DNA Substrate Requirements for Stable Joint Molecule Formation by

THEJOURNAL OF BIOLOGICAL CHEMISTRY (c)

Vol. 266,No. 16, Issue of June 5, pp. 10112-10121.1991 Printed in U.S.A.

1991 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA Substrate Requirementsfor Stable Joint Molecule Formation by the RecA and Single-stranded DNA-binding Proteins of Escherichia coli* (Received for publication, November 19, 1990)

Boyana B. KonfortiS and Ronald W.Davis From the Department of Biochemistry, Stanford University Medical Center, Stanford, California 94305-5307

In reactions between linear single-stranded DNAs bles primarily in the 5’ to 3‘ direction (9). Once formed, this (ssDNAs) andcirculardouble-stranded DNAs (ds- nucleoprotein complex becomes asequence-specific DNADNAs), stable joint molecule formation promoted by binding entity. During the second phase, binding andhomolthe recA protein (RecA) requires negative superhelic- ogous alignment of the two DNA molecules result in the ity, a homologous end, and an RecA-ssDNA complex. formation of a protein-stabilized paranemic jointmolecule (6, Linear ssDNAs with 3’-end homology react more effi- 10-13) in which the incoming strand is paired with its comciently than linear ssDNAs with 5‘-end homology. This 3’-end preference is explained by the finding that3‘- plement in the double-stranded DNA (dsDNA), but the two Conversion of a paranemic ends are more effectively coated RecA by than 5’-ends, strands are not stably interwound. as judged by exonuclease VI1 protection, and are thus joint into a stably interwoundplectonemic joint requires that more reactive. The ability of linear ssDNAs with 5’- the incoming ssDNA and its homologous partner are free to each other.Rotationandintertwining of end homology to react is improved by the presence of rotatearound strands require an end in the region of homology between the low concentrations of exonuclease VII. In reactions between ssDNAs and linear dsDNAs with end homol- two DNAs (10, 13, 14). The final phaseof strand exchange is ogy, stable joint molecule formation occurs more effi- branch migration, the exchange of the incoming ssDNA for ciently when thehomology is at the3’-end rather than the strand resident in thedsDNA molecule (8). at the5’-end of the complementary strand. In addition,Strand exchangebetween linear dsDNA and circular linear dsDNAs with homology at the 3‘-end of the ssDNAwith a short hybridized fragment (15) orcircular complementary strand react more efficiently with lin- ssDNA (16)shows an endpreference. Strand exchange occurs ear ssDNAs with 3’-end homology than with linear when the 3‘-end of the complementary strandof the dsDNA ssDNAs with 5‘-end homology. The ability of linear ssDNAs with 5’-end homology to react, in the absencepairs with the ssDNAcircle, while the 5‘-end of the identical of single-stranded DNA-binding protein, is improved strand of the dsDNA is displaced. Strand exchange does not by adding 33-46nucleotides of heterologous sequence occur when the 5’-end of the complementary strand of the to the5’-end of the linear ssDNA. The poor reactivity dsDNA is available to pair with the ssDNA circle. Using of linear ssDNAs with 5‘-end homology is explained completely homologous ssDNAcircles and linear dsDNAs, by a lack of RecA at the 5‘-ends of linear ssDNAs, Cox and Lehman (17) showed that the formation of heterowhich is a consequence of the polar association and duplex DNA as monitored by the susceptibility to restriction dissociation of RecA. enzyme cleavage occurs in a polar manner. Electron microscopy of reactions between ssDNA circles and linear dsDNAs with regions of nonhomologous DNAinserted at various positions from the end showed an accumulation of migrating The recA protein (RecA)-promoted strand exchange reac- branches at heterologous borders (18). Together, these data tion proceeds via a number of kinetically distinct phases (for have been interpreted tosuggest that RecA promotes branch reviews, see Refs. 1-3). In the first phase of this reaction, migration in the 5’ to 3’ direction relative to the incoming RecA binds cooperatively and stoichiometrically to single- ssDNA within the nucleoprotein complex. stranded DNA(ssDNA)’ to forma nucleoprotein filament (4, The directionalityof RecA-promoted branch migrationsug5). The single-stranded DNA-binding protein (SSB) facili- gests that the 5’-end of a linear ssDNA should be the initiating tates formationof this complex (4,6-8). RecA binds to ssDNA end in reactions with circular duplex DNA. Contrary to this as a sequence-independent DNA-binding protein and assemprediction, in the presence of SSB, linear ssDNAs with 3’molecules, whereas those with * This work was supported by National Institutes of Health Grant end homology form stable joint R37 HG00198-16 (to R. W. D.) and by grants from the Lucille P. 5’-end homology do not (19, 20). Moreover, in the absenceof Markey Charitable Trust and the MacArthur Foundation Fund (to SSB, linear ssDNAs with 3’-end homology are 5-10 times B. B. K.). The costs of publication of this article were defrayed in more reactive than those with 5‘-end homology (20). These part by the payment of page charges. This article must therefore be data suggest that the preference for 3’-end homology is inhereby marked “aduertisement” in accordance with18U.S.C. Section trinsic to RecA-promoted strand exchange. 1734 solely to indicate thisfact. The two sets of reactions described above differ most no$ To whom reprint requests should be addressed. Theabbreviations used are: ssDNA,single-strandedDNA; tably with regard to the structure of the interacting DNAs. dsDNA, double-stranded DNA; SSB, single-stranded DNA-binding Examining the ability of a variety of different sets of DNAs protein; ATPrS, adenosine 5’-(y-thio)triphosphate;Tricine, N - [ 2 to participate in stable joint molecule formation has provided hydroxy-1,l-bis(hydroxymethyl)ethyl]glycine;Hepes,4-(2-hydroxya set of rules that serve to predict the efficiency and end ethyl)-1-piperazineethanesulfonicacid kb, kilobase(s); SDS, sodium preference for any given set of DNA substrates. dodecyl sulfate.

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DNA Substrate Requirements Stable for

Joint Molecule Formation

10113

kb fragment containing1.3 kb of M I 3 sequence. Linear dsDNAswith M13 homology atthe5'-end of thecomplementarystrand were Enzymes and Chemicals-RecA was purified by the spermidine prepared by digesting pBR322-M13(3') with the single-site restriction precipitation method (21) as modified by Kowalczykowski,' and ali- enzyme EcoRI to generate linear dsDNAs with 1.1 kb of M13 sequots were stored at -70 "C in R buffer (20 mM Tris-HC1 (pH7.5), 1 quence. mM dithiothreitol, 0.1mM Na' EDTA) containing10% glycerol. RecA Circular ssDNAs-Ml3mpl8-lacZA was constructed by replacing concentration was determined using an extinction coefficient of 2.7 the EcoRIIHincII fragment of M13mp18 with an EcoRJ/HpaI fragX IO4M" cm" a t 280 nm (22). Additional RecA was purchased from ment of pMC874 (23) comprising 2 kb of lacZ sequences. M13mp18Pharmacia LKB Biotechnology Inc. lacZA/PL was constructed by inserting the 66-base pairEcoRI polySSB and gyrase A and B subunits were generous gifts of Dr. A. linker from PICEM-19R into theEcoRI site of Ml3mplS-lacZA. The Kornberg (Departmentof Biochemistry, Stanford University). EcoRI, orientation of the polylinker was determined by sequencing across HincII, proteinase K, and ATPyS were purchased from Boehringer the insert usinga 19-mer located20 bases from the EcoRI insert as a Mannheim. All other restriction endonucleases, calf intestinal alka- primer. '"P-Labeled viral (+)-strand ssDNA was prepared as previline phosphatase, T4 polynucleotide kinase, and Klenow fragment ously described (20). were purchased from New England Biolahs, Inc. Creatine phosphaLinear ssDNAs-Linear ssDNAs were generated from circular tase and type I creatine phosphokinase were purchased from Sigma. ssDNA by annealing a specific oligonucleotide to generate a unique Exonuclease VI1 and topoisomerase J (calf thymus) were purchased from Bethesda Research Laboratories. Pancreatic DNase I was pur- restriction site. After cleavage with the appropriate enzyme, linear chased from Worthington. %Labeled inorganic phosphate was pur- ssDNAs were purified by gel electrophoresis through 1%low-geling temperature SeaPlaque (FMC, Rockland, ME) agarose in TBE buffer chased from Du Pont-New England Nuclear. Nucleotides-ATP was purchased from Boehringer Mannheim.[a- (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA) at50 V for 12 h at '"PIdCTP and (y-"'PIATP were purchased from Du Pont-New Eng- 4 "C. DNA was visualized by ethidium bromide staining. The ssDNA was cut outof the gel; and theDNA wasisolated by phenol extraction, land Nuclear. Duplex DNAs-Closed circular duplex DNA (pMC874A) was con- followed by ethanol precipitation. Specifically, circular M13mp18structed by deleting a 1.1-kb BamHI/HpaI fragment from pMC874 IacZA ssDNA was annealed with a specific 16-, 18-, or 20-mer, which generated an EcoRI, HincII, or XmnI site, respectively. Linear ss(23). This derivative contains 2 kb of lacZ sequence, and the (+)strand of the lacZ sequence is identical to the 5'- or 3'-end of the DNAs generatedby HincJJ digestion contain 1956 nucleotides of lacZ linear Ml3mpl8-IacZA ssDNA generated by EcoRI or HincII cleav- sequence at the 3'-end and 7220 nucleotides of M13mp18 sequence at the 5'-end. Linear ssDNAs generatedby EcoRI digestion contain age, respectively. In pBR322-M13(3'), an EcoRJ/EcoRV fragment of 1956 nucleotides of lacZ sequence at the5'-end and 7220 nucleotides pBR322 is replaced by a 1.1-kb BalI/EcoRI fragment of M13mp18 of M13mp18 sequence at the 3'-end. Linear ssDNAs generated by sequence. The (+)-strand of the M13mp18 sequence in this dsDNA is identical tothe3'-end of thelinear M13mpl8-IacZA ssDNA XmnI digestion contain 1956 nucleotides of lacZ sequence flanked by generated byEcoRIcleavage. pBR322-M13(5')contains a 1.3-kb 3635 and 3585 nucleotides of M13mp18 sequence. Circular M13mp18HincII/XmnI fragmentof M13mp18 sequencecloned into theEcoRV lacZA/PL ssDNA was annealed witha specific 77-mer that generated 14 unique restriction sites. site of pBR322. The (+)-strand of the M13mp18 sequence in this dsDNA is identical to the 5'-end of the linear Ml3mpl8-lacZA ssDNA StableJoint Molecule Assay-Standard reactionmixturescongenerated by HincII cleavage.SupercoiledDNAwas isolatedas tained 10 p~ "P-labeled ssDNA in nucleotides, 20 p~ duplex DNA previously described (20) or was prepared by treating topoisomerase in nucleotides, 25 mM Tris acetate (pH 7.5), 1 mM MgOAc, 1 mM I-relaxed DNA with gyrase A and B. Topoisomerase I-relaxed DNA dithiothreitol, and5% glycerol. RecA was added to solutions contain(10 pg) was incubated with 300 ng of gyrase A subunit and 150 ng of ing 1 mM ATP, an ATP-regenerating system (4 mM creatine phosgyrase B subunit in 30 mM Tricine/KOH (pH 7.6), 8 mM MgOAc, 2 phate and 9.6 units/ml creatine kinase), and ssDNA and incubated mM ATP, 1 mM dithiothreitol, and 100 pg of bovine serum albumin/ for 5 min a t 37 "C. The magnesium concentration was raised to 10 ml for 1 h a t 30 "C. The reaction was stopped by the addition of SDS mM by addition of 1.0 M MgOAc at the time of addition of duplex t o 0.5%, Na,EDTA to10 mM, and proteinase K to 100 pg/ml; DNA. SSB (one monomer/l8 nucleotides of ssDNA) was omitted or incubated for 15 min at 37 "C and.for 2.5 min at 65 "C; extracted added, and the incubation was continued a t 37 "C for 30 min. The twice with phenol/CHC13 (1:l); precipitated with ethanol; and resus- reactions were stopped by the addition of Na2EDTA to 20 mM and pendedin 20 p1 of 10 mM Tris-HC1(pH 7.6), 1 mM Na,EDTA. SDS to1%. Stable jointmolecule formation in the presence of ATPyS Relaxed dsDNA was prepared by treatment with calf thymus topoi- was carried out in a solution of 6 p~ RecA, 0.9 p~ SSB, 10 p~ '"Psomerase J or DNase I. Topoisomerase I-relaxed DNA was prepared labeled ssDNA in nucleotides, 20 p~ duplex DNA in nucleotides, 1 as follows. Supercoiled pMC874A DNA (10 pg) was incubated with mM ATPyS, 4 mM MgOAc, 1 mM dithiothreitol, and 5% glycerol. 20 units of topoisomerase I in 20 mM Hepes/KOH (pH 8), 20 mM The ssDNA was preincubated with SSB for 10 min at 37 "C; RecA KC1, 5 mM MgC12, 100 pg of bovine serumalhumin/ml,and 5% and ATPyS were then added, and the mixture was incubated for 10 glycerol (volume = 100 pl) for 40 min a t 30 'C. The reaction was min at 37 "C. The reaction was initiated by the addition of dsDNA, stopped, and DNA was isolated as described above. Nicked plasmid and the incubationwas continued for 30 min at 37 "C. The reactions DNA was prepared as follows. DNA (10 pg) was incubated with 6 pg were stopped by the addition of Na'EDTA to 50 mM, SDS to 1%, of DNase in 50 mM Tris-HC1 (pH 8 ) , 50 mM NaC1, 10 mM MgC12, and 0.35 mg of ethidiumbromide/ml for 15 min at 30 "C. The and proteinase K to 100 pg/ml and were incubated at 37 "C for 10 min. The agarose gel assay for stable joint molecules was conducted ethidium bromide was subsequently removedby extractionwith water-saturated butanol before phenol extraction and DNA isolation as previously described (20). Quantitation of the data was performed using a Molecular Dynamics Model 300A computing densitometer. as described above. Agarose gel electrophoresis confirmed that the Susceptibility of End-labeled ssDNA to Exonuclease VII Digestion correct topologicalforms were producedbefore subsequentassays in Presence and Absence of SSB-Linear ssDNA was labeled at the were performed. Linear duplex DNAs were prepared bydigestion 5'-end with T4 polynucleotide kinase and [y-"P]ATP (24). Linear with an appropriate restrictionenzyme, followed by double extraction ssDNA was labeled at the 3'-end by annealing the 18-mer used to with phenol and ethanol precipitation. Specifically, linear dsDNAs filling in therecessed 3"terminus with Klenow with 2 kb of internal lacZ homology flanked by 0.6 and 5.5 kb of generate the end and presence of ATP,linear heterology were prepared by digesting pMC874A with the single-site fragmentand[a-"'P]dCTP(24).Inthe restriction enzyme HindJII. Linear dsDNAs with lacZ homology a t ssDNAs were preincubated for 10min a t 37 "Cinthestandard the 3'-end of the complementary strand were prepared by digesting reaction buffer (see above) containing 1 mM MgOAc and RecA; the pMC874 or pMC874A with the single-site restriction enzyme EcoRI. final concentration of MgOAc was increased to 10mM, and SSB was Linear dsDNAs withlacZ homology at the 5'-endof the complemen- either added or not. In the presence of ATPyS, linear ssDNAs were tary strand were prepared by digesting pMC874 with HpaI and gel- preincubated for 10 min a t 37 "C in the standard reactionbuffer (see isolating the8.1-kb fragment containing2 kb of lacZ sequence. Linear above) containing 4 mM MgOAc and SSB; RecA and ATPyS were dsDNAs with M13 homology a the 3'-end of the complementary then added, and the mixture was incubated for 10min a t 37 "C. strand were prepared by digesting pBR322-M13(3') and NaeI and Exonuclease VI1 was added to the reaction at a concentration of 50 gel-isolating the 3.7-kb fragment containing 0.6 kb of M13 sequence units/ml. Digestion was conducted for 1-2 min a t 37 "C and stopped or by digesting pBR322-M13(5') with PstI and gel-isolating the 2.1by the addition of 20 mM Na2EDTA and 1% SDS. The sample was analyzed by agarose gel electrophoresis as previously described (20). '' S. C. Kowalczykowski, personal communication. Quantitation of the data was performed as described above. MATERIALS AND METHODS

10114

DNA Substrate Requirements for Stable Joint Molecule Formation RESULTS

Homology at 3’-End of Linear ssDNA Is Sufficientfor Stable Joint Molecule Formation with Supercoiled DNA and Is Preferred over Homology at 5’-End-Our previous studies (19, 20) of joint molecule formation between linear ssDNAs and supercoiled DNAs demonstrated that in thepresence of SSB, 3‘-end homology is essential.Linear ssDNAs with 5‘-end homology do not form stable joint molecules. In the absence of SSB, linear ssDNAs with 3’-end homology are still more reactive than those with 5‘-end homology (20). The preference for a homologous 3’-end in the absence of SSB suggests that this is an intrinsic property of RecA-promoted strand exchange. To test the generality of these results, the new substrates schematically diagramed in Fig. lA were assayed for stable joint molecule formation. In these experiments, the region of homology between the two DNAs is either lacZ or M13 and is limited to only the 5’- or 3’-end of the linear ssDNA. Increasing amounts of RecAwere titrated into the reactions in the absence or presence of SSB (Fig. 1B). In thepresence of SSB, optimal joint molecule formation (58-62%) occurs at a concentration of one RecA monomer/three nucleotides of ssDNA and depends on homology at the3‘-end of the linear ssDNA. No stablejoint molecules (