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1.
When recA protein pairs linear duplex DNA with a homologous duplex molecule that has a single-stranded tail, it produces a recombination intermediate called the Holliday structure and causes reciprocal or symmetric strand exchange, whereas the pairing of a linear duplex molecule with fully single-stranded DNA leads to an asymmetric exchange. To study the location of recA protein on DNA molecules undergoing symmetric exchange, we labeled individually each end of the four strands involved and looked for protection against DNase I or restriction endonucleases. As expected, because of its preferred binding to single-stranded DNA, recA protein protected the single-stranded tails of either substrates, or products. In addition however, strong protection extended into the newly formed heteroduplex DNA along the strand to which recA protein was initially bound. Experiments with uniformly labeled DNA showed a corresponding homology-dependent asymmetry in the protection of the tailed substrate versus its fully duplex partner. Restriction experiments showed that protection extended 50-75 base pairs beyond the point where strand exchange was blocked by a long region of heterology. When compared with earlier observations (Chow, S. A., Honigberg, S. M., Bainton, R. J., and Radding, C. M. (1986) J. Biol. Chem. 261, 6961-6971), the present experiments reveal a pattern of association of recA protein with DNA that suggests a common mechanism of asymmetric and symmetric strand exchange.  相似文献   

2.
We have examined whether DNA strand exchange activities from nuclear extracts of HeLa cells or Drosophila melanogaster embryos have detectable helicase or melting activities. The partially purified recombinases have been shown to recognize homologous single strand and double strand DNA molecules and form joint molecules in a DNA strand exchange reaction. The joint molecule product consists of a linear duplex joined at one end by a region of DNA heteroduplex to a homologous single strand circular DNA. Using two different partially duplex helicase substrates, we are unable to detect any melting of duplex regions under conditions that promote joint molecule formation. One substrate consists of a 32P-labeled oligonucleotide 20 or 30 bases long annealed to M13mp18 circular single strand DNA. The second substrate consists of a linear single strand region flanked at each end by short duplex regions. We observe that even in the presence of excess recombinase protein or after prolonged incubation no helicase activity is apparent. Control experiments rule out the possibility that a helicase is masked by reannealing of displaced single strand fragments. Based on these findings and other data, we conclude that the human and D. melanogaster recombinases recognize and pair homologous sequences without significant melting of duplex DNA prior to strand exchange.  相似文献   

3.
An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.  相似文献   

4.
Summary An activity that can promote homologous pairing and strand transfer between suitable DNA substrates has been partially purified from human skin fibroblasts and from Hela cells. The strand transfer reaction was investigated with DNA substrates consisting of single-stranded circular and duplex linear phage DNA. It requires ATP, and under optimal conditions yields heteroduplex molecules containing one strand from each parental DNA substrate. The reactions appears to be of the same general nature as those mediated by RecA proteins of Escherichia coli and the Rec1 protein of Ustilago maydis.  相似文献   

5.
Two proteins encoded by bacteriophage T7, the gene 2.5 single-stranded DNA binding protein and the gene 4 helicase, mediate homologous DNA strand exchange. Gene 2.5 protein stimulates homologous base pairing of two DNA molecules containing complementary single-stranded regions. The formation of a joint molecule consisting of circular, single-stranded M13 DNA, annealed to homologous linear, duplex DNA having 3'- or 5'-single-stranded termini of approximately 100 nucleotides requires stoichiometric amounts of gene 2.5 protein. In the presence of gene 4 helicase, strand transfer proceeds at a rate of > 120 nucleotides/s in a polar 5' to 3' direction with respect to the invading strand, resulting in the production of circular duplex M13 DNA. Strand transfer is coupled to the hydrolysis of a nucleoside 5'-triphosphate. The reaction is dependent on specific interactions between gene 2.5 protein and gene 4 protein.  相似文献   

6.
RecA protein catalyzes homologous pairing of partially single-stranded duplex DNA and fully duplex DNA to form stable joint molecules. We constructed circular duplex DNA with various defined gap lengths and studied the pairing reaction between the gapped substrate with fully double-stranded DNA. The reaction required a stoichiometric amount of RecA protein, and the optimal reaction was achieved at a ratio of 1 RecA monomer per 4 base pairs. The length of the gap, ranging from 141 to 1158 nucleotides, had little effect on the efficiency of homologous pairing. By using a circular gapped duplex DNA prepared from the chimeric phage M13Gori1, we were able to show the formation of nonintertwined or paranemic joints in duplex regions between the gapped and fully duplex molecules. The formation of such paranemic joints occurred efficiently and included nearly all of the DNA in the reaction mixture. The reaction required negative superhelicity, and pairing was greatly reduced with linear or nicked circular DNA. We conclude that one functional role of the single-stranded gap is for facilitating the binding of RecA protein to the duplex region of the gapped DNA. Once the nucleoprotein filament is formed, homologous pairing between the gapped and fully duplex DNA can take place anywhere along the length of the nucleoprotein complex.  相似文献   

7.
The Rad3 ATPase/DNA helicase was purified to physical homogeneity from extracts of yeast cells containing overexpressed Rad3 protein. The DNA helicase can unwind duplex regions as short as 11 base pairs in a partially duplex circular DNA substrate and does so by a strictly processive mechanism. On partially duplex linear substrates, the enzyme has a strict 5'----3' polarity with respect to the single strand to which it binds. Nicked circular DNA is not utilized as a substrate, and the enzyme requires single-stranded gaps between 5 and 21 nucleotides long to unwind oligonucleotide fragments from partially duplex linear molecules. The enzyme also requires duplex regions at least 11 base pairs long when these are present at the ends of linear molecules. Rad3 DNA helicase activity is inhibited by the presence of ultraviolet-induced photoproducts in duplex regions of partially duplex circular molecules.  相似文献   

8.
Formation of nascent heteroduplex structures by RecA protein and DNA   总被引:13,自引:0,他引:13  
A M Wu  R Kahn  C DasGupta  C M Radding 《Cell》1982,30(1):37-44
E. coli RecA protein promotes homologous pairing in two distinguishable phases: synapsis and strand exchange. With circular single strands (plus strand only) and linear duplex DNA, polarized or unidirectional strand exchange appeared to cause heteroduplex joints to form and grow from a unique end of the duplex DNA. However, a variety of other pairs of substrates appeared to form joint molecules without regard to the polarity of the strands involved. This paradox has been resolved by observations that show that synapsis is fast, nonpolar and sensitive to inhibition by ADP, whereas strand exchange is slow, directional and relatively insensitive to inhibition by ADP. Thus a heteroduplex joint initiated at one end of the duplex DNA grows by continued strand exchange, whereas a joint initiated at the other end dissociates and is unable to start again because accumulating ADP inhibits synapsis. RecA protein appears to form a nascent protein-DNA structure, the RecA synaptic structure, in which at least 100-300 bp in the duplex molecule are held in an unwound configuration and in which the incoming strand is aligned with its complement.  相似文献   

9.
Underwinding of DNA associated with duplex-duplex pairing by RecA protein   总被引:3,自引:0,他引:3  
Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.  相似文献   

10.
recA protein, in the presence of ATP, polymerizes on single-stranded DNA (plus strand) to form a presynaptic nucleoprotein filament that pairs with linear duplex DNA and actively displaces the plus strand from the recipient molecule in a polarized fashion to form a new heteroduplex molecule. The interaction between recA protein and DNA during strand exchange was studied by labeling different strands and probing the intermediate with pancreatic deoxyribonuclease I (DNase I) or restriction endonuclease. The incoming single strand was resistant to DNase I in the original nucleoprotein filament and remained resistant even after extensive strand exchange had occurred. Both strands of the parental duplex molecule were sensitive to DNase I in the absence of joint molecule formation; but as strand exchange progressed following homologous pairing, increasing stretches of the parental plus strand became resistant, whereas the complementary parental minus strand remained sensitive to DNase I throughout the reaction. Except for a region of 50-100 base pairs at the end of the newly formed heteroduplex DNA where strand exchange was initiated, the rest of the heteroduplex region was resistant to cleavage by restriction endonucleases. The data suggest that recA protein promotes strand exchange by binding both the incoming and outgoing strands of the same polarity, whereas the complementary strand, which must switch pairing partners, is unhindered by direct contact with the protein.  相似文献   

11.
B J Rao  B Jwang  M Dutreix 《Biochimie》1991,73(4):363-370
During the directional strand exchange that is promoted by RecA protein between linear duplex DNA and circular single-stranded DNA, a triple-stranded DNA intermediate was formed and persisted even after the completion of strand transfer followed by deproteinization. In the deproteinized three-stranded DNA complexes, the sequestered linear third strand resisted digestion by E coli exonuclease I. In relation to polarity of strand exchange which defines the proximal and distal ends of the duplex DNA, when homology was restricted to the distal region of duplex substrate, the joints formed efficiently and were stable even upon complete deproteinization. Enzymatic probing of deproteinized distal joints with nuclease P1 revealed that the joints consist of long three-stranded structures that at neutral pH lack significant single-stranded character in any of the three strands. Instead of circular single-stranded DNA, when a linear single strand is recombined with partially homologous duplex DNA, in the presence of SSB, the formation of homologous joints by RecA protein, is significantly more efficient at distal end than at the proximal. Taken together, these observations suggest that with any single-stranded DNA (circular or linear), RecA protein efficiently promotes the formation of distal joints, from which, however, authentic strand exchange may not occur. Moreover, these joints might represent an intermediate which is trapped into a stable triple stranded state.  相似文献   

12.
In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.  相似文献   

13.
Using gapped circular DNA and homologous duplex DNA cut with restriction nucleases, we show that E. coli RecA protein promotes strand exchanges past double-strand breaks. The products of strand exchange are heteroduplex DNA molecules that contain nicks, which can be sealed by DNA ligase, thereby effecting the repair of double-strand breaks in vitro. These results show that RecA protein can promote pairing interactions between homologous DNA molecules at regions where both are duplex. Moreover, pairing leads to strand exchanges and the formation of heteroduplex DNA. In contrast, strand exchanges are unable to pass a double-strand break in the gapped substrate. This apparent paradox is discussed in terms of a model for RecA-DNA interactions in which we propose that each RecA monomer contains two nonequivalent DNA-binding sites.  相似文献   

14.
In the presence of 100 mM NaCl, the efficient exchange of strands between a circular single strand and an homologous DNA duplex promoted by the recA and single-stranded DNA binding proteins of Escherichia coli requires an unpaired 3' terminus. Of the duplex DNAs tested, only those with 4 unpaired bases at the 3' termini are effective. Without added NaCl, strand exchange proceeds efficiently with all duplex DNA termini examined including a nicked circular duplex. Thus, at approximately physiological salt concentrations, factors in addition to the recA and single-stranded DNA binding proteins are needed to promote efficient strand exchange. One such factor may be a DNA helicase(s).  相似文献   

15.
We studied the formation of linked circular DNA molecules promoted by the combined action of rec 1 protein and type I topoisomerase of Ustilago maydis. When ATP was added as cofactor to reactions containing rec 1 protein, pairs of homologous circular DNA molecules became linked after addition of topoisomerase. Closed circular duplex molecules could be joined at homologous sites with circular single-stranded molecules or with other circular duplex molecules, provided that homologous single-stranded DNA fragments or RNA polymerase and nucleoside triphosphates were also added. Complexes formed were topologically linked through regions of heteroduplex DNA. When the analog adenylyl-imidodiphosphate was substituted for ATP, nonhomologous pairs of circular DNA molecules became linked.  相似文献   

16.
Vaccinia virus infection induces expression of a protein which can catalyze joint molecule formation between a single-stranded circular DNA and a homologous linear duplex. The kinetics of appearance of the enzyme parallels that of vaccinia virus DNA polymerase and suggests it is an early viral gene product. Extracts were prepared from vaccinia virus-infected HeLa cells, and the strand exchange assay was used to follow purification of this activity through five chromatographic steps. The most highly purified fraction contained three major polypeptides of 110 +/- 10, 52 +/- 5, and 32 +/- 3 kDa. The purified protein requires Mg2+ for activity, and this requirement cannot be satisfied by Mn2+ or Ca2+. One end of the linear duplex substrate must share homology with the single-stranded circle, although this homology requirement is not very high, as 10% base substitutions had no effect on the overall efficiency of pairing. As with many other eukaryotic strand exchange proteins, there was no requirement for ATP, and ATP analogs were not inhibitors. Electron microscopy was used to show that the joint molecules formed in these reactions were composed of a partially duplex circle of DNA bearing a displaced single-strand and a duplex linear tail. The recovery of these structures shows that the enzyme catalyzes true strand exchange. There is also a unique polarity to the strand exchange reaction. The enzyme pairs the 3' end of the duplex minus strand with the plus-stranded homolog, thus extending hybrid DNA in a 3'-to-5' direction with respect to the minus strand. Which viral gene (if any) encodes the enzyme is not yet known, but analysis of temperature-sensitive mutants shows that activity does not require the D5R gene product. Curiously, v-SEP appears to copurify with vaccinia virus DNA polymerase, although the activities can be partially resolved on phosphocellulose columns.  相似文献   

17.
Purified human Rad51 protein (hRad51) catalyses ATP-dependent homologous pairing and strand transfer reactions, characteristic of a central role in homologous recombination and double-strand break repair. Using single-stranded circular and partially homologous linear duplex DNA, we found that the length of heteroduplex DNA formed by hRad51 was limited to approximately 1.3 kb, significantly less than that observed with Escherichia coli RecA and Saccharomyces cerevisiae Rad51 protein. Joint molecule formation required the presence of a 3' or 5'-overhang on the duplex DNA substrate and initiated preferentially at the 5'-end of the complementaryx strand. These results are consistent with a preference for strand transfer in the 3'-5' direction relative to the single-stranded DNA. The human single-strand DNA-binding protein, hRP-A, stimulated hRad51-mediated joint molecule formation by removing secondary structures from single-stranded DNA, a role similar to that played by E. coli single-strand DNA-binding protein in RecA-mediated strand exchange reactions. Indeed, E. coli single-strand DNA-binding protein could substitute for hRP-A in hRad51-mediated reactions. Joint molecule formation by hRad51 was stimulated or inhibited by hRad52, dependent upon the reaction conditions. The inhibitory effect could be overcome by the presence of hRP-A or excess heterologous DNA.  相似文献   

18.
When recA protein promotes homologous pairing and strand exchange involving circular single strands and linear duplex DNA, the protein first polymerizes on the single-stranded DNA to form a nucleoprotein filament which then binds naked duplex DNA to form nucleoprotein networks, the existence of which is independent of homology, but requires the continued presence of recA protein (Tsang, S. S., Chow, S. A., and Radding, C. M. (1985) Biochemistry 24, 3226-3232). Further experiments revealed that within a few minutes after the beginning of homologous pairing and strand exchange, these networks began to be replaced by a distinct set of networks with inverse properties: their formation depended upon homology, but they survived removal of recA protein by a variety of treatments. Formation of this second kind of network required that homology be present specifically at the end of the linear duplex molecule from which strand exchange begins. Escherichia coli single-stranded DNA-binding protein or phage T4 gene 32 protein largely suppressed the formation of this second population of networks by inactivating the newly formed heteroduplex DNA, which, however, could be reactivated when recA protein was dissociated by incubation at 0 degrees C. We interpret these observations as evidence of reinitiation of strand invasion when recA protein acts in the absence of auxiliary helix-destabilizing proteins. These observations indicate that the nature of the nucleoprotein products of strand exchange determines whether pairing and strand exchange are reversible or not, and they further suggest a new explanation for the way in which E. coli single-stranded DNA-binding protein and gene 32 protein accelerate the apparent forward rate of strand exchange promoted by recA protein, namely by suppressing initiation of the reverse reaction.  相似文献   

19.
M Bianchi  B Riboli    G Magni 《The EMBO journal》1985,4(11):3025-3030
RecA protein was found to catalyze the dissociation of the strands of a DNA substrate consisting of a 20-nucleotide primer annealed to circular single-stranded M13mp DNA. The strand separation reaction requires ATP hydrolysis and the presence of single-stranded DNA flanking the duplex DNA region to be unwound. RecA-catalyzed strand separation is effective only for very short duplexes, not exceeding 30 bp, and is not stimulated by single-stranded DNA-binding protein. These results are consistent with the ability of recA protein to disrupt regions of secondary structure in single-stranded DNA and to incorporate large non-homologies into heteroduplex DNA.  相似文献   

20.
Strand exchange protein 1 (Sep1) from Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA molecules from single-stranded circles and homologous linear duplex DNA in vitro. Previously, Sep1 was purified as a 132,000-Da species; however, DNA sequence analysis indicates that the SEP1 gene is capable of encoding a 175,000-Da protein (Tishkoff, D.X., Johnson, A.W., and Kolodner, R.D. (1991) Mol. Cell. Biol. 11, 2593-2608). The SEP1 gene was cloned into a GAL10 expression vector and expressed in a protease-deficient yeast strain. Intact Sep1, which migrated as a Mr-160,000 polypeptide during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was purified to apparent homogeneity and shown to have activities similar to those of the originally purified Mr = 132,000 fragment. We report here that, in addition to strand exchange activity, Sep1 contains an intrinsic exonuclease that is active on single- and double-stranded DNA with a severalfold preference for single-stranded DNA. The nuclease was induced in crude extracts upon induction with galactose, it co-purified with the strand exchange activity of Sep1, and the nuclease and strand exchange activities of Sep1 showed the same kinetics of heat inactivation. Sep1 nuclease, which requires Mg2+, can be functionally separated from the strand exchange activity by the substitution of Ca2+ for Mg2+. Under these conditions, the nuclease is inactive, and strand exchange activity is dependent on prior resection of the DNA ends by an exogenous exonuclease. Thus, the nuclease is necessary for synapsis but not strand exchange. Electron microscopic analysis revealed that true strand exchange products, alpha molecules and nicked double-stranded circular molecules, were formed. In addition, strand transfer proceeded to similar extents on 5'-resected and 3'-resected DNA. This result suggests that the polarity of strand transfer by Sep1 is determined by the polarity of its intrinsic nuclease.  相似文献   

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