Proc. Natl. Acad. Sci. USA 79 (1982) 5101

Correction. In the article "Recombinational bypass of pyrimi-dine dimers promoted by the recA protein ofEscherichia coli"by Zvi Livneh and I. R. Lehman, which appeared in number

-RFII-RFIlI

-RFl

-ss DNA

10, May 1982 ofProc. Natl. Acad. Sci. USA (79, 3171-3175), thereproduction of Fig. 2 did not do justice to the original pho-tograph. A version at less contrast is shown here.

Ss RAlI I N1

| i N id I gg g~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~vi' 1: i-- - .'t. f,'.'4' X - ~~~~R F

..-R..I':~ s DNAi...i , i.. 2,..;:-.

it a.-; -~~~~ssDNA

M 0 10 25 50 90 120 0 10 25 50 90 120 M M 0 10 25 50 90 120 0 10 25 50 90 120 M

No dimers 7 dimers molecule 21 dimersimolecule 70 dimers/molecules

FIG. 2. Analysis by agarose gel electrophoresis of strand exchange between UV-irradiated phage kX174 ss DNA preparations with 0, 7, 21,and 70 dimers per molecule and 4X174 linear duplex DNA. The reaction was carried out without an ATP-regenerating system. Lanes: M, DNAmarkers; 0-120, minutes of incubation.

Correction. In the article "Linkage disequilibrium due to ran-dom genetic drift in finite subdivided populations" by TomokoOhta, which appeared in number 6, March 1982 of Proc. Natl.Acad. Sci. USA (79, 1940-1944), the author requests that thefollowing be noted. The 16th line of the Abstract should read"natural selection and limited migration, showing the latter asthe main cause of the observed linkage disequilibrium. "

Correction. The article "Molecular dynamics of an a-helicalpolypeptide: Temperature dependence and deviation from har-monic behavior" by Ronald M. Levy, David Perahia, and Mar-tin Karplus, which appeared in number 4, February 1982 ofProc. Natl. Acad. Sci. USA (79, 1346-1350), should have ap-peared under the classification Biophysics.

Corrections

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Proc. Natl. Acad. Sci. USA 79 (1982) 5101

Correction. In the article "Recombinational bypass of pyrimi-dine dimers promoted by the recA protein ofEscherichia coli"by Zvi Livneh and I. R. Lehman, which appeared in number

-RFII-RFIlI

-RFl

-ss DNA

10, May 1982 ofProc. Natl. Acad. Sci. USA (79, 3171-3175), thereproduction of Fig. 2 did not do justice to the original pho-tograph. A version at less contrast is shown here.

Ss RAlI I N1

| i N id I gg g~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~vi' 1: i-- - .'t. f,'.'4' X - ~~~~R F

..-R..I':~ s DNAi...i , i.. 2,..;:-.

it a.-; -~~~~ssDNA

M 0 10 25 50 90 120 0 10 25 50 90 120 M M 0 10 25 50 90 120 0 10 25 50 90 120 M

No dimers 7 dimers molecule 21 dimersimolecule 70 dimers/molecules

FIG. 2. Analysis by agarose gel electrophoresis of strand exchange between UV-irradiated phage kX174 ss DNA preparations with 0, 7, 21,and 70 dimers per molecule and 4X174 linear duplex DNA. The reaction was carried out without an ATP-regenerating system. Lanes: M, DNAmarkers; 0-120, minutes of incubation.

Correction. In the article "Linkage disequilibrium due to ran-dom genetic drift in finite subdivided populations" by TomokoOhta, which appeared in number 6, March 1982 of Proc. Natl.Acad. Sci. USA (79, 1940-1944), the author requests that thefollowing be noted. The 16th line of the Abstract should read"natural selection and limited migration, showing the latter asthe main cause of the observed linkage disequilibrium. "

Correction. The article "Molecular dynamics of an a-helicalpolypeptide: Temperature dependence and deviation from har-monic behavior" by Ronald M. Levy, David Perahia, and Mar-tin Karplus, which appeared in number 4, February 1982 ofProc. Natl. Acad. Sci. USA (79, 1346-1350), should have ap-peared under the classification Biophysics.

Corrections

Proc. NatL Acad. Sci. USAVol. 79, pp. 3171-3175, May 1982Biochemistry

Recombinational bypass of pyrimidine dimers promoted by therecA protein of Escherichia coli

(postreplication repair/single-stranded DNA binding protein/T4 endonuclease V/UV-irradiated DNA)

Zvi LIVNEH AND I. R. LEHMANDepartment of Biochemistry, Stanford University School of Medicine, Stanford, California 94305

Contributed by I. Robert Lehman, February 25, 1982

ABSTRACT recA protein, in the presence of single-strandedDNA binding protein and ATP, promotes the complete exchangeof strands between circular single-stranded DNA containing py-rimidine dimers and a homologous linear duplex, converting thepyrimidine dimer-containing single-stranded DNA to a circularduplex. Bypass ofa pyrimidine dimer during the branch-migrationphase of the reaction requires Mo20 seconds, a rate 1/50th of thatin the absence of the dimer. The circular duplex product is spe-cifically incised by the pyrimidine dimer-specific T4 endonucleaseV, and the resulting 3' hydroxyl termini can serve as primers fordeoxynucleotide polymerization by DNA polymerase I. Thesefindings indicate that recA protein serves a direct role in recom-binational repair and demonstrate that the pyrimidine dimers thathave been bypassed can be processed by enzymes of the excision-repair pathway.

Exposure of cells to UV irradiation leads to the formation ofpyrimidine dimers and, to a lesser extent, other lesions in theirDNA (1). These lesions can be removed by excision of the dam-aged bases or by photoreactivation-a process that specificallymonomerizes pyrimidine dimers (2, 3). In most instances, how-ever, pyrimidine dimers are not entirely excised or mono-merized and present an obstacle to DNA replication. Replica-tion can resume at a site beyond the dimer; however, as aconsequence, a single-stranded gap is introduced (4-6). Pyrim-idine dimers in single-stranded regions of duplex DNA cannotbe excised (3). However, they can be bypassed and, thereby,become part of the chromosome.

At least two mechanisms for the bypass ofpyrimidine dimershave been identified in Escherichia coli; both are dependenton the product of the recA gene (recA protein). One of these,"error-prone repair," permits bypass of pyrimidine dimers atthe expense of replication fidelity and is responsible for mostUV-induced mutagenesis (7, 8). Although the mechanism oferror-prone repair is unknown, recA protein clearly plays a reg-ulatory role (9). The second type of bypass involves a recom-binational event that occurs through exchange of homologoussegments between sister DNA molecules (10). In this case recAprotein participates directly (11-14).

In this study we have enquired whether the recA protein canpromote strand exchange between single-stranded DNA (ssDNA) containing pyrimidine dimers and a homologous duplex,a reaction that is very likely part of recombinational repair invivo. We show that recA protein, in the presence of E. coli ssDNA binding protein (SSB), promotes the recombinational by-pass of pyrimidine dimers through branch migration. We alsoshow that the duplex DNA product containing the pyrimidinedimers can be processed by enzymes known to be involved inexcision repair.

MATERIALS AND METHODSMaterials. Bacteriophage 4X174 circular ss DNA was pre-

pared from OX174am3-infected E. coli C as described (15).4X174 replicative form I [RFI; supercoiled double-strandedDNA (ds DNA)] was prepared essentially as described (16) withtwo equilibrium sedimentations in CsCl/ethidium bromide re-placing the sucrose density gradient sedimentation. DNA la-beled with [3H]thymidine was prepared by the same procedurewith E. coli H502 (thy-endor-uvrA) as host. 4X174 linear du-plex DNA was prepared by cleavage of 4X174 RFI with re-striction endonuclease Pst I, followed by phenol extraction.

recA protein, purified as described (17), was the gift of Mi-chael Cox ofthis department. SSB and DNA polymerase I werethe gifts of J. Kaguni and S. Scherer, respectively, of this de-partment. T4 endonuclease V was the gift of P. C. Seawell andA. K. Ganesan, and T4 DNA ligase was the gift of R. Simoni,ofthe Biological Sciences Department, Stanford University. PstI restriction endonuclease was the product of New EnglandBioLabs; nuclease S1 and pyruvate kinase were purchased fromSigma.

ATP, ADP, and unlabeled dNTPs were from P. L. Biochem-icals; [3H]dTlP and [3H]thymidine were purchased fromAmersham.

Irradiation of DNA. 4X174 circular ss DNA in 10 mMTris HCl, pH 7.5/1 mM EDTA (400 ,ul at 50 jig/ml) was ir-radiated in 2-mm-width quartz spectrophotometric cells, witha germicidal UV lamp as the light source. The incident dose ratewas 3.8 J m-2 sect as determined by a Latarjet UV meter. Thenumber of pyrimidine dimers per molecule of [3H]thymidine-labeled ss DNA was determined by acid hydrolysis followed bypaper chromatography (18). DNA preparations used for thisstudy contained 7 pyrimidine dimers per molecule (irradiationfor 1 min), 21 dimers (3 min), 36 dimers (6 min), and 70 dimers(18 min).Measurement of DNA Strand Exchange. The standard re-

action mixture contained 25 mM Tris HCl (pH 7.2), 5% (vol/vol) glycerol, 10mM MgCl2, 1 mM dithiothreitol, 2.2mM ATP,6.7 ,AM UV-irradiated 4X174 circular ss DNA, 11.2 AM 4X174linear duplex DNA, 4.2 AM recA protein, and 0.37 AM SSB.When an ATP-regenerating system was included, it contained2.1 mM phosphoenolpyruvate and four units of pyruvate ki-nase. The reaction was started by addition ofSSB and ATP afterpreincubation of the other components at 370C. Strand ex-change was measured by agarose gel electrophoresis or the S1nuclease assay.

Agarose Gel Electrophoresis. Samples (25 jd) were removedfrom the reaction mixture, added to 2 ,l of 10% NaDodSO4,

Abbreviations: ss DNA, single-stranded DNA; ds DNA, double-stranded DNA; RF, replicative form; RFI, supercoiled ds DNA; RFII,circular ds DNA containing a single-strand break in one of the twostrands; SSB, E. coli ss DNA binding protein.

3171

The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

3172 Biochemistry: Livneh and Lehman

and kept on ice. They were adjusted to 10% glycerol and 0.01%bromphenol blue and subjected to electrophoresis in 0.7% agar-ose gels in 40 mM Tris acetate, pH 8.1/2 mM EDTA at a con-stant current of50 mA. Under these conditions, 4X174 circularss DNA and the RFI, RFII (circular ds DNA containing a single-strand break in one ofthe two strands), and linear duplex formsof 4X174 DNA are resolved.

SI Nuclease Assay. The assay was performed essentially asdescribed (19) with slight modifications. Samples (25 1d) wereremoved from the reaction mixture and added to 2 Al of 10%NaDodSO4 in an Eppendorf test tube. After at least 15 min onice, 380 A.l of 0.5 M NaCV50 mM sodium acetate, pH 4.6/1mM zinc acetate containing denatured calf thymus DNA (20,ug), and S1 nuclease (2.0 units/ml) was added. The mixtureswere incubated for 25 min at 3rC, then 40 tg ofdenatured calfthymus DNA was added, and the DNA was precipitated by theaddition of 1 ml of cold 10% trichloroacetic acid. After 35 minon ice, the mixtures were filtered through Whatman GF/C fil-ters, which were rinsed three times with 1-ml portions of cold10% trichloroacetic acid and once with 95% ethanol. They werethen dried and assayed for radioactivity. The total acid-precip-itable [3H]DNA was determined in the same way except thatS1 nuclease was omitted. To obtain the value for 100% heter-oduplex formation, this value was multiplied by a correctionfactor of 0.68 to allow for the slight excess of ss DNA (X0. 85)and to compensate for factors found to affect the efficiency ofthe S1 nuclease assay (xO.8) (unpublished data).ATPase Assay. ATP hydrolysis during strand exchange was

determined as described (20) with [3H]ATP at 80 uCi/ml (1 Ci= 3.7 X 1010 becquerels).

Isolation ofOX174 RFII Product. The RFII product ofstrandexchange was separated from other DNA species by preparativegel electrophoresis as described except that low-meltingSeaPlaque agarose (FMC, Rockland, ME) was used. The bandswere visualized under long-wavelength UV light, cut out,melted at 65°C, cooled to 37°C, and then extracted twice withphenol.

42-I recA proteinSSBATP j

Incision and Polymerization. The incision, reaction mixture(30 ,1) contained 20 mM Tris HCl (pH 7.5), 0.5 mM EDTA,0.1 M NaCl, 8 mM dithiothreitol, 4% sucrose, 80 yg of bovineserum albumin per ml, 0.06 ,ug of RFII product, either unir-radiated DNA or UV-irradiated DNA containing seven pyrim-idine dimers per molecule, and 400 units of T4 endonucleaseV (an amount that will incise 4.5 X 1011 dimers per min at3rC). The mixture was incubated at 37C for 10 min. Whena ligation step was included, the T4 endonuclease V was inac-tivated by heating (70°C for 4 min), and ATP was added to aconcentration of1 mM, followed byT4 DNA ligase. The mixturewas incubated at 37°C fdr 10 min and again heat inactivated.For the polymerization reaction, MgCl2 was added to a finalconcentration of 10 mM; dATP, dGTP, and dCTP were addedat 62 ,uM, and [3H]dTTP was added at 23 ,uM. The reaction wasstarted by addition of 0.75 ,ug of DNA polymerase I; incubationwas at 37°C. The reaction was stopped by addition of 1 ml of10% trichloroacetic acid/0. 1 M sodium pyrophosphate; after 15min on ice, the mixtures were filtered through GF/C filters,washedwith four portions (2 ml each) of10% trichloroacetic acid/0.1 M sodium pyrophosphate, once with 95% ethanol, and thendried for the radioactivity assay.

RESULTSPyrimidine Dimers Are Bypassed During recA Protein-Pro-

moted Branch Migration. recA protein, in the presence ofSSBand ATP, promoted the exchange of strands between UV-ir-radiated phage cX174 circular ss DNA and a homologous4X174 linear duplex. The reaction products were a RFII-likecircular duplex containing pyrimidine dimers and a full-lengthlinear single strand (Fig. 1). A series of UV-irradiated 4X174ss DNAs containing an average of 7, 21, and 70 pyrimidine di-mers per molecule, respectively, were incubated with #X174linear duplex DNA, and the reaction was observed by agarosegel electrophoresis under conditions that allowed resolution ofss DNA, RFII, and linear duplex DNA. recA protein promoted

ATP ADP + Pi

recA proteinSSB)

DNA polymerase IdNTPs

FIG. 1. Model for recA protein-promoted repair of DNA containing pyrimidine dimers. In the presence of recA protein, SSB, and ATP, UV-ir-radiated phage 4X174 ss DNA reacts with the homologous linear duplex DNA to produce a D loop, which is extended by branch migration coupledto ATP hydrolysis. The RFII product is incised at the site of the pyrimidine dimers by T4 endonuclease V, and the 3' hydroxyl termini formed serveas primers for DNA polymerase I.-, DNA strand that was originally in the duplex form; -, DNA strand that was in the single-stranded form;vww, DNA regions newly synthesized by DNA polymerase I; *, apyrimidinic sites created by T4 endonuclease V.

Proc. Nad Acad. Sci. USA 79 (1982)

j

4

Proc. NatL Acad. Sci. USA 79 (1982) 3173

-RFII- RFIII

-RFI

-ssDNW

-RFII-RFIII

-RFi

-ssDNA

M 0 10 25 50 90 120 0 10 25 50 90120 M M 0 10 25 50 90120 0 10 25 50 90 120 M

No dimers 7 dimers/molecule 21 dimers/molecule 70 dimers/molecule

FIG. 2. Analysis by agarose gel electrophoresis of strand exchange between UV-irradiated phage 4X174 ss DNA preparations with 0,7,21, and70 dimers per molecule and OX174 linear duplex DNA. The reaction was carried out without an ATP-regenerating system. Lanes: M, DNA markers;0-120, minutes of incubation.

the complete exchange ofstrands between the linear duplex andss DNAs containing 7 and 21 dimers (Fig. 2). In contrast, onlylimited strand exchange occurred with ss DNA containing 70dimers. In addition to RFII and the linear single strand, formsmigrating more slowly than RFII appeared at early reactiontimes (Fig. 2). These species are probably intermediates in thestrand-exchange reaction-for example, the extended D-loopstructures that have been observed by electron microscopy(11)-and disappear upon continued incubation, with a corre-sponding increase in RFII. The RFII originated from the inputss DNA because, when 3H-labeled ss DNA and unlabeled linearduplex were used, there was a decrease in the amount of ra-dioactivity in the ss DNA, accompanied by the parallel accu-mulation of label in RFII (data not shown).

Unirradiated ss DNA and ss DNA containing seven pyrim-idine dimers showed similar reaction patterns (Fig. 2), indicat-ing that the branch migration accompanying strand exchangeproceeded past the pyrimidine dimers. With ss DNA containing21 dimers, the rate of reaction was lower as judged by the per-sistence of slowly migrating species at longer reaction times.With ss DNA containing 70 dimers, the reaction was almostundetectable.

Branch migration also was observed by measuring hetero-duplex formation with the SI nuclease assay; 70% of the unir-radiated ss DNA was converted to heteroduplex within 35 min(Fig. 3). The rate of heteroduplex formation with ss DNA con-taining 7, 21, and 36 dimers was slower but neverthelessreached nearly the same extent as that observed with unirra-diated ss DNA (70%, 70%, and 65%, respectively). In the caseof ss DNA containing 70 dimers, heteroduplex formation pro-ceeded at a low rate and reached a level of only 40%.ATP Hydrolysis. ATP hydrolysis accompanies branch mi-

gration during recA protein-promoted strand exchange (19). Foreffective bypass ofpyrimidine dimers during branch migration,an ATP-regenerating system (phosphoenolpyruvate and pyru-vate kinase) was required. Its absence did not significantly in-fluence branch migration with unirradiated ss DNA or with ssDNA containing seven dimers; however, the rate and extentwere significantly diminished with ss DNA preparations that

contained 21, 36, or 70 dimers per molecule (data not shown).In contrast, ATP hydrolysis occurred at the same rate with bothUV-irradiated and with unirradiated ss DNA, even in the ab-sence ofan ATP-regenerating system (data not shown). A plau-sible interpretation of these effects is that in the absence of anATP-regenerating system, ADP accumulates to the point atwhich it inhibits the reaction (unpublished data). Inasmuch asthe rate of branch migration is diminished by pyrimidine di-

010 20 30 40 50 0 70

70 __

F 60 36b

n50

40

zC~ 30

4'X174 ss DNA and 4XX174 linear duplex DNA. An ATP-regenerationsystem was included in the reaction, and heteroduplex formation wasmeasured with the Si nuclease assay. ss DNA preparations: o, Unir-radiated; *, with 7 pyrimidine dimers; A, with 21 dimers; *, with 36dimers, and o, with 70 dimers.

Biochemistry: Livneh and Lehman

3174 Biochemistry: Livneh and Lehman

"300 60

200- ~~~~~~~40-

100l~o ;20-

0 20 40 60 0 20 40 60Time, min

FIG. 4. Formation of primers for DNA polymerase I by incision of the RFII product with T4 endonuclease V. The RFII was incubated with(.) or without (o) T4 endonuclease V, followed by incubation with DNA polymerase I. Unirradiated RFII was tested under similar conditions inthe presence (v) or in the absence (A) of T4 endonuclease V. The reactions were performed without (A) or with (B) treatment with T4 DNA ligaseprior to reaction with DNA polymerase I.

mers, whereas ATP hydrolysis is unaffected, an inhibitory levelofADP is reached at a lower extent of strand exchange.Enzymes of Excision Repair Act on the RFH Product Con-

taining Pyrimidine Dimers. The pyrimidine dimer-specific T4endonuclease V incised the RFII product of DNA strand ex-

change at the dimers, producing 3' hydroxyl termini that couldserve as primers for DNA polymerase I (Fig. 1). Incorporationof[3H]dTTP into the pyrimidine dimer-containing RFII incisedby T4 endonuclease V occurred to a greater extent than into theRFII that had not been treated with endonuclease V (Fig. 4).There was some [3H]dTTP incorporation when the RFII formedfrom unirradiated ss DNA was incubated with T4 endonucleaseV, presumably because of nonspecific single-strand breaks in-troduced by the T4 endonuclease V (Fig. 4A). Because T4 DNAligase does not catalyze the ligation of single-strand breaks in-troduced by T4 endonuclease V at pyrimidine dimers (unpub-lished data), the nonspecific breaks introduced by the T4 en-

donuclease V preparation could be specifically eliminated byligase treatment. Under these conditions, incorporation oc-curred only with pyrimidine dimer-containing RFII that hadbeen incised with T4 endonuclease V (Fig. 4B).The action of T4 endonuclease V on pyrimidine dimer-con-

taining DNA involves cleavage of the glycosylic bond of the 5'-pyrimidine of the dimer, followed by incision of the phospho-diester bond linking the two pyrimidines and leaving a 3'-apy-rimidinic site (21-24). The ability of such sites to serve as

primers for DNA polymerase I is increased after treatment withendonucleases IV or VI (22). Nevertheless, the RFII incised atthe site of the pyrimidine dimers clearly provided primers forDNA polymerase I, even in the absence ofadded endonucleaseIV or VI, and polymerization was readily detectable (Fig. 4B;refs. 22 and 25).

DISCUSSIONOur basic finding is that recA protein can promote branch mi-gration through a DNA strand containing pyrimidine dimers.The initial rate of branch migration with unirradiated DNA is4 or 5 base pairs per second (19). Assuming that the decreasein rate observed with DNA containing 7, 21, or 36 pyrimidinedimers is the result of an impediment to branch migration atthe sites of the pyrimidine dimers, it can be calculated that-20 seconds are required to bypass a dimer (an average valueobtained from the 5- and 10-min points of Fig. 3). This repre-

sents a decrease by a factor of 50 in the rate of the branch mi-gration. Nevertheless, strand exchange does proceed to com-pletion, even with heavily irradiated ss DNA containing 36pyrimidine dimers. This finding implies that damaged bases inaddition to pyrimidine dimers may be bypassed. Thus, a mol-ecule of ss DNA irradiated with 1.37 kJ m-2 contains the fol-lowing lesions: 22 thymine-thymine and 12 cytosine-thyminedimers, both in the cis-syn conformation; 2 thymine-thyminedimers in the less frequent trans-syn conformation; 6 cytosinehydrates; and about 1.5 pyrimidine adducts (64'-[pyrimidine-2'-one]-pyrimidine) (ref. 2; unpublished data). It is thereforelikely that recA protein-promoted branch migration can alsoproceed through a limited number of mismatched bases.

The finding that pyrimidine dimers can be bypassed suggeststhat branch migration does not necessarily proceed through aconcerted mechanism in which the unwinding of one base pairin the duplex occurs simultaneously with the winding of theprevious base into the heteroduplex. Although this may be thecase for undamaged DNA, our findings suggest that whenbranch migration encounters a dimer, several base pairs withinthe duplex are unwound before rewinding of the duplex canoccur. In fact, the decrease in rate at the site of a pyrimidinedimer may indicate that the normal pathway of branch migra-tion does indeed proceed through a concerted mechanism.

Both SSB and ATP hydrolysis are required for branch mi-gration with UV-irradiated DNA. In the absence of SSB, therate is much slower and reaches an extent of 35-40% after 60min (data not shown). Although the precise role of SSB is notyet clear, it appears to be involved in the formation ofacomplexwith recA protein and ss DNA (unpublished data).

Once dimers have been bypassed, they become part of aduplex DNA structure and therefore, should, be removable byexcision repair. Indeed, the RFII product of strand exchangebetween pyrimidine dimer-containing ss DNA and linear du-plex DNA is specifically incised by the pyrimidine dimer-spe-cific T4 endonuclease V, with the formation of 3' hydroxyl ter-mini that can then serve as primers for DNA polymerase I. Thelatter is believed to be the principal enzyme involved in theexcision and repolymerization steps of the excision-repair path-way in E. coli (Fig 1; refs. 2 and 3).

West et al. (26) have shown that recA protein can promotestrand exchange between a gapped DNA molecule and a ho-mologous duplex, if the latter contains a single-strand break

I I I

B

Proc. Nad Acad. Sci. USA 79 (1982)

Proc. Natl. Acad. Sci. USA 79 (1982) 3175

-< -A.TT r A- TT

bBranchmigration

=Strandseparation

Repairreplication

e d

FIG. 5. A model describing the sequence of events in postreplication recombinational repair in E. coli. (a) Replication stop at a pyrimidine dimer.(b) Endonucleolytic incision opposite the single-stranded gap. (c) D-loop formation. (d) Branch migration and bypass of the dimer. (e) Strand sep-aration, followed by repair replication of single-stranded regions and ligation.

opposite the gap. This observation and the other known prop-erties of recA protein were used as the basis of a model for re-combinational repair. A similar scheme (Fig. 5) incorporates ourfinding that recA protein-promoted branch migration can pro-ceed past a pyrimidine dimer. The following steps are dia-grammed in Fig. 5: DNA replication is blocked at a pyrimidinedimer but then resumes at a point beyond the dimer (a); thehomologous parental strand on the sister duplex is cleaved bya nuclease opposite the single-stranded gap (b); recA proteinunwinds the sister duplex at the nick and forms a D loop at thesingle-stranded region, past the pyrimidine dimer (c); recA pro-tein, in the presence of SSB, promotes branch migration cou-pled to ATP hydrolysis, leading to strand exchange and bypassof the dimer (d); and nuclease cleavage resolves the X structureinto two sister duplexes, and repair replication followed by li-gation completes the process (e). The bypassed dimers now canbe removed either by excision repair or by photoreactivation.Alternatively, they may simply persist during additional roundsof DNA replication.

Because it is likely that other types of lesions in DNA arebypassed by recombinational repair, it would be of interest toexamine the effects of bases modified by chemical carcinogensor by ionizing radiation on recA protein promoted branchmigration.We are grateful to Dr. Michael M. Cox for his interest, helpful sug-

gestions, and thoughtful criticism ofthe manuscript. We also thank Drs.P. C. Seawell and A. K. Ganesan for T4 endonuclease V and for helpfuldiscussions. This work was supported by grants from the National In-stitutes of Health (GM 06196) and the National Science Foundation(PCM 79-04638). Z. L. was supported by a Dr. Chaim Weizmann Post-doctoral Fellowship for Scientific Research.

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Biochemistry: Livneh and Lehman