Haijema, Bert Jan

ARTS repository - University of Groningen

   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. 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: 08-06-2022Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

English

Function and regulation of the bacillus subtilis addAB genes in homologous recombination

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Proceedings - University of Groningen

   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. 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: 30-10-2023Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

Dissertations of the University of Groningen

   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. 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: 17-07-2023Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. 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: 03-06-2022Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. 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: 09-09-2023Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

University of Groningen Research Database

  
 University of Groningen
Function and regulation of the bacillus subtilis addAB genes in homologous recombination
Haijema, Bert Jan
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Publisher's PDF, also known as Version of record
Publication date:
1996
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologous
recombination Groningen: s.n.
Copyright
Other 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 the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately
and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum.
Download date: 11-02-2018
Chapter lX Chapter lX
Summary and general discussion
Homologous recombination is a biological process essential to allorganisms,
prokaryotes and eukaryotes, because it is involved in the generation of genetic
diversity, the maintenance of genomic integrity and the proper segregation oÍ
chromosomes. The first part of chapter 1 gives a brief overview of our knowledge
of the molecular mechanisms of homologous recombination derived from studies in
the prokaryote Escherichia coli. This process has only been studied to some degree
in the species Bacillis subtilis, as described in the second part of chapter 1. To date,
in this Gram-positive, spore forming soil bacterium, 13 genes, including addAand
addB, which are involved in the complex process of homologous recombination,
have been identified. The products of these genes are involved in many cellular
processes such as natural transformation, DNA repair, bacteriophage-mediated
transduction and DNA repair.
The work, described in chapter 2 to 6, is mainly focused on the B. subtilis
addAB genes and their gene products. The B. subtilis addA and addB genes encode
the two subunits of the ATP-dependent deoxyribonuclease nzyme complex, which
is involved homologous recombination and DNA repair. The heterodimeric enzyme
is an ATP-dependent helicase and exonuclease.
Chapter 2 showg that the B. subtilis AddAB enzyme is the functional
equivalent of the E. coli RecBCD enzyme, although no extended amino acid
sequence similarity exists between the proteins. The introduction of the addAB
genes into an E. coli recBCD- strain nearly completely restored the viability, the
ability to repair U.V. damaged DNA and recombination in conjugation. However, the
B. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.
The individual B. subtilis Add proteins neither complemented recB nor recC
mutations.
Chapter 3 and 4 communicate the characterization of AddAB enzymes,
mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 shows
that the lysine to glycine substitution in the ATP binding site of AddA led to
significantly reduced transformation with chromosomal DNA, cell viability, the
capacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,
and bacteriophage PBS-1 mediated transduction. These effects could be related to
significantly reduced rates of ATP-dependent exonuclease- and helicase activities,
1 8 6
suggesting that at least onr
recombination. A similar muta
marginal effects on these prr
characterization of the wild type
demonstrated that the lysine ol
the helicase- and exonuclease
hydrolysis of ATP and not fot t
the corresponding lysine of su
Chapter 5 describes th
AddA. In this chapter it is shc
activity but not for the helicar
exonuclease activity, an AddAl
A, could almost fully compleme
with respect to homologous rer
with those obtained in chal
exonuclease- activity, is esser
AddA shows a high degree of I
of the E. coli RecBCD enzym(
similar function in exonuclease
Chapter 6 deals with thr
addB genes are located in ar
overlap between the two open
type promoter with a low, r
competence development, a cr
observed. A direct interaction I
and the promoter region of ac
assays. These findings indici
establishment of the comoete
recombination.
The transcription of re
chapter 7 demonstrates that C
to its promoter upstream regic
8. in which it was demonstral
promoter region, forcing the rr
analyses demonstrated that C
; essential to all organisms,
the generation of genetic
the proper segregation of
overview of our knowledge
tíon derived from studies in
rcn studied to some degree
I part of chapter 1. To date,
genes, includíng addA and
omologous recombination.
r involved in many cellular
r, bacteriophage_medrated
focused on the B. subtilis
tA and addB genes encode
e enzyme complex, which
the heterodímeric enzyme
)nzyme is the functional
no extended amino acid
rtroduction of the addAB
restored the viability, the
;onjugation. However, the
le lambda recombination.
rmented recB nor recC
on of AddAB enzymes,
:tively. Chapter 3 shows
ng site of AddA led to
DNA, ceil viabitity, the
r mitomycin C treatment,
fects could be related to
. and helicase activities,
Chapter lX
suggesting that at least one of these activities is involved in homologous
recombination. A similar mutation in the ATP-binding site of subunit B had only
marginal effects on these processes. Chapter 4 describes the purification and
characterization of the wild type AddAB enzyme and the two mutant enzymes. lt was
demonstrated that the lysine of the ATP binding site of subunit AddA is essential for
the helicase- and exonuclease activities of the enzyme, because it is required for the
hydrolysis of ATP and not fot the binding of ATP. No function could be attributed to
the corresponding lysine of subunit B.
Chapter 5 describes the examination of the C-terminal region of subunit
AddA. In this chapter it is shown that this region is required for the exonuclease
activity but not for the helicase activity of the enzyme. Despite the lack of the
exonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunit
A, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutant
with respect o homologous recombination and DNA repair. These results, together
with those obtained in chapter 3, suggest that the helicase-, but not the
exonuclease- activity, is essential for these processess. The C-terminal region of
AddA shows a high degree of homology with the C-terminal region of subunit RecB
of the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting a
similar function in exonuclease activity for this region in RecB.
Chapter 6 deals with the transcriptional regulation of addAB. The addA and
addB genes are located in an operon structure as was suggested by the limited
overlap between the two open reading frames. The genes are transcribed from a oÉ
type promoter with a low, constitutive level of expression. However, during
competence development, a comK-dependent induction of addAB transcriptíon was
observed. A direct interaction between ComK, the competence transcription factor,
and the promoter region of addAB was demonstrated by means of gel retardation
assays. These findings indicate that a direct regulatory link exists between the
establishment of the competent state and the synthesis of AddAB, required for
recombination.
The transcription of recA is regulated in a similar way as that of addAB:
chapter 7 demonstrates that ComK also stimulates the expression of recA by binding
to its promoter upstream region. This binding was studied in more detail in chapter
8, in which it was demonstrated that at least 4 ComK molecules bind to the recA
promoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotection
analyses demonstrated that ComK covers an extended region of the recApromoter
187
--...-
Chapter lX
region, spanning almost 100 bp. Chapter 7 shows that the expression of recA is also
regulated by DinR, the transcriptional repressor of the sos DNA repair system. lt
was demonstrated that comK and DinR can bind simultaneously to the recA
promoter egion, which suggest hat the ComK-dependent transcriptional ctivation
of recA during competence development is not caused by competition for binding
between ComK and DinR.
In conclusion, the present results indicate that AddAB plays an important role
in homologous recombination and DNA repair for which the helicase activity, but not
the exonuclease activity, seems essential. Possibly, the enzyme plays an important
role in the initiation stage of recombination: the unwinding of dsDNA molecules to
generate ssDNA tails, which are essential substrates for further processing by the
recombination machinery. However, the exact role of AddAB, in, for instance, the
transformation process with chromosomal DNA, still remains to be established. This
process can be examined by studying the fate of internalized DNA in various AddAB
mutants.
Chapter lX
Samenvatting en algement
Homologe recombinat
eukaryoten. Het is betrokke
handhaven van de integriteil
chromosomen. Het eerste
mechanisme van homologt
Escheichia coli. Hettweede
bekend is over dit proces in i
vormende grondbacterie. T
gevonden die betrokken zijn
z'rjn betrokken b'rj verscheide
schade, transformatie, trans
Het werk, zoals dat be
gericht op de B. subÍi/ts
specificeren de twee subee
complex. Het complex is be
DNA schade.
Hoofdstuk 2laalzien
E. coli RecBCD complex i
gebracht in een E. colirecBr
om DNA schade te herste
hersteld. De AddAB eiwi
bacteriofaag lambda recoml
staat een E. coli recBCD- s'
H o o f d s t u k 3 e n 4 b e
ofwel gemuteerd zijn in de I
3 laat zien dat de substituti<
bindings plaats van subeet
transformatie efficientie met
herstellen en transductie me
sterk verlaagde helicase en
van deze activiteiten van i
mutatie in de ATP bindings
1 8 8


   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. 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: 04-06-2022Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

University of Groningen

   University of GroningenFunction and regulation of the bacillus subtilis addAB genes in homologous recombinationHaijema, Bert JanIMPORTANT 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:1996Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Haijema, B. J. (1996). Function and regulation of the bacillus subtilis addAB genes in homologousrecombination. Groningen: 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: 12-11-2019Chapter lX Chapter lXSummary and general discussionHomologous recombination is a biological process essential to allorganisms,prokaryotes and eukaryotes, because it is involved in the generation of geneticdiversity, the maintenance of genomic integrity and the proper segregation oÍchromosomes. The first part of chapter 1 gives a brief overview of our knowledgeof the molecular mechanisms of homologous recombination derived from studies inthe prokaryote Escherichia coli. This process has only been studied to some degreein the species Bacillis subtilis, as described in the second part of chapter 1. To date,in this Gram-positive, spore forming soil bacterium, 13 genes, including addAandaddB, which are involved in the complex process of homologous recombination,have been identified. The products of these genes are involved in many cellularprocesses such as natural transformation, DNA repair, bacteriophage-mediatedtransduction and DNA repair.The work, described in chapter 2 to 6, is mainly focused on the B. subtilisaddAB genes and their gene products. The B. subtilis addA and addB genes encodethe two subunits of the ATP-dependent deoxyribonuclease nzyme complex, whichis involved homologous recombination and DNA repair. The heterodimeric enzymeis an ATP-dependent helicase and exonuclease.Chapter 2 showg that the B. subtilis AddAB enzyme is the functionalequivalent of the E. coli RecBCD enzyme, although no extended amino acidsequence similarity exists between the proteins. The introduction of the addABgenes into an E. coli recBCD- strain nearly completely restored the viability, theability to repair U.V. damaged DNA and recombination in conjugation. However, theB. subtilis AddAB enzyme did not show Chi-activity in phage lambda recombination.The individual B. subtilis Add proteins neither complemented recB nor recCmutations.Chapter 3 and 4 communicate the characterization of AddAB enzymes,mutated in the ATP binding site of subunit A and B, respectively. Chapter 3 showsthat the lysine to glycine substitution in the ATP binding site of AddA led tosignificantly reduced transformation with chromosomal DNA, cell viability, thecapacity to repair DNA damage inflicted by U.V irradiation or mitomycin C treatment,and bacteriophage PBS-1 mediated transduction. These effects could be related tosignificantly reduced rates of ATP-dependent exonuclease- and helicase activities,1 8 6suggesting that at least onrrecombination. A similar mutamarginal effects on these prrcharacterization of the wild typedemonstrated that the lysine olthe helicase- and exonucleasehydrolysis of ATP and not fot tthe corresponding lysine of suChapter 5 describes thAddA. In this chapter it is shcactivity but not for the helicarexonuclease activity, an AddAlA, could almost fully complemewith respect to homologous rerwith those obtained in chalexonuclease- activity, is esserAddA shows a high degree of Iof the E. coli RecBCD enzym(similar function in exonucleaseChapter 6 deals with thraddB genes are located in aroverlap between the two opentype promoter with a low, rcompetence development, a crobserved. A direct interaction Iand the promoter region of acassays. These findings indiciestablishment of the comoeterecombination.The transcription of rechapter 7 demonstrates that Cto its promoter upstream regic8. in which it was demonstralpromoter region, forcing the rranalyses demonstrated that C; essential to all organisms,the generation of geneticthe proper segregation ofoverview of our knowledgetíon derived from studies inrcn studied to some degreeI part of chapter 1. To date,genes, includíng addA andomologous recombination.r involved in many cellularr, bacteriophage_medratedfocused on the B. subtilistA and addB genes encodee enzyme complex, whichthe heterodímeric enzyme)nzyme is the functionalno extended amino acidrtroduction of the addABrestored the viability, the;onjugation. However, thele lambda recombination.rmented recB nor recCon of AddAB enzymes,:tively. Chapter 3 showsng site of AddA led toDNA, ceil viabitity, ther mitomycin C treatment,fects could be related to. and helicase activities,Chapter lXsuggesting that at least one of these activities is involved in homologousrecombination. A similar mutation in the ATP-binding site of subunit B had onlymarginal effects on these processes. Chapter 4 describes the purification andcharacterization of the wild type AddAB enzyme and the two mutant enzymes. lt wasdemonstrated that the lysine of the ATP binding site of subunit AddA is essential forthe helicase- and exonuclease activities of the enzyme, because it is required for thehydrolysis of ATP and not fot the binding of ATP. No function could be attributed tothe corresponding lysine of subunit B.Chapter 5 describes the examination of the C-terminal region of subunitAddA. In this chapter it is shown that this region is required for the exonucleaseactivity but not for the helicase activity of the enzyme. Despite the lack of theexonuclease activity, an AddAB enzyme, containing a C-terminal truncated subunitA, could almost fully complement an E. coli recBCD and a B. subf/ls recBCD mutantwith respect o homologous recombination and DNA repair. These results, togetherwith those obtained in chapter 3, suggest that the helicase-, but not theexonuclease- activity, is essential for these processess. The C-terminal region ofAddA shows a high degree of homology with the C-terminal region of subunit RecBof the E coli RecBCD enzyme, the functional equivalent of AddAB, suggesting asimilar function in exonuclease activity for this region in RecB.Chapter 6 deals with the transcriptional regulation of addAB. The addA andaddB genes are located in an operon structure as was suggested by the limitedoverlap between the two open reading frames. The genes are transcribed from a oÉtype promoter with a low, constitutive level of expression. However, duringcompetence development, a comK-dependent induction of addAB transcriptíon wasobserved. A direct interaction between ComK, the competence transcription factor,and the promoter region of addAB was demonstrated by means of gel retardationassays. These findings indicate that a direct regulatory link exists between theestablishment of the competent state and the synthesis of AddAB, required forrecombination.The transcription of recA is regulated in a similar way as that of addAB:chapter 7 demonstrates that ComK also stimulates the expression of recA by bindingto its promoter upstream region. This binding was studied in more detail in chapter8, in which it was demonstrated that at least 4 ComK molecules bind to the recApromoter egion, forcing the region to bend. DNAsel and hydroxyl radicalprotectionanalyses demonstrated that ComK covers an extended region of the recApromoter187--...-Chapter lXregion, spanning almost 100 bp. Chapter 7 shows that the expression of recA is alsoregulated by DinR, the transcriptional repressor of the sos DNA repair system. ltwas demonstrated that comK and DinR can bind simultaneously to the recApromoter egion, which suggest hat the ComK-dependent transcriptional ctivationof recA during competence development is not caused by competition for bindingbetween ComK and DinR.In conclusion, the present results indicate that AddAB plays an important rolein homologous recombination and DNA repair for which the helicase activity, but notthe exonuclease activity, seems essential. Possibly, the enzyme plays an importantrole in the initiation stage of recombination: the unwinding of dsDNA molecules togenerate ssDNA tails, which are essential substrates for further processing by therecombination machinery. However, the exact role of AddAB, in, for instance, thetransformation process with chromosomal DNA, still remains to be established. Thisprocess can be examined by studying the fate of internalized DNA in various AddABmutants.Chapter lXSamenvatting en algementHomologe recombinateukaryoten. Het is betrokkehandhaven van de integriteilchromosomen. Het eerstemechanisme van homologtEscheichia coli. Hettweedebekend is over dit proces in ivormende grondbacterie. Tgevonden die betrokken zijnz'rjn betrokken b'rj verscheideschade, transformatie, transHet werk, zoals dat begericht op de B. subÍi/tsspecificeren de twee subeecomplex. Het complex is beDNA schade.Hoofdstuk 2laalzienE. coli RecBCD complex igebracht in een E. colirecBrom DNA schade te herstehersteld. De AddAB eiwibacteriofaag lambda recomlstaat een E. coli recBCD- s'H o o f d s t u k 3 e n 4 b eofwel gemuteerd zijn in de I3 laat zien dat de substituti<bindings plaats van subeettransformatie efficientie metherstellen en transductie mesterk verlaagde helicase envan deze activiteiten van imutatie in de ATP bindings1 8 8

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Function and regulation of the bacillus subtilis addAB genes in homologous recombination

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