An investigation of the mechanism of the polAex1 mutation in vitro suggested that the excision repair deficiency observed in vivo does not result from an inability of the enzyme to nick translate. The defect appears to reside in the inability of the enzyme to effectively generate a nick structure to serve as a substrate for DNA ligase

Bambara, R A

Hockensmith, J W

Kowalski, S

Wahl, A F

PubMed

An investigation of the mechanism of the polAexl mutation in vitro suggested that the excision repair deficiency observed in vivo does not result from an inability of the enzyme to nick translate. The defect appears to reside in the inability of the enzyme to effectively generate a nick structure to serve as a substrate for DNA ligase

Wahl, Alan F.

Hockensmith, Joel W.

Kowalski, Stanley

Bambara, Robert A.

UNH Scholars' Repository

University of New HampshireUniversity of New Hampshire Scholars' RepositoryLaw Faculty Scholarship University of New Hampshire – School of Law1-1-1983Alternative Explanation for Excision RepairDeficiency Caused by the polAex1 MutationAlan F. WahlJoel W. HockensmithStanley KowalskiUniversity of New Hampshire School of Law, stanley.kowalski@law.unh.eduRobert A. BambaraFollow this and additional works at: https://scholars.unh.edu/law_facpubPart of the Life Sciences CommonsThis Article is brought to you for free and open access by the University of New Hampshire – School of Law at University of New Hampshire Scholars'Repository. It has been accepted for inclusion in Law Faculty Scholarship by an authorized administrator of University of New Hampshire Scholars'Repository. For more information, please contact ellen.phillips@law.unh.edu.Recommended CitationAlan F. Wahl, Joel W. Hockensmith, Stanley P. Kowalski & Robert A. Bambara, "Alternative explanation for excision repair deficiencycaused by the polAex1 mutation,” 155 J. Bacteriol 922 (1983), available at http://www.ncbi.nlm.nih.gov/pmc/articles/PMC217772/Vol. 155, No. 2JOURNAL OF BACTERIOLOGY, Aug. 1983, p. 922-9250021-9193/83/080922-04$02.00/0Copyright 0 1983, American Society for MicrobiologyAlternative Explanation for Excision Repair DeficiencyCaused by the polAexl MutationALAN F. WAHL, JOEL W. HOCKENSMITH,t STANLEY KOWALSKI, AND ROBERT A. BAMBARA*Department ofBiochemistry and Cancer Center, University of Rochester Medical Center, Rochester, NewYork 14642Received 7 February 1983/Accepted 3 May 1983An investigation of the mechanism of the polAexl mutation in vitro suggestedthat the excision repair deficiency observed in vivo does not result from aninability of the enzyme to nick translate. The defect appears to reside in theinability of the enzyme to effectively generate a nick structure to serve as asubstrate for DNA ligase.Traditional models of excision repair in Esch-erichia coli have suggested that an incision stepintroduces a nick into the DNA 5' to a thymidinedimer. DNA polymerase I binds at this site andsynthesizes from the 3'-hydroxyl terminus,while simultaneously degrading with 5' -* 3'exonuclease activity (nick translation) throughthe dimer, removing it, and then dissociatingbefore ligation (1). Therefore, it was not surpris-ing that E. coli containing the polAexl mutation,a temperature-sensitive defect in 5' -> 3' exonu-clease activity, was found to be excision repairdeficient (3).However, some recent work of Sancar andRupp (7a) has demonstrated that the E. coliincision enzyme complex (uvrABC) makes twocleavages, one at the eighth phosphodiester 5' tothe pyrimidine dimer and one at the fourth orfifth phosphodiester 3' to the dimer. The result-ant short DNA segment containing the dimer isincompletely base paired and is easily dissocia-ble from the larger DNA, leaving a 12- to 13-nucleotide gap. Therefore, nick translationshould not be necessary for repair; the polymer-ase need only fill in this short gap to form aligatable substrate. Why then does a defect inthe 5' -+ 3' exonuclease activity ofDNA polym-erase I impair excision repair? One explanationis that the polymerase section of the moleculealone is not sufficient for gap closure and thepresence of the 5' -- 3' exonuclease in its native,active conformation is somehow required togenerate a nick that can be a substratefor DNA ligase. To test this hypothesis, weconstructed a tritiated ColEl plasmid DNA tem-plate containing an average of 0.6 gap 20 nucleo-tides long per DNA molecule to examine thet Present address: Institute of Molecular Biology, Universi-ty of Oregon, Eugene, OR 97403.repair process. The number of 3'-hydroxyl ter-mini and the amount of single-strand templateper 3'-hydroxyl terminus were determined bythe method of Hockensmith and Bambara (2).The plasmid was first nicked with pancreaticDNase (1 ng/300 nmol of DNA) for 8 min at23°C, and the number of available 3' termini wasdetermined by treating the preparation with ex-cess E. coli DNA polymerase 1, 10 ,uM dCTP, 10,uM dATP, and 10 ,uM [3H] dTTP (77 Ci/mmol)to add an average of three nucleotides per 3'end. These nicks were then widened to gaps bythe action of E. coli exonuclease III (a gift fromL. Loeb), which sequentially degrades from the3' end. The size of these gaps was determined byincorporating radiolabeled nucleotides with T4DNA polymerase, which lacks a 5' -- 3' exonu-clease and is able to fill gaps exactly to ligatableends without causing strand displacement (5).The gap length which we calculated varied from18 to 22 nucleotides depending on the method ofdetermination, and we used an average value of20 nucleotides. We tested the abilities of purifiedE. coli DNA polymerase I, mutant polAexlDNA polymerase I, and the DNA polymerase Ilarge fragment (in which the 5' -- 3' exonucleasewas proteolytically removed) to function with E.coli DNA ligase to convert the DNA template toa closed circular duplex. Conversion was detect-ed by subsequent sedimentation on alkaline su-crose density gradients to determine the fractionof DNA converted to a closed circular form.Figure 1 shows the results obtained with reac-tion mixtures containing DNA polymerase andDNA ligase with the gap-containing DNA tem-plate. The relative areas under the closed circu-lar DNA and open circular DNA peaks could beused to assess the number of breaks in themolecules (6). The polymerases were all adjust-ed to equivalent activities at both 30 and 43°C ona ColEl DNA template with 200 nucleotide gaps922NOTES 923Lu"I.)I....Q..0qS JOBOTOMIS 20 15 20 25 30TOPFRACTION NUMBERFIG. 1. Alkaline sucrose sedimentation of 20-nucleotide, gap-containing, 3H-labeled ColEl DNA that wasnot treated (0) or was treated with DNA polymerase I and DNA ligase (0) at 30°C (A); treated with DNApolymerase I large fragment and DNA ligase at 30°C (0) or 43°C (0) (B); treated with polAexl polymerase andDNA ligase at 30C (0) and 430C (0) (C); and treated with polAexl DNA polymerase at 43°C (0) or DNApolymerase large fragment at 43°C (0), each followed by heat inactivation and subsequent incubation with T4polymerase and DNA ligase (D). Each standard reaction mixture (0.045 ml) contained 50mM Tris-hydrochloride(pH 8.0), 8 mM MgCl2, deoxynucleoside triphosphates (30 FjM each), 25 FM NAD, 5 mM ,B-mercaptoethanol,150 mM NH4C1, 15 gLM 3H-labeled ColEl DNA, and 0.15 Fg of DNA ligase (a gift from I. R. Lehman). Eachform of DNA polymerase I used was present at a concentration of 10 U/reaction. DNA polymerase I waspurchased from New England Biolabs, Inc. The DNA polymerase I large fragment (Klenow fragment) waspurchased from New England Nuclear Corp. polAexl mutant polymerase was prepared by the method ofUyemura et al. (8). T4 DNA polymerase (Miles Laboratories, Inc.) was present at a concentration of 0.4U/reaction. The reaction time was 30 min, and reactions were terminated by centrifugation through Sephadex G-50 (Pharmacia) to remove unreacted deoxyribonucleoside triphosphates (7).per molecule, so that polymerase activity differ-ences could not affect the results. Figure 1Ashows the results with untreated DNA and DNAreacted with DNA polymerase I in the presenceof DNA ligase. DNA reacted with DNA poly-merase I in-the absence of DNA ligase resultedin a sucrose gradient sedimentation profile thatwaas indistinguishable from the profile of un-treated DNA. When both enzymes were presentat 30°C, 85% of the original gaps were repaired.Similar results were obtained at 43°C (data notshown). The polymerase large fragment and thepolAexl polymerase were also tested with DNAligase for the ability to repair at 30 and 43°C (Fig.1B and C). The mutant polymerase was lessefficient than the wild type at generating a ligata-ble substrate, repairing 34% of the original gapsat 30°C. When the temperature was increased to43°C, only 22% repair was observed. The pro-teolyzed polymerase I large fragment was inef-fective at either temperature, repairing only 21and 22% of the gaps at 30 and 43°C, respectively.With respect to the polymerase large fragmentour results are consistent with those of Lund-quist and Olivera (4), who showed that thispolymerase fragment displaces nucleotides atthe 5' terminus of a nick to produce single-stranded tails that resist ligation.There are two reasons why the ligase sub-strate may not be generated by the polAexlpolymerase: (i) the defective polymerase cannotclose the gap down to a nick; and (ii) thedefective polymerase immediately begins to dis-place the strand ahead of it once the gap hasbeen closed, providing no ligatable intermediate.If the first alternative were predominant, subse-quent synthesis at a 3' terminus would generatea DNA ligase substrate.We designed a series of experiments to test forthese mechanisms. Under identical conditionsT4 DNA polymerase, wild-type DNA polymer-ase I, polAexl mutant DNA polymerase I, andthe DNA polymerase I large fragment wereallowed to synthesize for 30 min at 43°C in the30 I I I I' 1.A B20I0.20 CD-'0VOL. 1S5, 198325 3 5 10TOPI BOTTOM924 NOTESabsence ofDNA ligase on the 20-nucleotide gap-containing template by using [t-32P]dATP (NewEngland Nuclear Corp.) and three unlabeleddeoxyribonucleoside triphosphates. Nucleotideincorporation was determined from the 32Pincorporation corrected for counting efficiency.Maximum synthesis by T4 DNA polymeraseyielded 21 nucleotides per 3' terminus, DNApolymerase I added an average of 67 nucleotidesper 3' terminus, the polymerase large fragmentadded an average of 55 nucleotides per 3' ter-minues, and polAexl polymerase added an aver-age of 36 nucleotides per 3' terminus. Therefore,each form of DNA polymerase I added morethan enough nucleotides to completely fill thegap. The addition ofDNA ligase to the reactionmixtures reduced the average number of nucleo-tides added by the wild-type polymerase, thepolAexl mutant polymerase, and the polymer-ase large fragment by 30, 1, and 4%, respective-ly. However, synthesis remained well in excessof gap length.To detect the products evolved as a result of5' -+ 3' exonuclease activity, the polymeraseassay was repeated, this time in the presence ofunlabeled deoxynucleoside triphosphates. Thereactions were terminated, and the product sizeswere determined by column chromatography onBio-Gel A5m (Bio-Rad Laboratories). The elu-tion profiles of the template reacted with thewild-type polymerase showed a peak of radioac-tivity corresponding to the single nucleotidesevolved as a result of 5' 3' exonucleaseactivity (nick translation). The relative size ofthe single nucleotide peak represented 0.3% ofthe total DNA. The elution profiles of the tem-plate reacted with either the polymerase largefragment or the polAexl mutant polymerasefailed to show any products less than 100 nucleo-tides long (i.e., smaller than the exclusion limitof the column [data not shown]), implying thatpolymerization in excess of gap length by eitherof these enzymes occurs solely by strand dis-placement.To examine the extent of strand displacement,the 5' termini of the gap-containing templateswere labeled with 32p. Each enzyme was al-lowed to polymerize on this template, and thereactions were terminated by heating the mix-tures to 70°C for 5 min. A sample of eachreaction preparation was then treated with S1nuclease (Sigma Chemical Co.) to degrade thesingle-stranded 5' ends displaced by polymeriza-tion. Under these conditions, the maximum re-lease of acid-soluble 32P at 30 or 43°C occurredafter reaction with wild-type polymerase I fol-lowed by S1 nuclease; we defined this release asthe 100lo value for this experiment. At both 30and 43°C, the amount of 32P released afterreaction with the polAexl polymerase or thepolymerase large fragment without S1 nucleasewas not significantly different from the amountreleased by Si nuclease alone (considered the0o value for this experiment). However, afterreaction with the polymerase large fragment at43°C, subsequent treatment with Si nucleasereleased 82% of the 32p. At 30 and 430C reactionwith the polAExl polymerase followed by Sinuclease released 53 and 44% of the 32p, respec-tively. These results indicate that somewhat lessthan one-half of the 5' ends were strand dis-placed by the action of the polAexl polymeraseat the restrictive temperature.To detect the presence of residual gap struc-tures after polymerization, the polymerase assaywas repeated at 43°C in the absence of DNAligase. After 30 min, the reactions were termi-nated by heating the mixtures for 10 min at 70°C.Bacteriophage T4 DNA polymerase and DNAligase were then added, and the reaction wascontinued for 30 min at 37°C. When the wild-type polymerase I reaction was terminated andreconstituted with T4 DNA polymerase andDNA ligase, 76% of the original gaps wererepaired (data not shown). When the polymeraseI large fragment was reacted, inactivated, andreconstituted with T4 DNA polymerase andDNA ligase, only 15% of the original gaps wererepaired (Fig. ID). In contrast, when thepolAexl polymerase was used in this manner,62% of the original gaps were repaired (Fig. 1D).This result indicates that in most gaps synthesisby the polAexl polymerase leaves a residual gapstructure which is not a substrate for ligation.To determine the sizes of these residual gaps,the polAexl DNA polymerase was reacted withthe template and four deoxynucleoside triphos-phates for 30 min at 30 and 43°C. The reactionmixtures were heat inactivated and then centri-fuged through Sephadex G-50 (Pharmacia FineChemicals, Inc.) to remove unincorporated nu-cleotides. The reaction was reconstituted withT4 DNA polymerase and a-32P-labeled deoxy-nucleoside triphosphates and further reacted for30 min at 37°C. Approximately one additionalnucleotide was added by the T4 polymeraseafter initial incorporation by the polAexl poly-merase at either temperature.In conclusion, the polAexl DNA polymeraseis expected to function poorly in excision repairat elevated temperatures based on the propertiesof the pure enzyme in vitro. We examined theprocess of gap filling by this enzyme and foundthat the 5' -- 3' exonuclease dysfunction pre-vents the formation of a ligatable substrate aftera gap is filled. At restrictive temperatures onlynominal repair can be obtained with polAexlpolymerase and DNA ligase, although anamount of nucleotides well in excess of gap sizehas been added. In direct contrast to resultsJ. BACTERIOL.NOTES 925obtained with the polymerase I large fragment,heat inactivation of polAexl reactions followedby treatment with T4 polymerase and DNAligase did result in significant gap repair. Ananalysis of products on sizing columns showedno mono- or oligonucleotide products evolvedfrom the polAexl polymerase reaction. Stranddisplacement of 5' termini was observed on afraction of the gaps. We conclude that with thepolAexl polymerase, synthesis on a large popu-lation of 3' termini stops short of the 5' terminus,leaving a residual template that can be extendedby T4 polymerase and DNA ligase for completerepair. This limited synthesis is masked by asmaller population of 3' ends which have sus-tained significant synthesis via strand displace-ment, resulting in a total incorporation per gapthat is much greater than the gap size.Our results suggest that at restrictive tempera-tures in vivo the polAexl DNA polymerase doesnot efficiently close most gaps to nicks and that,with those which are closed, begins to stranddisplace before ligation can occur.ACKNOWLEDGMENTWe gatefully acknowledge W. Dean Rupp for access todata before publication.This work was supported by Public Health Service grantsGM 24441 and T32-GM07102 from the National Institutes ofHealth.LITERATURE CITED1. Hanawalt, P. C., P. K. Cooper, A. K. Ganesan, and C. A.Smith. 1979. DNA repair in bacteria and mammalian cells.Annu. Rev. Biochem. 48:783-836.2. Hockensmith, J. W., and R. A. Bambara. 1981. Kineticcharacteristics which distinguish two forms of calf thymusDNA polymerase alpha. Biochemistry 20:227-232.3. Konrad, E. B., and I. R. Lehman. 1974. A conditionallethal mutant of Escherichia coli K12 defective in the 5' -*3' exonuclease associated with DNA polymerase I. Proc.Natl. Acad. Sci. U.S.A. 71:2048-2051.4. Lundquist, R. C., and B. M. Ollvera. 1982. Transient gen-eration of displaced single stranded DNA during nicktranslation. Cell 31:53-0.5. Masamune, Y., and C. C. Rkhardson. 1971. Strand dis-placement during deoxyribonucleic acid synthesis at singlestrand breaks. J. Biol. Chem. 246:2692-2701.6. Matson, S., and R. A. Bambara. 1981. Short deoxyribonu-cleic acid repair patch length in Escherichia coli is deter-mined by the processive mechanism of deoxyribonucleicacid polymerase I. J. Bacteriol. 146:275-284.7. Penefsky, H. S. 1977. Reversable binding of Pi by beef heartmitochondrial adenosine triphosphatase. J. Biol. Chem.252:2891-2899.7a.Saogar, A., and W. D. Rupp. 1983. A novel repair enzyme:UVRABC excision nuclease of Escherichia coli cuts aDNA strand on both sides of the damaged region. Cell33:249-260.8. Uyemura, D., D. C. Eichler, and I. R. Lehman. 1976.Biochemical characterization of mutant forms of DNApolymerase I from Escherichia coli. J. Biol. Chem.251:4085-4089.VOL. 155, 1983

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Alternative Explanation for Excision Repair Deficiency Caused by the polAex1 Mutation

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Alternative explanation for excision repair deficiency caused by the polAex1 mutation.

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