurar ions tud ics ,  markcrs  rc la t i vc ly  r i ch  in  thyrn inc  usua l ly  a rc  rc la r ivc ly  scns i t i ve  toi r rad ia t ion  and,  convcrsa ly ,  tha t  tnarkers  rc la t i ve ly  poor  in  thyrn ine  usua l lyare  rc la t i vc lv  rcs is tan t  to  i r rad ia t ion .Cot r ipar ison  c , f  thc  rcsu l ts  ob ta i r - rcd  in  thc  acc tophcnonc sys tc rn  w i th  thoseobta incd  in  thc  254 nrn  svs tc rn  o f  i r rad ia t ion ,  shows tha t  bo th  t l - rc  rc la t i vcordcrs , .> f  n ta rkcr  sc r rs i t i v i t i t : s  and o f  rnarkcr  rcpa i rab i l i t i cs  a rc  c lc - rse ly  s imi la rrn  thc  two svs tcnrs  o f  i r rad ia t ion .  Wc cons idcr  t i r i s  as  cv idcncc  t l ia t  a lso  in  thcl 92 5 4  tmarkare t lThethe  efirstllthe  kof  U'lo l lo rrecipiand sof d:abscnjuclue+uvrtranslis intgratelirradicxc luconcl iatnouT h c  Istr-rditstrai  nThc  Iandtnolc,rc iso liÀ int20-4 !for fdatnar e e x tS C C  O TThc 1was  Ihypot h c  ct igatrt h c  cl igascsigni lt y  c cvVhen in v ivo photo-y working under dimrt, differential markerhich supporrs rhe hy-r l  lesions determinesred with strain 8G-5f inact ivat ing lesions: size of this fractiong that excision-repairDNA. On the average'ker inact ivat ion andi way. Transformingof  ace tophenone asu ld  be  induceda 2  ,43  .:  thar ,  in  add i t ion  tore  a lso  produced.  Ined DNA al lowed usI  the s ingle-strandedrne break occurs pers account for  aboutrepair  werc also ob-I ts c le morlstrate that ,t randcd DNA breaLsr th is obscrvat ion i tí c rc r r t ia l  markcr  in -rcy of ff di.n"rs inr  rcpair  is  e xclusivclycrs.  Support  for  thehc nrarked scgmentsorcact ivat ion exper-r  the f requency ofs i t iv i ty of  the mar-rPporr  for  th is con-r  l icat  dcnaturat ionat ivc ly  scns i t i ve  ton  thyminc  usua l lysys tcm wi th  thoseboth  thc  rc la t i vearc c loscly s i rn i larrcc  tha t  a lso  in  thc254 nm system of irradiation TT dimers are the main cause for differentialmarker inactivation and that differences in the efficiency of repair "f fT dimersare the main cause for differential marker repair.The fate of UV lesions in transforming DNA was studied more detailed inthe exper iments descr ibed in paper IV.  The pr incipal  a ims were to invest igate:f i rst ly,  genet ic integrat ion of  UV-irradiated transforming DNA, and secondly,the k inet ics of  in v ivo repair  of  Uv- inact ivated transforming DNA. Tl-re ef fectof  UV lesions in t ransÍorming DNA on gener ic intcgrat ion was studied in theÍc. ' l lowing way. UV-irradiated DNA was used to t ransform a repair less uvr-recipient.  Af tcr  a l lowing t ime for intcgrat ion to occur,  DNA was reextractedand subsequent ly assayed on uvr-  and uvr-  second recipicnts for  the prcsenceof dark-repairable damage in rccombinant type moleculcs.  " ihe presence orabsence of  dark-repairable darnage in thc recombinant type molccules wasjudged by the number of  recornbinant typc t ransformants obt: i incd on theuvr*  re la t i ve  to  the  number  on  the  uvr -  second rcc ip ien t .  Thc  rcsu l ts  show tha ttransfortning DNA irradiatcd wi th modcrate doscs of  UV (2,000 erg/mrn2)is integrated almost equal ly ef f ic , icnt ly : rs non- i r radiarcd DNA, and that inrc-grated UV lesions account alrnost exclusivcly for  t l ie inact iv:r t ion.  Howcver,i r rad ia t ion  o f  thc  DNA wi th  20 .000 " .g / - -2  o f  uV rcsu l ts  in  a  s ign i f i can texclusion of  darnagcd DNA frorn th" p.oc"ss of  intcgrar ion.  Under thcsecond i t ions ,  the  cont r ibu t ion  o f  thc  exc lus ion  to  to ta l  markcr  inac t iva t ionamounts  to  approx i rna te ly  1 }o / , t  in  uvr *  and to  less  than 7oh in  uvr -  rcc ip icn ts .The kinet ics of  in v ivo cxcis ion-repair  of  Uv- i r radiated rransforming DNA we rcs tud icd  by  a  c losc ly  s imi la r  method.  ln  th is  case.  e r  rcpa i r -p ro f i c icn t  uvr *s t ra in  sc rvcd  as  thc  f i rs t  rcc ip ien t  in  t rans format ion  w i th  UV- i r rad ia rcd  DNA.The DNA was rccx t rac tcd  a t  var ious  in te rva ls  a f te r  n r i r ing  cc l l s  ; inc i  donor  DNA,and the rcsidual  amount of  dark-repairablc damage in recombin: int  ry pctno iccu les  in  the  rcex t rac ted  DNA was de tc rmincd as  a  func t ion  o f  t i rnc  o fre iso la t ion .  Thc  rcsu l ts  ind ica te  tha t  repa i r  on ly  s ta r ts  a f - tc r  the  donor  DNAi i  i r - r tcgra tcd  in to  the  rcc ip ien t  gcnomc.  Cornp le t ion  o f  repa i r  rcqLr i res  f ro r r r20-45  rn in  fo r  DNA i r rad ia tcd  w i th  2 ,000 c rg /n r rn l ,  and approx i r r ia tc ly  2  hfo r  DNA i r rad i : r ted  w i th  15 ,000 erg lmrn2.  T l i c  rcsu l ts  a lso  i r - rd ica tc  rha t  UV-danragcd cc l ipse  phase DNA (DNA tha t  i s  taken up  bu t  nor  in tcgra tcd)reex t rac tcd  f rom rec ip icn t  ce l l s ,  i s  no t  sub jcc t  to  cxc is ion- rcpa i r  in  uur*s c c o n d  r c c i p i e n t  s t r a i n s .The poss ib i l i t y  o f  in  v i t ro  cxc is ion- rcpa i r  o f  UV- inac t iva tcd  t rans for r r i ing  DNAwas s tud ied  in  thc  expcr i rnents  dcscr ibcd  in  thc  las t  papcr  o i  th is  t l r cs is .  Thchypothes is  pu t  fo rwardby  KELLY e t  a l .a0  th r t  DNA po lynrerasc  pcr fc , r r r rs  bo ththe  cxc is ion  and the  rep l i ca t ion  s tep  in  exc is ion- repa i r ,  p ron lp rcd  us  ro  invcs-t iga tc  whcthcr  UV- i r rad ia ted  t rans forming  DNA can bc  rcpa i rcd  in  v i t ro  bythe  combined ac t ion  o f  UV-spcc i f i c  cndonuc lcasc ,  DNA po ly rncrasc  and DNAl igasc. Thc resul ts show th:r t  the biological  : rct iv i ty of  inact ivarcd DNA incrc: : rsess ign i f i can t ly  fo l low ing  incubat ion  w i th  the  th rec  cnzyrnes .  Thc  rcs to rcd  acr iv i -ty  can  no t  bc  fu r thcr  inc reascd by  photoreac t iva t ion  in  v i t ro .  ind ica t ing  r l ra t20-photorepairable dimcrs are no longer present.  I t  could be demonstrated thatái-"rr  àre speci f ical ly excised upon incubat ion of  the i r radiated DNA withthe thre e enzymes, and that under our condi t ions approximatciy 50' /o of  thcinduccdlesionsare succesful ly repaired. These rcsul ts s l - row that,  at  lcast  in v i t ro,KELLY's40 mode l  app l i cs  to  repa i r  o f  UV les ions  in  DNA.  Whether  th is  mode lalso appl ics io repair  in v ivo,  remains to be establ ished. I t  may also be envisagedthat al tcrnat ive pathwat 's of  excis ion-re pair  exist .Dit proefsclheid voorsubt i l is ,  deherstel vanEén van drvan inaktivwerd een nDe isolerinschreven inmerkers wckregen. Hcbehoren detransformervan merkerbij transforgekoppeldstam werdeDe inaktiveen I I I  bes ,bestraald nmee te t rarongelijke nàeze differwaalmee P(deze hyportweetal  korgevoelighedDNA, die rheden trandan met overzadigencongemerktegreerd danmeer PoteÍkeli jke gev<opgelost dctraties DNIkatie ortStrmerkers inkomen,  wet l- l

Dissertations of the University of Groningen

   University of GroningenUltraviolet inactivation and excision-repair in bacillus subtilusBron, SierdIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1972Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bron, S. (1972). Ultraviolet inactivation and excision-repair in bacillus subtilus. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 28-12-2022SUMMARYThis  thcs is  cons is ts  o f  a  co l l cc t ion  o f  5  papcrs  dca l ing  w i th  u l t rav io lc r  inac t iva t ionat rd  cxc is ion- rcpa i r  in  the  t r : rns fo r rnab le  bac ter iunr  Bac i l lus  subt i l i s ,  spcc ia l l y  w i thresPcc t  to  t ra t ts fo rn ' r ing  DNA.  Onc o f  thc  m: r in  ob lcc t rvcs  was to  s t r - rdy  UVi r r a c i i v a t i o r r  o f  a  n u t n b e r  o f  t r a r r s f o r r n i n g  n r : r r k c r s .  T o  t h a t  p u r p ( ) s c . : l  n r u l t i p l caL lxo i rc - 'ph i t  t - ' ru tá r t r t .  s r : . r i r  BG-5 ,  w i l s  cons t ru lc tcd .  wh ich  scrvcd  as  thc  rcc ip icn ts t ra i r r .  Thc  procedurc  o f  i so la t ion  and the  ch : r rac tc r iza t ion  o f  s t ra in  BG-5 arcdescr ibc ,d  in  papcr  l .  Th is  s t ra in  t rans forms c lu i te  sz r t i s fac to ry :  s ing le  n rarkcrt ra t ts fo r tna t io t r  f i ' cquenc ics  o f  : i  few pcrccn t  a rc  usua l l t ,  ob ta incc l .  A l thouchthc v:rr ious mark"r ,  " . "  s l ight ly di f fercnt  wi th rcspccr to " f f i . i . , r .1, . r f  r r , , , i r -fo r - tna t io t r ,  t l i cy  p robab ly  bc long to  on ly  onc  c lass .  a l l  bc ing  o f  thc  h ish-c f f i c ic t rcy  typc .  Thc  markers  were  loca tcd  re la t i vc  to  cach o thcr  by  , r r " r , . , ,o f  rnarkcr  f rcc luency  ar - ra lys is .  Of  the  B nrarkers  s tud icd .  t rp , , ,  a r rc i  t y r í  Arcc losc ly  l inkcd  in  t rans format ion .  Thc  r ib  rnarkcr  wá ls  shown-  to  be  wcak lyl inked to  thc  t rp  r - ty r r  pa i r  o f  n rarkers .  No fu r ther  l inkagcs  c -x is t  bcrwccnthc  r la rkcrs  in  s t r : r in  BG-5 .UV inac t iv : r t io t r  o f  thc  B  rnarkcrs  i s  dcscr ibcd  in  papcrs  l l  anc l  I l t .  DNA iso la tcc lf ro tn  p ro to t roph ic  cc l l s  w; rs  i r rad ia ted  w i t l i  var ious  doscs  o f  UV: rnd  subscc lucn t lyuscd to  t rans for tn  8G-5  rec ip icn t  cc l l s .  Thc  rcsu l ts  dcnrons t ra tc  tha t  thc  vur iousnt : r rkc rs  a rc  inac t iva tcd  a t  d i f f c rcn t  ra tcs .  Or - r r  work ing  hypothcs is  was th r r td i f fe rcn t ia l  markcr  inac t iva t ion  is  duc  to  d i f f c rcnccs  in  t l - rc  f rcc lucncv , - , ipo tcn t ia l  photochcrn ica l  les ions  in  the  rnarkcd  segmcnts  o f  ONA.  Howcvcr .in tc rpre ta t ion  o f  the  resu l ts  o f  our  marker  inac t iva t ion  cxper in ren ts  accord i r rgto  th is  hypothes is  w: rs  compl ica ted  fo r  a t  l cas t  two rcasons .  F i rs t l y .  thc  rcsu l tsshow tha t  marker  scns i t i v i t i cs  to  UV depend on  thc  DNA conccnt ra r lo l l  c iu r ingt rans for tna t ion .  A t  sa tura t ing  amounts  o f  DNA,  thc  rnarkcrs  a rc  : rpparcn t lyn lo re  scns i t i vc  to  UV than á i t  non-sa tura t ing  ár ïnounts .  Th is  i s  p robab ly  duct o  t h e  f a c t  t h a t  a t  s a t u r a t i n g  a m o u n t s  o f  D N A ,  i n  a c i d i t i o n  t o  t h c  m o l c c u l c scar ry ing  se lec tcd  nrarkcrs .  morc  non-sc lec tcd  danragcc l  rno lccu lcs  : r rc  in tcgra tcdin to  thê  rec ip icn t  genomc th : rn  a t  non-s i l tu r l r t ing  iu r louuts .  Thcrc fo rc .  r r - lo rcpoten t ia l  t rans fornran ts  a rc  k i l l cd  undcr  thc  fo rnrc r  cond i t ions .  wh ich  rcsu l tsin  overes t in la t ion  o f  n rarkcr  scns i t i v i t i cs .  Th is  compl icu t io r . r  was  avo idcd  bydeter rn in ing  the  markcr  scns i t i v i t i cs  to  UV a t  DNA conccnt r r r t io r " rs  fa r  bc lowsatura t ion .  Second ly ,  cornp l i ca t ions  w i l l  a lso  ar isc ,  i f  a f t " r  up takc  l . r r  rcc ip ic r r tce l l s ,  the  inac t iva tcd  mzt rkers  a rc  rcpa i rcd  to  d i f f c rcn t  dcgrccs .  To  uvo id  t l r i scompl icat ion,  an excis ion-repair-def ic ient  c ler ivat ivc w;rs iÀolatcd f rorn strainB G - 5 .  T h c  p r o p c r t i c s  o f  t h i s  s t r : r i n : r r c  a l s o  d c s c r i b e d  i n  p a p c r  l .  T h i s  s r n r i n .B G - 1  1 ,  i s  u n a b l c  t o  c x c i s e  p y r i m i c l i n c  d i n r c r s .  t o  r c a s s u r r r c  D N A  s v n t h c s i s  a f t c rU V  i r r a d i a t i o r - r ,  a n d  t o  r c a c t i v u t c  U V - i n a c t i v a t c d  b a c t c r i o p h a q c .  A s  c o r n p l r c c lt t t  s t r : t i t - t  I JG-5 ,  i t  t rans for r r rs  nornra l l v  r ,v i r l r  t rn i r r l c l i l t c r ]  I )NA.  bu t  1 ' , . . r , , r l vw i t h  U V - i r r a c l i : r t c c l  l ) N A .  B c c : r r - r s c  o f  t l r c s c  p r o p c r t i c s  s r r i r i n  S ( ;  l l  . 1 , , n  t r . ,1 8-classi f ied as being def ic ient  in excis ion-repair  (uvr-) .  When in v ivo photo-react ivat ion of  UV-irradiated donor DNA is prevented by working undcr diml ight ,  ancl  excis ion-repair  by using strain 8G-11 as recipient,  d i f ferent ia l  markerinact ivat ion by UV of 254 nm st i l l  exists (paper I I ) ,  which supports thc hy-pothesis that  the f requency of  potent ia l  photochemical  lesions determinesthe sensi t iv i ty to UV of  the marked segmenrs of  ONA.Comparison of the results on marker inactivation obtained with strain BG-5and strain BG-11 al lowed us to compute the f ract ion of  inact ivat ing lesionsrvhich is not repaired by the 8G-5 (uvr+) recipient.  The size of  th is f ract ionis marker-specific and varies from 13t/o to 26o/o, indicating that excision-repairis not equally efficient for different markers in irradiated DNA. On the averageabout 80' /o of  the induced lesions are succesful ly repaired.The involvement "f fr dimers in both differeniial rnarker inactivation anddifferential marker repair was studied in the following way. TransformingDNA was i r radiated with 313 nm l ight  in the presence of  acerophenone assensi t izer.  Under these condi t ions exclusively TT dimers should be induc "642,43 .H^owever,  our resul ts (presented in paper I I I )  demonstrate that ,  in addi t ion toTT dimers,  non repairable s ingle-stranded DNA breaks are : r lso produced. Invi t ro photoreact ivat ion of  the TT dimers in the i r radiated DNA al lowed usto colnPute the relat ive contr ibut ions of  the dirners and the single-strandedDNA breaks to total  marker sensi t iv i t ies.  Al though only one break occurs perapproximately 90 f f  d imers,  i t  appears that  the breaks account for  abàut20' /o of  the observed marker sensi t iv i t ies.Di f ferent ia l  marker inact ivat ion and di f ferent ia l  markcr repair  werc also ob-served in thc acetophenone systein of  i r radiat ion.  Thc resul ts dernonstrate thar,i r respect ive of  the uvr genotype of  the recipicnt ,  thc s inglc-strandcd DNA brcaLsinact ivate the var ious markers to the same extcnt.  From this observat ion i tcan be concluded that in the acetophenonc system di f ferent ia l  marker in-act ivat ion is exclusively duc to di f fercnces in t i re f requency " f  f f  d i rncrs inthe marked segments of  DNA. In addi t ion.  d i f fe rcnt ia i  markcr rcpair  is  exclusivelydue to di f ferenccs in thc ef f ic iency of  repair  of  r r  d imcrs.  Support  for  thcconclusion that di f ference s in the f requency " f  f r  d imers in thc rrr" . t "d scgmenrsof DNA causc di f fcrcnt ia l  rnarkcr inact ivat ion is that  photoreact ivat ion exper-imcnts  ind ica tc  tha t  exce l l cn t  cor re la t ion  ex is ts  be tween the  f requency  o fTT dirners in thc rnarked scgmcnts of  DNA arrd thc scnsi t iv i ty , r f  th.  '^^r-kc rs  to  i r rad ia t ion  in  thc  acc tophenonc sys tem.  Fur thcr  suppor t  fo r  th is  con-c lus ion  is  p rov idcc l  by  thc  obscrva t ion  tha t ,  as  judge d  f ro rn  heat  dcnarurar ions tud ics ,  markcrs  rc la t i vc ly  r i ch  in  thyrn inc  usua l ly  a rc  rc la r ivc ly  scns i t i ve  toi r rad ia t ion  and,  convcrsa ly ,  tha t  tnarkers  rc la t i ve ly  poor  in  thyrn ine  usua l lyare  rc la t i vc lv  rcs is tan t  to  i r rad ia t ion .Cot r ipar ison  c , f  thc  rcsu l ts  ob ta i r - rcd  in  thc  acc tophcnonc sys tc rn  w i th  thoseobta incd  in  thc  254 nrn  svs tc rn  o f  i r rad ia t ion ,  shows tha t  bo th  t l - rc  rc la t i vcordcrs , .> f  n ta rkcr  sc r rs i t i v i t i t : s  and o f  rnarkcr  rcpa i rab i l i t i cs  a rc  c lc - rse ly  s imi la rrn  thc  two svs tcnrs  o f  i r rad ia t ion .  Wc cons idcr  t i r i s  as  cv idcncc  t l ia t  a lso  in  thcl 92 5 4  tmarkare t lThethe  efirstllthe  kof  U'lo l lo rrecipiand sof d:abscnjuclue+uvrtranslis intgratelirradicxc luconcl iatnouT h c  Istr-rditstrai  nThc  Iandtnolc,rc iso liÀ int20-4 !for fdatnar e e x tS C C  O TThc 1was  Ihypot h c  ct igatrt h c  cl igascsigni lt y  c cvVhen in v ivo photo-y working under dimrt, differential markerhich supporrs rhe hy-r l  lesions determinesred with strain 8G-5f inact ivat ing lesions: size of this fractiong that excision-repairDNA. On the average'ker inact ivat ion andi way. Transformingof  ace tophenone asu ld  be  induceda 2  ,43  .:  thar ,  in  add i t ion  tore  a lso  produced.  Ined DNA al lowed usI  the s ingle-strandedrne break occurs pers account for  aboutrepair  werc also ob-I ts c le morlstrate that ,t randcd DNA breaLsr th is obscrvat ion i tí c rc r r t ia l  markcr  in -rcy of ff di.n"rs inr  rcpair  is  e xclusivclycrs.  Support  for  thehc nrarked scgmentsorcact ivat ion exper-r  the f requency ofs i t iv i ty of  the mar-rPporr  for  th is con-r  l icat  dcnaturat ionat ivc ly  scns i t i ve  ton  thyminc  usua l lysys tcm wi th  thoseboth  thc  rc la t i vearc c loscly s i rn i larrcc  tha t  a lso  in  thc254 nm system of irradiation TT dimers are the main cause for differentialmarker inactivation and that differences in the efficiency of repair "f fT dimersare the main cause for differential marker repair.The fate of UV lesions in transforming DNA was studied more detailed inthe exper iments descr ibed in paper IV.  The pr incipal  a ims were to invest igate:f i rst ly,  genet ic integrat ion of  UV-irradiated transforming DNA, and secondly,the k inet ics of  in v ivo repair  of  Uv- inact ivated transforming DNA. Tl-re ef fectof  UV lesions in t ransÍorming DNA on gener ic intcgrat ion was studied in theÍc. ' l lowing way. UV-irradiated DNA was used to t ransform a repair less uvr-recipient.  Af tcr  a l lowing t ime for intcgrat ion to occur,  DNA was reextractedand subsequent ly assayed on uvr-  and uvr-  second recipicnts for  the prcsenceof dark-repairable damage in rccombinant type moleculcs.  " ihe presence orabsence of  dark-repairable darnage in thc recombinant type molccules wasjudged by the number of  recornbinant typc t ransformants obt: i incd on theuvr*  re la t i ve  to  the  number  on  the  uvr -  second rcc ip ien t .  Thc  rcsu l ts  show tha ttransfortning DNA irradiatcd wi th modcrate doscs of  UV (2,000 erg/mrn2)is integrated almost equal ly ef f ic , icnt ly : rs non- i r radiarcd DNA, and that inrc-grated UV lesions account alrnost exclusivcly for  t l ie inact iv:r t ion.  Howcver,i r rad ia t ion  o f  thc  DNA wi th  20 .000 " .g / - -2  o f  uV rcsu l ts  in  a  s ign i f i can texclusion of  darnagcd DNA frorn th" p.oc"ss of  intcgrar ion.  Under thcsecond i t ions ,  the  cont r ibu t ion  o f  thc  exc lus ion  to  to ta l  markcr  inac t iva t ionamounts  to  approx i rna te ly  1 }o / , t  in  uvr *  and to  less  than 7oh in  uvr -  rcc ip icn ts .The kinet ics of  in v ivo cxcis ion-repair  of  Uv- i r radiated rransforming DNA we rcs tud icd  by  a  c losc ly  s imi la r  method.  ln  th is  case.  e r  rcpa i r -p ro f i c icn t  uvr *s t ra in  sc rvcd  as  thc  f i rs t  rcc ip ien t  in  t rans format ion  w i th  UV- i r rad ia rcd  DNA.The DNA was rccx t rac tcd  a t  var ious  in te rva ls  a f te r  n r i r ing  cc l l s  ; inc i  donor  DNA,and the rcsidual  amount of  dark-repairablc damage in recombin: int  ry pctno iccu les  in  the  rcex t rac ted  DNA was de tc rmincd as  a  func t ion  o f  t i rnc  o fre iso la t ion .  Thc  rcsu l ts  ind ica te  tha t  repa i r  on ly  s ta r ts  a f - tc r  the  donor  DNAi i  i r - r tcgra tcd  in to  the  rcc ip ien t  gcnomc.  Cornp le t ion  o f  repa i r  rcqLr i res  f ro r r r20-45  rn in  fo r  DNA i r rad ia tcd  w i th  2 ,000 c rg /n r rn l ,  and approx i r r ia tc ly  2  hfo r  DNA i r rad i : r ted  w i th  15 ,000 erg lmrn2.  T l i c  rcsu l ts  a lso  i r - rd ica tc  rha t  UV-danragcd cc l ipse  phase DNA (DNA tha t  i s  taken up  bu t  nor  in tcgra tcd)reex t rac tcd  f rom rec ip icn t  ce l l s ,  i s  no t  sub jcc t  to  cxc is ion- rcpa i r  in  uur*s c c o n d  r c c i p i e n t  s t r a i n s .The poss ib i l i t y  o f  in  v i t ro  cxc is ion- rcpa i r  o f  UV- inac t iva tcd  t rans for r r i ing  DNAwas s tud ied  in  thc  expcr i rnents  dcscr ibcd  in  thc  las t  papcr  o i  th is  t l r cs is .  Thchypothes is  pu t  fo rwardby  KELLY e t  a l .a0  th r t  DNA po lynrerasc  pcr fc , r r r rs  bo ththe  cxc is ion  and the  rep l i ca t ion  s tep  in  exc is ion- repa i r ,  p ron lp rcd  us  ro  invcs-t iga tc  whcthcr  UV- i r rad ia ted  t rans forming DNA can bc  rcpa i rcd  in  v i t ro  bythe  combined ac t ion  o f  UV-spcc i f i c  cndonuc lcasc ,  DNA po ly rncrasc  and DNAl igasc. Thc resul ts show th:r t  the biological  : rct iv i ty of  inact ivarcd DNA incrc: : rsess ign i f i can t ly  fo l low ing  incubat ion  w i th  the  th rec  cnzyrnes .  Thc  rcs to rcd  acr iv i -ty  can  no t  bc  fu r thcr  inc reascd by  photoreac t iva t ion  in  v i t ro .  ind ica t ing  r l ra t20-photorepairable dimcrs are no longer present.  I t  could be demonstrated thatái-"rr  àre speci f ical ly excised upon incubat ion of  the i r radiated DNA withthe thre e enzymes, and that under our condi t ions approximatciy 50' /o of  thcinduccdlesionsare succesful ly repaired. These rcsul ts s l - row that,  at  lcast  in v i t ro,KELLY's40 mode l  app l i cs  to  repa i r  o f  UV les ions  in  DNA.  Whether  th is  mode lalso appl ics io repair  in v ivo,  remains to be establ ished. I t  may also be envisagedthat al tcrnat ive pathwat 's of  excis ion-re pair  exist .Dit proefsclheid voorsubt i l is ,  deherstel vanEén van drvan inaktivwerd een nDe isolerinschreven inmerkers wckregen. Hcbehoren detransformervan merkerbij transforgekoppeldstam werdeDe inaktiveen I I I  bes ,bestraald nmee te t rarongelijke nàeze differwaalmee P(deze hyportweetal  korgevoelighedDNA, die rheden trandan met overzadigencongemerktegreerd danmeer PoteÍkeli jke gev<opgelost dctraties DNIkatie ortStrmerkers inkomen,  wet l- l

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 University of Groningen
Ultraviolet inactivation and excision-repair in bacillus subtilus
Bron, Sierd
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1972
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Citation for published version (APA):
Bron, S. (1972). Ultraviolet inactivation and excision-repair in bacillus subtilus s.n.
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SUMMARY
This  thcs is  cons is ts  o f  a  co l l cc t ion  o f  5  papcrs  dca l ing  w i th  u l t rav io lc r  inac t iva t ion
at rd  cxc is ion- rcpa i r  in  the  t r : rns fo r rnab le  bac ter iunr  Bac i l lus  subt i l i s ,  spcc ia l l y  w i th
resPcc t  to  t ra t ts fo rn ' r ing  DNA.  Onc o f  thc  m: r in  ob lcc t rvcs  was to  s t r - rdy  UV
i r r a c i i v a t i o r r  o f  a  n u t n b e r  o f  t r a r r s f o r r n i n g  n r : r r k c r s .  T o  t h a t  p u r p ( ) s c . : l  n r u l t i p l c
aL lxo i rc - 'ph i t  t - ' ru tá r t r t .  s r : . r i r  BG-5 ,  w i l s  cons t ru lc tcd .  wh ich  scrvcd  as  thc  rcc ip icn t
s t ra i r r .  Thc  procedurc  o f  i so la t ion  and the  ch : r rac tc r iza t ion  o f  s t ra in  BG-5 arc
descr ibc ,d  in  papcr  l .  Th is  s t ra in  t rans forms c lu i te  sz r t i s fac to ry :  s ing le  n rarkcr
t ra t ts fo r tna t io t r  f i ' cquenc ics  o f  : i  few pcrccn t  a rc  usua l l t ,  ob ta incc l .  A l thouch
thc v:rr ious mark"r ,  
" . "  
s l ight ly di f fercnt  wi th rcspccr to 
" f f i . i . , r .1, . r f  r r , , , i r -fo r - tna t io t r ,  t l i cy  p robab ly  bc long to  on ly  onc  c lass .  a l l  bc ing  o f  thc  h ish-
c f f i c ic t rcy  typc .  Thc  markers  were  loca tcd  re la t i vc  to  cach o thcr  by  , r r " r , . , ,
o f  rnarkcr  f rcc luency  ar - ra lys is .  Of  the  B nrarkers  s tud icd .  t rp , , ,  a r rc i  t y r í  Arc
c losc ly  l inkcd  in  t rans format ion .  Thc  r ib  rnarkcr  wá ls  shown-  to  be  wcak ly
l inked to  thc  t rp  r - ty r r  pa i r  o f  n rarkers .  No fu r ther  l inkagcs  c -x is t  bcrwccn
thc  r la rkcrs  in  s t r : r in  BG-5 .
UV inac t iv : r t io t r  o f  thc  B  rnarkcrs  i s  dcscr ibcd  in  papcrs  l l  anc l  I l t .  DNA iso la tcc l
f ro tn  p ro to t roph ic  cc l l s  w; rs  i r rad ia ted  w i t l i  var ious  doscs  o f  UV: rnd  subscc lucn t ly
uscd to  t rans for tn  8G-5  rec ip icn t  cc l l s .  Thc  rcsu l ts  dcnrons t ra tc  tha t  thc  vur ious
nt : r rkc rs  a rc  inac t iva tcd  a t  d i f f c rcn t  ra tcs .  Or - r r  work ing  hypothcs is  was th r r t
d i f fe rcn t ia l  markcr  inac t iva t ion  is  duc  to  d i f f c rcnccs  in  t l - rc  f rcc lucncv , - , i
po tcn t ia l  photochcrn ica l  les ions  in  the  rnarkcd  segmcnts  o f  ONA.  Howcvcr .
in tc rpre ta t ion  o f  the  resu l ts  o f  our  marker  inac t iva t ion  cxper in ren ts  accord i r rg
to  th is  hypothes is  w: rs  compl ica ted  fo r  a t  l cas t  two rcasons .  F i rs t l y .  thc  rcsu l ts
show tha t  marker  scns i t i v i t i cs  to  UV depend on  thc  DNA conccnt ra r lo l l  c iu r ing
t rans for tna t ion .  A t  sa tura t ing  amounts  o f  DNA,  thc  rnarkcrs  a rc  : rpparcn t ly
n lo re  scns i t i vc  to  UV than á i t  non-sa tura t ing  ár ïnounts .  Th is  i s  p robab ly  duc
t o  t h e  f a c t  t h a t  a t  s a t u r a t i n g  a m o u n t s  o f  D N A ,  i n  a c i d i t i o n  t o  t h c  m o l c c u l c s
car ry ing  se lec tcd  nrarkcrs .  morc  non-sc lec tcd  danragcc l  rno lccu lcs  : r rc  in tcgra tcd
in to  thê  rec ip icn t  genomc th : rn  a t  non-s i l tu r l r t ing  iu r louuts .  Thcrc fo rc .  r r - lo rc
poten t ia l  t rans fornran ts  a rc  k i l l cd  undcr  thc  fo rnrc r  cond i t ions .  wh ich  rcsu l ts
in  overes t in la t ion  o f  n rarkcr  scns i t i v i t i cs .  Th is  compl icu t io r . r  was  avo idcd  by
deter rn in ing  the  markcr  scns i t i v i t i cs  to  UV a t  DNA conccnt r r r t io r " rs  fa r  bc low
satura t ion .  Second ly ,  cornp l i ca t ions  w i l l  a lso  ar isc ,  i f  a f t " r  up takc  l . r r  rcc ip ic r r t
ce l l s ,  the  inac t iva tcd  mzt rkers  a rc  rcpa i rcd  to  d i f f c rcn t  dcgrccs .  To  uvo id  t l r i s
compl icat ion,  an excis ion-repair-def ic ient  c ler ivat ivc w;rs iÀolatcd f rorn strain
B G - 5 .  T h c  p r o p c r t i c s  o f  t h i s  s t r : r i n : r r c  a l s o  d c s c r i b e d  i n  p a p c r  l .  T h i s  s r n r i n .
B G - 1  1 ,  i s  u n a b l c  t o  c x c i s e  p y r i m i c l i n c  d i n r c r s .  t o  r c a s s u r r r c  D N A  s v n t h c s i s  a f t c r
U V  i r r a d i a t i o r - r ,  a n d  t o  r c a c t i v u t c  U V - i n a c t i v a t c d  b a c t c r i o p h a q c .  A s  c o r n p l r c c l
t t t  s t r : t i t - t  I JG-5 ,  i t  t rans for r r rs  nornra l l v  r ,v i r l r  t rn i r r l c l i l t c r ]  I )NA.  bu t  1 ' , . . r , , r l v
w i t h  U V - i r r a c l i : r t c c l  l ) N A .  B c c : r r - r s c  o f  t l r c s c  p r o p c r t i c s  s r r i r i n  S ( ;  l l  . 1 , , n  t r . ,
1 8
-classi f ied as being def ic ient  in excis ion-repair  (uvr-) .  When in v ivo photo-
react ivat ion of  UV-irradiated donor DNA is prevented by working undcr dim
l ight ,  ancl  excis ion-repair  by using strain 8G-11 as recipient,  d i f ferent ia l  marker
inact ivat ion by UV of 254 nm st i l l  exists (paper I I ) ,  which supports thc hy-
pothesis that  the f requency of  potent ia l  photochemical  lesions determines
the sensi t iv i ty to UV of  the marked segmenrs of  ONA.
Comparison of the results on marker inactivation obtained with strain BG-5
and strain BG-11 al lowed us to compute the f ract ion of  inact ivat ing lesions
rvhich is not repaired by the 8G-5 (uvr+) recipient.  The size of  th is f ract ion
is marker-specific and varies from 13t/o to 26o/o, indicating that excision-repair
is not equally efficient for different markers in irradiated DNA. On the average
about 80' /o of  the induced lesions are succesful ly repaired.
The involvement 
"f 
fr dimers in both differeniial rnarker inactivation and
differential marker repair was studied in the following way. Transforming
DNA was i r radiated with 313 nm l ight  in the presence of  acerophenone as
sensi t izer.  Under these condi t ions exclusively TT dimers should be induc 
"642,43 
.
H^owever,  our resul ts (presented in paper I I I )  demonstrate that ,  in addi t ion to
TT dimers,  non repairable s ingle-stranded DNA breaks are : r lso produced. In
vi t ro photoreact ivat ion of  the TT dimers in the i r radiated DNA al lowed us
to colnPute the relat ive contr ibut ions of  the dirners and the single-stranded
DNA breaks to total  marker sensi t iv i t ies.  Al though only one break occurs per
approximately 90 f f  d imers,  i t  appears that  the breaks account for  abàut
20' /o of  the observed marker sensi t iv i t ies.
Di f ferent ia l  marker inact ivat ion and di f ferent ia l  markcr repair  werc also ob-
served in thc acetophenone systein of  i r radiat ion.  Thc resul ts dernonstrate thar,
i r respect ive of  the uvr genotype of  the recipicnt ,  thc s inglc-strandcd DNA brcaLs
inact ivate the var ious markers to the same extcnt.  From this observat ion i t
can be concluded that in the acetophenonc system di f ferent ia l  marker in-
act ivat ion is exclusively duc to di f fercnces in t i re f requency 
" f  
f f  d i rncrs in
the marked segments of  DNA. In addi t ion.  d i f fe rcnt ia i  markcr rcpair  is  exclusively
due to di f ferenccs in thc ef f ic iency of  repair  of  r r  d imcrs.  Support  for  thc
conclusion that di f ference s in the f requency 
" f  
f r  d imers in thc rrr" . t "d scgmenrs
of DNA causc di f fcrcnt ia l  rnarkcr inact ivat ion is that  photoreact ivat ion exper-
imcnts  ind ica tc  tha t  exce l l cn t  cor re la t ion  ex is ts  be tween the  f requency  o f
TT dirners in thc rnarked scgmcnts of  DNA arrd thc scnsi t iv i ty , r f  th.  
'^^r-
kc rs  to  i r rad ia t ion  in  thc  acc tophenonc sys tem.  Fur thcr  suppor t  fo r  th is  con-
c lus ion  is  p rov idcc l  by  thc  obscrva t ion  tha t ,  as  judge d  f ro rn  heat  dcnarurar ion
s tud ics ,  markcrs  rc la t i vc ly  r i ch  in  thyrn inc  usua l ly  a rc  rc la r ivc ly  scns i t i ve  to
i r rad ia t ion  and,  convcrsa ly ,  tha t  tnarkers  rc la t i ve ly  poor  in  thyrn ine  usua l ly
are  rc la t i vc lv  rcs is tan t  to  i r rad ia t ion .
Cot r ipar ison  c , f  thc  rcsu l ts  ob ta i r - rcd  in  thc  acc tophcnonc sys tc rn  w i th  those
obta incd  in  thc  254 nrn  svs tc rn  o f  i r rad ia t ion ,  shows tha t  bo th  t l - rc  rc la t i vc
ordcrs , .> f  n ta rkcr  sc r rs i t i v i t i t : s  and o f  rnarkcr  rcpa i rab i l i t i cs  a rc  c lc - rse ly  s imi la r
rn  thc  two svs tcnrs  o f  i r rad ia t ion .  Wc cons idcr  t i r i s  as  cv idcncc  t l ia t  a lso  in  thc
l 9
2 5 4  t
mark
are t l
The
the  e
firstll
the  k
of  U'
lo l lo r
recipi
and s
of d:
abscn
juclue
+uvr
transl
is int
gratel
irradi
cxc lu
concl i
atnou
T h c  I
str-rdit
strai  n
Thc  I
and
tnolc,
rc iso l
iÀ int
20-4 !
for f
datna
r e e x t
S C C  O T
Thc 1
was  I
hypo
t h c  c
t igatr
t h c  c
l igasc
signi l
t y  c c
vVhen in v ivo photo-
y working under dim
rt, differential marker
hich supporrs rhe hy-
r l  lesions determines
red with strain 8G-5
f inact ivat ing lesions
: size of this fraction
g that excision-repair
DNA. On the average
'ker inact ivat ion and
i way. Transforming
of  ace tophenone as
u ld  be  induceda 2  ,43  
.
:  thar ,  in  add i t ion  to
re  a lso  produced.  In
ed DNA al lowed us
I  the s ingle-stranded
rne break occurs per
s account for  about
repair  werc also ob-
I ts c le morlstrate that ,
t randcd DNA breaLs
r th is obscrvat ion i t
í c rc r r t ia l  markcr  in -
rcy of ff di.n"rs in
r  rcpair  is  e xclusivcly
crs.  Support  for  the
hc nrarked scgments
orcact ivat ion exper-
r  the f requency of
s i t iv i ty of  the mar-
rPporr  for  th is con-
r  l icat  dcnaturat ion
at ivc ly  scns i t i ve  to
n  thyminc  usua l ly
sys tcm wi th  those
both  thc  rc la t i ve
arc c loscly s i rn i lar
rcc  tha t  a lso  in  thc
254 nm system of irradiation TT dimers are the main cause for differential
marker inactivation and that differences in the efficiency of repair 
"f 
fT dimers
are the main cause for differential marker repair.
The fate of UV lesions in transforming DNA was studied more detailed in
the exper iments descr ibed in paper IV.  The pr incipal  a ims were to invest igate:
f i rst ly,  genet ic integrat ion of  UV-irradiated transforming DNA, and secondly,
the k inet ics of  in v ivo repair  of  Uv- inact ivated transforming DNA. Tl-re ef fect
of  UV lesions in t ransÍorming DNA on gener ic intcgrat ion was studied in the
Íc. ' l lowing way. UV-irradiated DNA was used to t ransform a repair less uvr-
recipient.  Af tcr  a l lowing t ime for intcgrat ion to occur,  DNA was reextracted
and subsequent ly assayed on uvr-  and uvr-  second recipicnts for  the prcsence
of dark-repairable damage in rccombinant type moleculcs.  " ihe presence or
absence of  dark-repairable darnage in thc recombinant type molccules was
judged by the number of  recornbinant typc t ransformants obt: i incd on the
uvr*  re la t i ve  to  the  number  on  the  uvr -  second rcc ip ien t .  Thc  rcsu l ts  show tha t
transfortning DNA irradiatcd wi th modcrate doscs of  UV (2,000 erg/mrn2)
is integrated almost equal ly ef f ic , icnt ly : rs non- i r radiarcd DNA, and that inrc-
grated UV lesions account alrnost exclusivcly for  t l ie inact iv:r t ion.  Howcver,
i r rad ia t ion  o f  thc  DNA wi th  20 .000 
" .g / - -2  o f  uV rcsu l ts  in  a  s ign i f i can t
exclusion of  darnagcd DNA frorn th" p.oc"ss of  intcgrar ion.  Under thcse
cond i t ions ,  the  cont r ibu t ion  o f  thc  exc lus ion  to  to ta l  markcr  inac t iva t ion
amounts  to  approx i rna te ly  1 }o / , t  in  uvr *  and to  less  than 7oh in  uvr -  rcc ip icn ts .
The kinet ics of  in v ivo cxcis ion-repair  of  Uv- i r radiated rransforming DNA we rc
s tud icd  by  a  c losc ly  s imi la r  method.  ln  th is  case.  e r  rcpa i r -p ro f i c icn t  uvr *
s t ra in  sc rvcd  as  thc  f i rs t  rcc ip ien t  in  t rans format ion  w i th  UV- i r rad ia rcd  DNA.
The DNA was rccx t rac tcd  a t  var ious  in te rva ls  a f te r  n r i r ing  cc l l s  ; inc i  donor  DNA,
and the rcsidual  amount of  dark-repairablc damage in recombin: int  ry pc
tno iccu les  in  the  rcex t rac ted  DNA was de tc rmincd as  a  func t ion  o f  t i rnc  o f
re iso la t ion .  Thc  rcsu l ts  ind ica te  tha t  repa i r  on ly  s ta r ts  a f - tc r  the  donor  DNA
i i  i r - r tcgra tcd  in to  the  rcc ip ien t  gcnomc.  Cornp le t ion  o f  repa i r  rcqLr i res  f ro r r r
20-45  rn in  fo r  DNA i r rad ia tcd  w i th  2 ,000 c rg /n r rn l ,  and approx i r r ia tc ly  2  h
fo r  DNA i r rad i : r ted  w i th  15 ,000 erg lmrn2.  T l i c  rcsu l ts  a lso  i r - rd ica tc  rha t  UV-
danragcd cc l ipse  phase DNA (DNA tha t  i s  taken up  bu t  nor  in tcgra tcd)
reex t rac tcd  f rom rec ip icn t  ce l l s ,  i s  no t  sub jcc t  to  cxc is ion- rcpa i r  in  uur*
s c c o n d  r c c i p i e n t  s t r a i n s .
The poss ib i l i t y  o f  in  v i t ro  cxc is ion- rcpa i r  o f  UV- inac t iva tcd  t rans for r r i ing  DNA
was s tud ied  in  thc  expcr i rnents  dcscr ibcd  in  thc  las t  papcr  o i  th is  t l r cs is .  Thc
hypothes is  pu t  fo rwardby  KELLY e t  a l .a0  th r t  DNA po lynrerasc  pcr fc , r r r rs  bo th
the  cxc is ion  and the  rep l i ca t ion  s tep  in  exc is ion- repa i r ,  p ron lp rcd  us  ro  invcs-
t iga tc  whcthcr  UV- i r rad ia ted  t rans forming  DNA can bc  rcpa i rcd  in  v i t ro  by
the  combined ac t ion  o f  UV-spcc i f i c  cndonuc lcasc ,  DNA po ly rncrasc  and DNA
l igasc. Thc resul ts show th:r t  the biological  : rct iv i ty of  inact ivarcd DNA incrc: : rses
s ign i f i can t ly  fo l low ing  incubat ion  w i th  the  th rec  cnzyrnes .  Thc  rcs to rcd  acr iv i -
ty  can  no t  bc  fu r thcr  inc reascd by  photoreac t iva t ion  in  v i t ro .  ind ica t ing  r l ra t
20
-photorepairable dimcrs are no longer present.  I t  could be demonstrated that
ái-"rr  àre speci f ical ly excised upon incubat ion of  the i r radiated DNA with
the thre e enzymes, and that under our condi t ions approximatciy 50' /o of  thc
induccdlesionsare succesful ly repaired. These rcsul ts s l - row that,  at  lcast  in v i t ro,
KELLY's40 mode l  app l i cs  to  repa i r  o f  UV les ions  in  DNA.  Whether  th is  mode l
also appl ics io repair  in v ivo,  remains to be establ ished. I t  may also be envisaged
that al tcrnat ive pathwat 's of  excis ion-re pair  exist .
Dit proefscl
heid voor
subt i l is ,  de
herstel van
Eén van dr
van inaktiv
werd een n
De isolerin
schreven in
merkers wc
kregen. Hc
behoren de
transformer
van merker
bij transfor
gekoppeld
stam werde
De inaktive
en I I I  bes ,
bestraald n
mee te t rar
ongelijke n
àeze differ
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DNA, die r
heden tran
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- l


   University of GroningenUltraviolet inactivation and excision-repair in bacillus subtilusBron, SierdIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1972Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bron, S. (1972). Ultraviolet inactivation and excision-repair in bacillus subtilus. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 30-10-2023SUMMARYThis  thcs is  cons is ts  o f  a  co l l cc t ion  o f  5  papcrs  dca l ing  w i th  u l t rav io lc r  inac t iva t ionat rd  cxc is ion- rcpa i r  in  the  t r : rns fo r rnab le  bac ter iunr  Bac i l lus  subt i l i s ,  spcc ia l l y  w i thresPcc t  to  t ra t ts fo rn ' r ing  DNA.  Onc o f  thc  m: r in  ob lcc t rvcs  was to  s t r - rdy  UVi r r a c i i v a t i o r r  o f  a  n u t n b e r  o f  t r a r r s f o r r n i n g  n r : r r k c r s .  T o  t h a t  p u r p ( ) s c . : l  n r u l t i p l caL lxo i rc - 'ph i t  t - ' ru tá r t r t .  s r : . r i r  BG-5 ,  w i l s  cons t ru lc tcd .  wh ich  scrvcd  as  thc  rcc ip icn ts t ra i r r .  Thc  procedurc  o f  i so la t ion  and the  ch : r rac tc r iza t ion  o f  s t ra in  BG-5 arcdescr ibc ,d  in  papcr  l .  Th is  s t ra in  t rans forms c lu i te  sz r t i s fac to ry :  s ing le  n rarkcrt ra t ts fo r tna t io t r  f i ' cquenc ics  o f  : i  few pcrccn t  a rc  usua l l t ,  ob ta incc l .  A l thouchthc v:rr ious mark"r ,  " . "  s l ight ly di f fercnt  wi th rcspccr to " f f i . i . , r .1, . r f  r r , , , i r -fo r - tna t io t r ,  t l i cy  p robab ly  bc long to  on ly  onc  c lass .  a l l  bc ing  o f  thc  h ish-c f f i c ic t rcy  typc .  Thc  markers  were  loca tcd  re la t i vc  to  cach o thcr  by  , r r " r , . , ,o f  rnarkcr  f rcc luency  ar - ra lys is .  Of  the  B nrarkers  s tud icd .  t rp , , ,  a r rc i  t y r í  Arcc losc ly  l inkcd  in  t rans format ion .  Thc  r ib  rnarkcr  wá ls  shown-  to  be  wcak lyl inked to  thc  t rp  r - ty r r  pa i r  o f  n rarkers .  No fu r ther  l inkagcs  c -x is t  bcrwccnthc  r la rkcrs  in  s t r : r in  BG-5 .UV inac t iv : r t io t r  o f  thc  B  rnarkcrs  i s  dcscr ibcd  in  papcrs  l l  anc l  I l t .  DNA iso la tcc lf ro tn  p ro to t roph ic  cc l l s  w; rs  i r rad ia ted  w i t l i  var ious  doscs  o f  UV: rnd  subscc lucn t lyuscd to  t rans for tn  8G-5  rec ip icn t  cc l l s .  Thc  rcsu l ts  dcnrons t ra tc  tha t  thc  vur iousnt : r rkc rs  a rc  inac t iva tcd  a t  d i f f c rcn t  ra tcs .  Or - r r  work ing  hypothcs is  was th r r td i f fe rcn t ia l  markcr  inac t iva t ion  is  duc  to  d i f f c rcnccs  in  t l - rc  f rcc lucncv , - , ipo tcn t ia l  photochcrn ica l  les ions  in  the  rnarkcd  segmcnts  o f  ONA.  Howcvcr .in tc rpre ta t ion  o f  the  resu l ts  o f  our  marker  inac t iva t ion  cxper in ren ts  accord i r rgto  th is  hypothes is  w: rs  compl ica ted  fo r  a t  l cas t  two rcasons .  F i rs t l y .  thc  rcsu l tsshow tha t  marker  scns i t i v i t i cs  to  UV depend on  thc  DNA conccnt ra r lo l l  c iu r ingt rans for tna t ion .  A t  sa tura t ing  amounts  o f  DNA,  thc  rnarkcrs  a rc  : rpparcn t lyn lo re  scns i t i vc  to  UV than á i t  non-sa tura t ing  ár ïnounts .  Th is  i s  p robab ly  duct o  t h e  f a c t  t h a t  a t  s a t u r a t i n g  a m o u n t s  o f  D N A ,  i n  a c i d i t i o n  t o  t h c  m o l c c u l c scar ry ing  se lec tcd  nrarkcrs .  morc  non-sc lec tcd  danragcc l  rno lccu lcs  : r rc  in tcgra tcdin to  thê  rec ip icn t  genomc th : rn  a t  non-s i l tu r l r t ing  iu r louuts .  Thcrc fo rc .  r r - lo rcpoten t ia l  t rans fornran ts  a rc  k i l l cd  undcr  thc  fo rnrc r  cond i t ions .  wh ich  rcsu l tsin  overes t in la t ion  o f  n rarkcr  scns i t i v i t i cs .  Th is  compl icu t io r . r  was  avo idcd  bydeter rn in ing  the  markcr  scns i t i v i t i cs  to  UV a t  DNA conccnt r r r t io r " rs  fa r  bc lowsatura t ion .  Second ly ,  cornp l i ca t ions  w i l l  a lso  ar isc ,  i f  a f t " r  up takc  l . r r  rcc ip ic r r tce l l s ,  the  inac t iva tcd  mzt rkers  a rc  rcpa i rcd  to  d i f f c rcn t  dcgrccs .  To  uvo id  t l r i scompl icat ion,  an excis ion-repair-def ic ient  c ler ivat ivc w;rs iÀolatcd f rorn strainB G - 5 .  T h c  p r o p c r t i c s  o f  t h i s  s t r : r i n : r r c  a l s o  d c s c r i b e d  i n  p a p c r  l .  T h i s  s r n r i n .B G - 1  1 ,  i s  u n a b l c  t o  c x c i s e  p y r i m i c l i n c  d i n r c r s .  t o  r c a s s u r r r c  D N A  s v n t h c s i s  a f t c rU V  i r r a d i a t i o r - r ,  a n d  t o  r c a c t i v u t c  U V - i n a c t i v a t c d  b a c t c r i o p h a q c .  A s  c o r n p l r c c lt t t  s t r : t i t - t  I JG-5 ,  i t  t rans for r r rs  nornra l l v  r ,v i r l r  t rn i r r l c l i l t c r ]  I )NA.  bu t  1 ' , . . r , , r l vw i t h  U V - i r r a c l i : r t c c l  l ) N A .  B c c : r r - r s c  o f  t l r c s c  p r o p c r t i c s  s r r i r i n  S ( ;  l l  . 1 , , n  t r . ,1 8-classi f ied as being def ic ient  in excis ion-repair  (uvr-) .  When in v ivo photo-react ivat ion of  UV-irradiated donor DNA is prevented by working undcr diml ight ,  ancl  excis ion-repair  by using strain 8G-11 as recipient,  d i f ferent ia l  markerinact ivat ion by UV of 254 nm st i l l  exists (paper I I ) ,  which supports thc hy-pothesis that  the f requency of  potent ia l  photochemical  lesions determinesthe sensi t iv i ty to UV of  the marked segmenrs of  ONA.Comparison of the results on marker inactivation obtained with strain BG-5and strain BG-11 al lowed us to compute the f ract ion of  inact ivat ing lesionsrvhich is not repaired by the 8G-5 (uvr+) recipient.  The size of  th is f ract ionis marker-specific and varies from 13t/o to 26o/o, indicating that excision-repairis not equally efficient for different markers in irradiated DNA. On the averageabout 80' /o of  the induced lesions are succesful ly repaired.The involvement "f fr dimers in both differeniial rnarker inactivation anddifferential marker repair was studied in the following way. TransformingDNA was i r radiated with 313 nm l ight  in the presence of  acerophenone assensi t izer.  Under these condi t ions exclusively TT dimers should be induc "642,43 .H^owever,  our resul ts (presented in paper I I I )  demonstrate that ,  in addi t ion toTT dimers,  non repairable s ingle-stranded DNA breaks are : r lso produced. Invi t ro photoreact ivat ion of  the TT dimers in the i r radiated DNA al lowed usto colnPute the relat ive contr ibut ions of  the dirners and the single-strandedDNA breaks to total  marker sensi t iv i t ies.  Al though only one break occurs perapproximately 90 f f  d imers,  i t  appears that  the breaks account for  abàut20' /o of  the observed marker sensi t iv i t ies.Di f ferent ia l  marker inact ivat ion and di f ferent ia l  markcr repair  werc also ob-served in thc acetophenone systein of  i r radiat ion.  Thc resul ts dernonstrate thar,i r respect ive of  the uvr genotype of  the recipicnt ,  thc s inglc-strandcd DNA brcaLsinact ivate the var ious markers to the same extcnt.  From this observat ion i tcan be concluded that in the acetophenonc system di f ferent ia l  marker in-act ivat ion is exclusively duc to di f fercnces in t i re f requency " f  f f  d i rncrs inthe marked segments of  DNA. In addi t ion.  d i f fe rcnt ia i  markcr rcpair  is  exclusivelydue to di f ferenccs in thc ef f ic iency of  repair  of  r r  d imcrs.  Support  for  thcconclusion that di f ference s in the f requency " f  f r  d imers in thc rrr" . t "d scgmenrsof DNA causc di f fcrcnt ia l  rnarkcr inact ivat ion is that  photoreact ivat ion exper-imcnts  ind ica tc  tha t  exce l l cn t  cor re la t ion  ex is ts  be tween the  f requency  o fTT dirners in thc rnarked scgmcnts of  DNA arrd thc scnsi t iv i ty , r f  th.  '^^r-kc rs  to  i r rad ia t ion  in  thc  acc tophenonc sys tem.  Fur thcr  suppor t  fo r  th is  con-c lus ion  is  p rov idcc l  by  thc  obscrva t ion  tha t ,  as  judge d  f ro rn  heat  dcnarurar ions tud ics ,  markcrs  rc la t i vc ly  r i ch  in  thyrn inc  usua l ly  a rc  rc la r ivc ly  scns i t i ve  toi r rad ia t ion  and,  convcrsa ly ,  tha t  tnarkers  rc la t i ve ly  poor  in  thyrn ine  usua l lyare  rc la t i vc lv  rcs is tan t  to  i r rad ia t ion .Cot r ipar ison  c , f  thc  rcsu l ts  ob ta i r - rcd  in  thc  acc tophcnonc sys tc rn  w i th  thoseobta incd  in  thc  254 nrn  svs tc rn  o f  i r rad ia t ion ,  shows tha t  bo th  t l - rc  rc la t i vcordcrs , .> f  n ta rkcr  sc r rs i t i v i t i t : s  and o f  rnarkcr  rcpa i rab i l i t i cs  a rc  c lc - rse ly  s imi la rrn  thc  two svs tcnrs  o f  i r rad ia t ion .  Wc cons idcr  t i r i s  as  cv idcncc  t l ia t  a lso  in  thcl 92 5 4  tmarkare t lThethe  efirstllthe  kof  U'lo l lo rrecipiand sof d:abscnjuclue+uvrtranslis intgratelirradicxc luconcl iatnouT h c  Istr-rditstrai  nThc  Iandtnolc,rc iso liÀ int20-4 !for fdatnar e e x tS C C  O TThc 1was  Ihypot h c  ct igatrt h c  cl igascsigni lt y  c cvVhen in v ivo photo-y working under dimrt, differential markerhich supporrs rhe hy-r l  lesions determinesred with strain 8G-5f inact ivat ing lesions: size of this fractiong that excision-repairDNA. On the average'ker inact ivat ion andi way. Transformingof  ace tophenone asu ld  be  induceda 2  ,43  .:  thar ,  in  add i t ion  tore  a lso  produced.  Ined DNA al lowed usI  the s ingle-strandedrne break occurs pers account for  aboutrepair  werc also ob-I ts c le morlstrate that ,t randcd DNA breaLsr th is obscrvat ion i tí c rc r r t ia l  markcr  in -rcy of ff di.n"rs inr  rcpair  is  e xclusivclycrs.  Support  for  thehc nrarked scgmentsorcact ivat ion exper-r  the f requency ofs i t iv i ty of  the mar-rPporr  for  th is con-r  l icat  dcnaturat ionat ivc ly  scns i t i ve  ton  thyminc  usua l lysys tcm wi th  thoseboth  thc  rc la t i vearc c loscly s i rn i larrcc  tha t  a lso  in  thc254 nm system of irradiation TT dimers are the main cause for differentialmarker inactivation and that differences in the efficiency of repair "f fT dimersare the main cause for differential marker repair.The fate of UV lesions in transforming DNA was studied more detailed inthe exper iments descr ibed in paper IV.  The pr incipal  a ims were to invest igate:f i rst ly,  genet ic integrat ion of  UV-irradiated transforming DNA, and secondly,the k inet ics of  in v ivo repair  of  Uv- inact ivated transforming DNA. Tl-re ef fectof  UV lesions in t ransÍorming DNA on gener ic intcgrat ion was studied in theÍc. ' l lowing way. UV-irradiated DNA was used to t ransform a repair less uvr-recipient.  Af tcr  a l lowing t ime for intcgrat ion to occur,  DNA was reextractedand subsequent ly assayed on uvr-  and uvr-  second recipicnts for  the prcsenceof dark-repairable damage in rccombinant type moleculcs.  " ihe presence orabsence of  dark-repairable darnage in thc recombinant type molccules wasjudged by the number of  recornbinant typc t ransformants obt: i incd on theuvr*  re la t i ve  to  the  number  on  the  uvr -  second rcc ip ien t .  Thc  rcsu l ts  show tha ttransfortning DNA irradiatcd wi th modcrate doscs of  UV (2,000 erg/mrn2)is integrated almost equal ly ef f ic , icnt ly : rs non- i r radiarcd DNA, and that inrc-grated UV lesions account alrnost exclusivcly for  t l ie inact iv:r t ion.  Howcver,i r rad ia t ion  o f  thc  DNA wi th  20 .000 " .g / - -2  o f  uV rcsu l ts  in  a  s ign i f i can texclusion of  darnagcd DNA frorn th" p.oc"ss of  intcgrar ion.  Under thcsecond i t ions ,  the  cont r ibu t ion  o f  thc  exc lus ion  to  to ta l  markcr  inac t iva t ionamounts  to  approx i rna te ly  1 }o / , t  in  uvr *  and to  less  than 7oh in  uvr -  rcc ip icn ts .The kinet ics of  in v ivo cxcis ion-repair  of  Uv- i r radiated rransforming DNA we rcs tud icd  by  a  c losc ly  s imi la r  method.  ln  th is  case.  e r  rcpa i r -p ro f i c icn t  uvr *s t ra in  sc rvcd  as  thc  f i rs t  rcc ip ien t  in  t rans format ion  w i th  UV- i r rad ia rcd  DNA.The DNA was rccx t rac tcd  a t  var ious  in te rva ls  a f te r  n r i r ing  cc l l s  ; inc i  donor  DNA,and the rcsidual  amount of  dark-repairablc damage in recombin: int  ry pctno iccu les  in  the  rcex t rac ted  DNA was de tc rmincd as  a  func t ion  o f  t i rnc  o fre iso la t ion .  Thc  rcsu l ts  ind ica te  tha t  repa i r  on ly  s ta r ts  a f - tc r  the  donor  DNAi i  i r - r tcgra tcd  in to  the  rcc ip ien t  gcnomc.  Cornp le t ion  o f  repa i r  rcqLr i res  f ro r r r20-45  rn in  fo r  DNA i r rad ia tcd  w i th  2 ,000 c rg /n r rn l ,  and approx i r r ia tc ly  2  hfo r  DNA i r rad i : r ted  w i th  15 ,000 erg lmrn2.  T l i c  rcsu l ts  a lso  i r - rd ica tc  rha t  UV-danragcd cc l ipse  phase DNA (DNA tha t  i s  taken up  bu t  nor  in tcgra tcd)reex t rac tcd  f rom rec ip icn t  ce l l s ,  i s  no t  sub jcc t  to  cxc is ion- rcpa i r  in  uur*s c c o n d  r c c i p i e n t  s t r a i n s .The poss ib i l i t y  o f  in  v i t ro  cxc is ion- rcpa i r  o f  UV- inac t iva tcd  t rans for r r i ing  DNAwas s tud ied  in  thc  expcr i rnents  dcscr ibcd  in  thc  las t  papcr  o i  th is  t l r cs is .  Thchypothes is  pu t  fo rwardby  KELLY e t  a l .a0  th r t  DNA po lynrerasc  pcr fc , r r r rs  bo ththe  cxc is ion  and the  rep l i ca t ion  s tep  in  exc is ion- repa i r ,  p ron lp rcd  us  ro  invcs-t iga tc  whcthcr  UV- i r rad ia ted  t rans forming DNA can bc  rcpa i rcd  in  v i t ro  bythe  combined ac t ion  o f  UV-spcc i f i c  cndonuc lcasc ,  DNA po ly rncrasc  and DNAl igasc. Thc resul ts show th:r t  the biological  : rct iv i ty of  inact ivarcd DNA incrc: : rsess ign i f i can t ly  fo l low ing  incubat ion  w i th  the  th rec  cnzyrnes .  Thc  rcs to rcd  acr iv i -ty  can  no t  bc  fu r thcr  inc reascd by  photoreac t iva t ion  in  v i t ro .  ind ica t ing  r l ra t20-photorepairable dimcrs are no longer present.  I t  could be demonstrated thatái-"rr  àre speci f ical ly excised upon incubat ion of  the i r radiated DNA withthe thre e enzymes, and that under our condi t ions approximatciy 50' /o of  thcinduccdlesionsare succesful ly repaired. These rcsul ts s l - row that,  at  lcast  in v i t ro,KELLY's40 mode l  app l i cs  to  repa i r  o f  UV les ions  in  DNA.  Whether  th is  mode lalso appl ics io repair  in v ivo,  remains to be establ ished. I t  may also be envisagedthat al tcrnat ive pathwat 's of  excis ion-re pair  exist .Dit proefsclheid voorsubt i l is ,  deherstel vanEén van drvan inaktivwerd een nDe isolerinschreven inmerkers wckregen. Hcbehoren detransformervan merkerbij transforgekoppeldstam werdeDe inaktiveen I I I  bes ,bestraald nmee te t rarongelijke nàeze differwaalmee P(deze hyportweetal  korgevoelighedDNA, die rheden trandan met overzadigencongemerktegreerd danmeer PoteÍkeli jke gev<opgelost dctraties DNIkatie ortStrmerkers inkomen,  wet l- l

   University of GroningenUltraviolet inactivation and excision-repair in bacillus subtilusBron, SierdIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1972Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Bron, S. (1972). Ultraviolet inactivation and excision-repair in bacillus subtilus. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 09-09-2023SUMMARYThis  thcs is  cons is ts  o f  a  co l l cc t ion  o f  5  papcrs  dca l ing  w i th  u l t rav io lc r  inac t iva t ionat rd  cxc is ion- rcpa i r  in  the  t r : rns fo r rnab le  bac ter iunr  Bac i l lus  subt i l i s ,  spcc ia l l y  w i thresPcc t  to  t ra t ts fo rn ' r ing  DNA.  Onc o f  thc  m: r in  ob lcc t rvcs  was to  s t r - rdy  UVi r r a c i i v a t i o r r  o f  a  n u t n b e r  o f  t r a r r s f o r r n i n g  n r : r r k c r s .  T o  t h a t  p u r p ( ) s c . : l  n r u l t i p l caL lxo i rc - 'ph i t  t - ' ru tá r t r t .  s r : . r i r  BG-5 ,  w i l s  cons t ru lc tcd .  wh ich  scrvcd  as  thc  rcc ip icn ts t ra i r r .  Thc  procedurc  o f  i so la t ion  and the  ch : r rac tc r iza t ion  o f  s t ra in  BG-5 arcdescr ibc ,d  in  papcr  l .  Th is  s t ra in  t rans forms c lu i te  sz r t i s fac to ry :  s ing le  n rarkcrt ra t ts fo r tna t io t r  f i ' cquenc ics  o f  : i  few pcrccn t  a rc  usua l l t ,  ob ta incc l .  A l thouchthc v:rr ious mark"r ,  " . "  s l ight ly di f fercnt  wi th rcspccr to " f f i . i . , r .1, . r f  r r , , , i r -fo r - tna t io t r ,  t l i cy  p robab ly  bc long to  on ly  onc  c lass .  a l l  bc ing  o f  thc  h ish-c f f i c ic t rcy  typc .  Thc  markers  were  loca tcd  re la t i vc  to  cach o thcr  by  , r r " r , . , ,o f  rnarkcr  f rcc luency  ar - ra lys is .  Of  the  B nrarkers  s tud icd .  t rp , , ,  a r rc i  t y r í  Arcc losc ly  l inkcd  in  t rans format ion .  Thc  r ib  rnarkcr  wá ls  shown-  to  be  wcak lyl inked to  thc  t rp  r - ty r r  pa i r  o f  n rarkers .  No fu r ther  l inkagcs  c -x is t  bcrwccnthc  r la rkcrs  in  s t r : r in  BG-5 .UV inac t iv : r t io t r  o f  thc  B  rnarkcrs  i s  dcscr ibcd  in  papcrs  l l  anc l  I l t .  DNA iso la tcc lf ro tn  p ro to t roph ic  cc l l s  w; rs  i r rad ia ted  w i t l i  var ious  doscs  o f  UV: rnd  subscc lucn t lyuscd to  t rans for tn  8G-5  rec ip icn t  cc l l s .  Thc  rcsu l ts  dcnrons t ra tc  tha t  thc  vur iousnt : r rkc rs  a rc  inac t iva tcd  a t  d i f f c rcn t  ra tcs .  Or - r r  work ing  hypothcs is  was th r r td i f fe rcn t ia l  markcr  inac t iva t ion  is  duc  to  d i f f c rcnccs  in  t l - rc  f rcc lucncv , - , ipo tcn t ia l  photochcrn ica l  les ions  in  the  rnarkcd  segmcnts  o f  ONA.  Howcvcr .in tc rpre ta t ion  o f  the  resu l ts  o f  our  marker  inac t iva t ion  cxper in ren ts  accord i r rgto  th is  hypothes is  w: rs  compl ica ted  fo r  a t  l cas t  two rcasons .  F i rs t l y .  thc  rcsu l tsshow tha t  marker  scns i t i v i t i cs  to  UV depend on  thc  DNA conccnt ra r lo l l  c iu r ingt rans for tna t ion .  A t  sa tura t ing  amounts  o f  DNA,  thc  rnarkcrs  a rc  : rpparcn t lyn lo re  scns i t i vc  to  UV than á i t  non-sa tura t ing  ár ïnounts .  Th is  i s  p robab ly  duct o  t h e  f a c t  t h a t  a t  s a t u r a t i n g  a m o u n t s  o f  D N A ,  i n  a c i d i t i o n  t o  t h c  m o l c c u l c scar ry ing  se lec tcd  nrarkcrs .  morc  non-sc lec tcd  danragcc l  rno lccu lcs  : r rc  in tcgra tcdin to  thê  rec ip icn t  genomc th : rn  a t  non-s i l tu r l r t ing  iu r louuts .  Thcrc fo rc .  r r - lo rcpoten t ia l  t rans fornran ts  a rc  k i l l cd  undcr  thc  fo rnrc r  cond i t ions .  wh ich  rcsu l tsin  overes t in la t ion  o f  n rarkcr  scns i t i v i t i cs .  Th is  compl icu t io r . r  was  avo idcd  bydeter rn in ing  the  markcr  scns i t i v i t i cs  to  UV a t  DNA conccnt r r r t io r " rs  fa r  bc lowsatura t ion .  Second ly ,  cornp l i ca t ions  w i l l  a lso  ar isc ,  i f  a f t " r  up takc  l . r r  rcc ip ic r r tce l l s ,  the  inac t iva tcd  mzt rkers  a rc  rcpa i rcd  to  d i f f c rcn t  dcgrccs .  To  uvo id  t l r i scompl icat ion,  an excis ion-repair-def ic ient  c ler ivat ivc w;rs iÀolatcd f rorn strainB G - 5 .  T h c  p r o p c r t i c s  o f  t h i s  s t r : r i n : r r c  a l s o  d c s c r i b e d  i n  p a p c r  l .  T h i s  s r n r i n .B G - 1  1 ,  i s  u n a b l c  t o  c x c i s e  p y r i m i c l i n c  d i n r c r s .  t o  r c a s s u r r r c  D N A  s v n t h c s i s  a f t c rU V  i r r a d i a t i o r - r ,  a n d  t o  r c a c t i v u t c  U V - i n a c t i v a t c d  b a c t c r i o p h a q c .  A s  c o r n p l r c c lt t t  s t r : t i t - t  I JG-5 ,  i t  t rans for r r rs  nornra l l v  r ,v i r l r  t rn i r r l c l i l t c r ]  I )NA.  bu t  1 ' , . . r , , r l vw i t h  U V - i r r a c l i : r t c c l  l ) N A .  B c c : r r - r s c  o f  t l r c s c  p r o p c r t i c s  s r r i r i n  S ( ;  l l  . 1 , , n  t r . ,1 8-classi f ied as being def ic ient  in excis ion-repair  (uvr-) .  When in v ivo photo-react ivat ion of  UV-irradiated donor DNA is prevented by working undcr diml ight ,  ancl  excis ion-repair  by using strain 8G-11 as recipient,  d i f ferent ia l  markerinact ivat ion by UV of 254 nm st i l l  exists (paper I I ) ,  which supports thc hy-pothesis that  the f requency of  potent ia l  photochemical  lesions determinesthe sensi t iv i ty to UV of  the marked segmenrs of  ONA.Comparison of the results on marker inactivation obtained with strain BG-5and strain BG-11 al lowed us to compute the f ract ion of  inact ivat ing lesionsrvhich is not repaired by the 8G-5 (uvr+) recipient.  The size of  th is f ract ionis marker-specific and varies from 13t/o to 26o/o, indicating that excision-repairis not equally efficient for different markers in irradiated DNA. On the averageabout 80' /o of  the induced lesions are succesful ly repaired.The involvement "f fr dimers in both differeniial rnarker inactivation anddifferential marker repair was studied in the following way. TransformingDNA was i r radiated with 313 nm l ight  in the presence of  acerophenone assensi t izer.  Under these condi t ions exclusively TT dimers should be induc "642,43 .H^owever,  our resul ts (presented in paper I I I )  demonstrate that ,  in addi t ion toTT dimers,  non repairable s ingle-stranded DNA breaks are : r lso produced. Invi t ro photoreact ivat ion of  the TT dimers in the i r radiated DNA al lowed usto colnPute the relat ive contr ibut ions of  the dirners and the single-strandedDNA breaks to total  marker sensi t iv i t ies.  Al though only one break occurs perapproximately 90 f f  d imers,  i t  appears that  the breaks account for  abàut20' /o of  the observed marker sensi t iv i t ies.Di f ferent ia l  marker inact ivat ion and di f ferent ia l  markcr repair  werc also ob-served in thc acetophenone systein of  i r radiat ion.  Thc resul ts dernonstrate thar,i r respect ive of  the uvr genotype of  the recipicnt ,  thc s inglc-strandcd DNA brcaLsinact ivate the var ious markers to the same extcnt.  From this observat ion i tcan be concluded that in the acetophenonc system di f ferent ia l  marker in-act ivat ion is exclusively duc to di f fercnces in t i re f requency " f  f f  d i rncrs inthe marked segments of  DNA. In addi t ion.  d i f fe rcnt ia i  markcr rcpair  is  exclusivelydue to di f ferenccs in thc ef f ic iency of  repair  of  r r  d imcrs.  Support  for  thcconclusion that di f ference s in the f requency " f  f r  d imers in thc rrr" . t "d scgmenrsof DNA causc di f fcrcnt ia l  rnarkcr inact ivat ion is that  photoreact ivat ion exper-imcnts  ind ica tc  tha t  exce l l cn t  cor re la t ion  ex is ts  be tween the  f requency  o fTT dirners in thc rnarked scgmcnts of  DNA arrd thc scnsi t iv i ty , r f  th.  '^^r-kc rs  to  i r rad ia t ion  in  thc  acc tophenonc sys tem.  Fur thcr  suppor t  fo r  th is  con-c lus ion  is  p rov idcc l  by  thc  obscrva t ion  tha t ,  as  judge d  f ro rn  heat  dcnarurar ions tud ics ,  markcrs  rc la t i vc ly  r i ch  in  thyrn inc  usua l ly  a rc  rc la r ivc ly  scns i t i ve  toi r rad ia t ion  and,  convcrsa ly ,  tha t  tnarkers  rc la t i ve ly  poor  in  thyrn ine  usua l lyare  rc la t i vc lv  rcs is tan t  to  i r rad ia t ion .Cot r ipar ison  c , f  thc  rcsu l ts  ob ta i r - rcd  in  thc  acc tophcnonc sys tc rn  w i th  thoseobta incd  in  thc  254 nrn  svs tc rn  o f  i r rad ia t ion ,  shows tha t  bo th  t l - rc  rc la t i vcordcrs , .> f  n ta rkcr  sc r rs i t i v i t i t : s  and o f  rnarkcr  rcpa i rab i l i t i cs  a rc  c lc - rse ly  s imi la rrn  thc  two svs tcnrs  o f  i r rad ia t ion .  Wc cons idcr  t i r i s  as  cv idcncc  t l ia t  a lso  in  thcl 92 5 4  tmarkare t lThethe  efirstllthe  kof  U'lo l lo rrecipiand sof d:abscnjuclue+uvrtranslis intgratelirradicxc luconcl iatnouT h c  Istr-rditstrai  nThc  Iandtnolc,rc iso liÀ int20-4 !for fdatnar e e x tS C C  O TThc 1was  Ihypot h c  ct igatrt h c  cl igascsigni lt y  c cvVhen in v ivo photo-y working under dimrt, differential markerhich supporrs rhe hy-r l  lesions determinesred with strain 8G-5f inact ivat ing lesions: size of this fractiong that excision-repairDNA. On the average'ker inact ivat ion andi way. Transformingof  ace tophenone asu ld  be  induceda 2  ,43  .:  thar ,  in  add i t ion  tore  a lso  produced.  Ined DNA al lowed usI  the s ingle-strandedrne break occurs pers account for  aboutrepair  werc also ob-I ts c le morlstrate that ,t randcd DNA breaLsr th is obscrvat ion i tí c rc r r t ia l  markcr  in -rcy of ff di.n"rs inr  rcpair  is  e xclusivclycrs.  Support  for  thehc nrarked scgmentsorcact ivat ion exper-r  the f requency ofs i t iv i ty of  the mar-rPporr  for  th is con-r  l icat  dcnaturat ionat ivc ly  scns i t i ve  ton  thyminc  usua l lysys tcm wi th  thoseboth  thc  rc la t i vearc c loscly s i rn i larrcc  tha t  a lso  in  thc254 nm system of irradiation TT dimers are the main cause for differentialmarker inactivation and that differences in the efficiency of repair "f fT dimersare the main cause for differential marker repair.The fate of UV lesions in transforming DNA was studied more detailed inthe exper iments descr ibed in paper IV.  The pr incipal  a ims were to invest igate:f i rst ly,  genet ic integrat ion of  UV-irradiated transforming DNA, and secondly,the k inet ics of  in v ivo repair  of  Uv- inact ivated transforming DNA. Tl-re ef fectof  UV lesions in t ransÍorming DNA on gener ic intcgrat ion was studied in theÍc. ' l lowing way. UV-irradiated DNA was used to t ransform a repair less uvr-recipient.  Af tcr  a l lowing t ime for intcgrat ion to occur,  DNA was reextractedand subsequent ly assayed on uvr-  and uvr-  second recipicnts for  the prcsenceof dark-repairable damage in rccombinant type moleculcs.  " ihe presence orabsence of  dark-repairable darnage in thc recombinant type molccules wasjudged by the number of  recornbinant typc t ransformants obt: i incd on theuvr*  re la t i ve  to  the  number  on  the  uvr -  second rcc ip ien t .  Thc  rcsu l ts  show tha ttransfortning DNA irradiatcd wi th modcrate doscs of  UV (2,000 erg/mrn2)is integrated almost equal ly ef f ic , icnt ly : rs non- i r radiarcd DNA, and that inrc-grated UV lesions account alrnost exclusivcly for  t l ie inact iv:r t ion.  Howcver,i r rad ia t ion  o f  thc  DNA wi th  20 .000 " .g / - -2  o f  uV rcsu l ts  in  a  s ign i f i can texclusion of  darnagcd DNA frorn th" p.oc"ss of  intcgrar ion.  Under thcsecond i t ions ,  the  cont r ibu t ion  o f  thc  exc lus ion  to  to ta l  markcr  inac t iva t ionamounts  to  approx i rna te ly  1 }o / , t  in  uvr *  and to  less  than 7oh in  uvr -  rcc ip icn ts .The kinet ics of  in v ivo cxcis ion-repair  of  Uv- i r radiated rransforming DNA we rcs tud icd  by  a  c losc ly  s imi la r  method.  ln  th is  case.  e r  rcpa i r -p ro f i c icn t  uvr *s t ra in  sc rvcd  as  thc  f i rs t  rcc ip ien t  in  t rans format ion  w i th  UV- i r rad ia rcd  DNA.The DNA was rccx t rac tcd  a t  var ious  in te rva ls  a f te r  n r i r ing  cc l l s  ; inc i  donor  DNA,and the rcsidual  amount of  dark-repairablc damage in recombin: int  ry pctno iccu les  in  the  rcex t rac ted  DNA was de tc rmincd as  a  func t ion  o f  t i rnc  o fre iso la t ion .  Thc  rcsu l ts  ind ica te  tha t  repa i r  on ly  s ta r ts  a f - tc r  the  donor  DNAi i  i r - r tcgra tcd  in to  the  rcc ip ien t  gcnomc.  Cornp le t ion  o f  repa i r  rcqLr i res  f ro r r r20-45  rn in  fo r  DNA i r rad ia tcd  w i th  2 ,000 c rg /n r rn l ,  and approx i r r ia tc ly  2  hfo r  DNA i r rad i : r ted  w i th  15 ,000 erg lmrn2.  T l i c  rcsu l ts  a lso  i r - rd ica tc  rha t  UV-danragcd cc l ipse  phase DNA (DNA tha t  i s  taken up  bu t  nor  in tcgra tcd)reex t rac tcd  f rom rec ip icn t  ce l l s ,  i s  no t  sub jcc t  to  cxc is ion- rcpa i r  in  uur*s c c o n d  r c c i p i e n t  s t r a i n s .The poss ib i l i t y  o f  in  v i t ro  cxc is ion- rcpa i r  o f  UV- inac t iva tcd  t rans for r r i ing  DNAwas s tud ied  in  thc  expcr i rnents  dcscr ibcd  in  thc  las t  papcr  o i  th is  t l r cs is .  Thchypothes is  pu t  fo rwardby  KELLY e t  a l .a0  th r t  DNA po lynrerasc  pcr fc , r r r rs  bo ththe  cxc is ion  and the  rep l i ca t ion  s tep  in  exc is ion- repa i r ,  p ron lp rcd  us  ro  invcs-t iga tc  whcthcr  UV- i r rad ia ted  t rans forming DNA can bc  rcpa i rcd  in  v i t ro  bythe  combined ac t ion  o f  UV-spcc i f i c  cndonuc lcasc ,  DNA po ly rncrasc  and DNAl igasc. Thc resul ts show th:r t  the biological  : rct iv i ty of  inact ivarcd DNA incrc: : rsess ign i f i can t ly  fo l low ing  incubat ion  w i th  the  th rec  cnzyrnes .  Thc  rcs to rcd  acr iv i -ty  can  no t  bc  fu r thcr  inc reascd by  photoreac t iva t ion  in  v i t ro .  ind ica t ing  r l ra t20-photorepairable dimcrs are no longer present.  I t  could be demonstrated thatái-"rr  àre speci f ical ly excised upon incubat ion of  the i r radiated DNA withthe thre e enzymes, and that under our condi t ions approximatciy 50' /o of  thcinduccdlesionsare succesful ly repaired. These rcsul ts s l - row that,  at  lcast  in v i t ro,KELLY's40 mode l  app l i cs  to  repa i r  o f  UV les ions  in  DNA.  Whether  th is  mode lalso appl ics io repair  in v ivo,  remains to be establ ished. I t  may also be envisagedthat al tcrnat ive pathwat 's of  excis ion-re pair  exist .Dit proefsclheid voorsubt i l is ,  deherstel vanEén van drvan inaktivwerd een nDe isolerinschreven inmerkers wckregen. Hcbehoren detransformervan merkerbij transforgekoppeldstam werdeDe inaktiveen I I I  bes ,bestraald nmee te t rarongelijke nàeze differwaalmee P(deze hyportweetal  korgevoelighedDNA, die rheden trandan met overzadigencongemerktegreerd danmeer PoteÍkeli jke gev<opgelost dctraties DNIkatie ortStrmerkers inkomen,  wet l- l

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Ultraviolet inactivation and excision-repair in bacillus subtilus

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