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1.
Helical RecA nucleoprotein filaments mediate homologous pairing and strand exchange 总被引:11,自引:0,他引:11
C M Radding 《Biochimica et biophysica acta》1989,1008(2):131-145
2.
The effects on strand exchange of 5' versus 3' ends of single-stranded DNA in RecA nucleoprotein filaments 总被引:2,自引:0,他引:2
Since the ends of DNA chains are thought to be important in homologous recombination, the way in which RecA protein and similar recombination enzymes process ends is important. We analyzed the effects of ends both on the formation of joints, and the progression of strand exchange. When the only homologous end was provided by a single strand, there was no significant difference between the formation of joints at a 5' end or a 3' end; but in agreement with the report of Konforti & Davis, Escherichia coli single-stranded DNA binding protein (SSB) selectively inhibited the activity of 5' ends. Complete strand exchange, assessed by study of linear single-stranded and double-stranded substrates, took place only in the 5' to 3' direction relative to DNA in the nucleoprotein filament. These observations pose a paradox: in the presence of SSB, of which there are about 800 tetramers per cell, the formation of homologous joints by RecA protein is favored at a 3' end, from which, however, authentic strand exchange appears not to occur. Since observations reported here and elsewhere show that joints have different properties when formed at a 5' versus a 3' end, we suggest that they may be processed differently in vivo. 相似文献
3.
To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein. The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values. In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks. At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity. Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange. Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion. Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner. Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA. Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected. We discuss how these results relate to inefficient allele exchange in mycobacteria. 相似文献
4.
The RecA binding locus of RecBCD is a general domain for recruitment of DNA strand exchange proteins
RecBCD enzyme facilitates loading of RecA protein onto ssDNA produced by its helicase/nuclease activity. This process is essential for RecBCD-mediated homologous recombination. Here, we establish that the C-terminal nuclease domain of the RecB subunit (RecBnuc) forms stable complexes with RecA. Interestingly, RecBnuc also interacts with and loads noncognate DNA strand exchange proteins. Interaction is with a conserved element of the RecA-fold, but because the binding to noncognate proteins decreases in a phylogenetically consistent way, species-specific interactions are also present. RecBnuc does not impede activities of RecA that are important to DNA strand exchange, consistent with its role in targeting of RecA. Modeling predicts the interaction interface for the RecA-RecBCD complex. Because a similar interface is involved in the binding of human Rad51 to the conserved BRC repeat of BRCA2 protein, the RecB-domain may be one of several structural domains that interact with and recruit DNA strand exchange proteins to DNA. 相似文献
5.
Forget AL Kudron MM McGrew DA Calmann MA Schiffer CA Knight KL 《Biochemistry》2006,45(45):13537-13542
All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments and for their coordinated activity during the catalysis of homologous recombination. 相似文献
6.
The RecA-like proteins constitute a group of DNA strand transfer proteins ubiquitous in eubacteria, eukarya, and archaea. However, the functional relationship among RecA proteins is poorly understood. For instance, Mycobacterium tuberculosis RecA is synthesized as a large precursor, which undergoes an unusual protein-splicing reaction to generate an active form. Whereas the precursor was inactive, the active form promoted DNA strand transfer less efficiently compared to EcRecA. Furthermore, gene disruption studies have indicated that the frequencies of allele exchange are relatively lower in Mycobacterium tuberculosis compared to Mycobacterium smegmatis. The mechanistic basis and the factors that contribute to differences in allele exchange remain to be understood. Here, we show that the extent of DNA strand transfer promoted by the M. smegmatis RecA in vitro differs significantly from that of M. tuberculosis RecA. Importantly, M. smegmatis RecA by itself was unable to promote strand transfer, but cognate or noncognate SSBs rendered it efficient even when added prior to RecA. In the presence of SSB, MsRecA or MtRecA catalyzed strand transfer between ssDNA and varying lengths of linear duplex DNA with distinctly different pH profiles. The factors that were able to suppress the formation of DNA networks greatly stimulated strand transfer reactions promoted by MsRecA or MtRecA. Although the rate and pH profiles of dATP hydrolysis catalyzed by MtRecA and MsRecA were similar, only MsRecA was able to couple dATP hydrolysis to DNA strand transfer. Together, these results provide insights into the functional diversity in DNA strand transfer promoted by RecA proteins of pathogenic and nonpathogenic species of mycobacteria. 相似文献
7.
Assembly and disassembly of RecA protein filaments occur at opposite filament ends. Relationship to DNA strand exchange 总被引:3,自引:0,他引:3
RecA protein primarily associates with and dissociates from opposite ends of nucleoprotein filaments formed on linear duplex DNA. RecA nucleoprotein filaments that are hydrolyzing ATP therefore engage in a dynamic process under some conditions that has some of the properties of treadmilling. We have also investigated whether the net polymerization of recA protein at one end of the filament and/or a net depolymerization at the other end drives unidirectional strand exchange. There is no demonstrable correlation between recA protein association/dissociation and the strand exchange reaction. RecA protein-mediated DNA strand exchange is affected minimally by changes in reaction conditions (dilution, pH shift, or addition of small amounts of adenosine-5'-O-(3-thiotriphosphate) that have large and demonstrable effects on recA protein association, dissociation, or both. Rather than driving strand exchange, these assembly and disassembly processes may simply represent the mechanism by which recA nucleoprotein filaments are recycled in the cell. 相似文献
8.
DNA binding, annealing, and strand exchange activities of Brh2 protein from Ustilago maydis 总被引:2,自引:0,他引:2
Brh2 is the Ustilago maydis ortholog of the BRCA2 tumor suppressor. It functions in repair of DNA by homologous recombination by controlling the action of Rad51. A critical aspect in the control appears to be the recruitment of Rad51 to single-stranded DNA regions exposed as lesions after damage or following a disturbance in DNA synthesis. In previous experimentation, Brh2 was shown to nucleate formation of the Rad51 nucleoprotein filament that becomes the active element in promoting homologous pairing and DNA strand exchange. Nucleation was found to be initiated at junctions of double-stranded and single-stranded DNA. Here we investigated the DNA binding specificity of Brh2 in more detail using oligonucleotide substrates. We observed that Brh2 prefers partially duplex structures with single-stranded branches, flaps, or D-loops. We found also that Brh2 has an inherent ability to promote DNA annealing and strand exchange reactions on free as well as RPA-coated substrates. Unlike Rad51, Brh2 was able to promote DNA strand exchange when preincubated with double-stranded DNA. These findings raise the notion that Brh2 may have roles in homologous recombination beyond the previously established Rad51 mediator activity. 相似文献
9.
Genetic evidence suggests that the RecF, RecO, and RecR (RecFOR) proteins participate in a common step of DNA recombination and repair, yet the biochemical event requiring collaboration of all three proteins is unknown. Here, we show that the concerted action of the RecFOR complex directs the loading of RecA protein specifically onto gapped DNA that is coated with single-stranded DNA binding (SSB) protein, thereby accelerating DNA strand exchange. The RecFOR complex recognizes the junction between the ssDNA and dsDNA regions and requires a base-paired 5' terminus at the junction. Thus, the RecFOR complex is a structure-specific mediator that targets recombinational repair to ssDNA-dsDNA junctions. This reaction reconstitutes the initial steps of recombinational gapped DNA repair and uncovers an event also common to the repair of ssDNA-tailed intermediates of dsDNA-break repair. We propose that the behavior of the RecFOR proteins is mimicked by functional counterparts that exist in all organisms. 相似文献
10.
The ability of E coli recA protein to promote homologous pairing with linear duplex DNA bound to HU protein (Nucleosome cores) was found to be differentially affected. The formation of paranemic joint molecules was not affected whereas the formation of plectomic joint molecules was inhibited from the start of the reaction. The formation of paranemic joint molecules between nucleoprotein filaments of recA protein-circular single stranded DNA and closed circular duplex DNA is believed to generate positive supercoiling in the duplex DNA. We found that the positively superhelical duplex DNA was inert in the formation of joint molecules but could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. These observations initiate an understanding of the structural features of E coli chromosome such as DNA supercoiling and nucleosome-like structures in homologous recombination. 相似文献
11.
The RecA protein of Escherichia coli has been used in vitro to mediate a strand-exchange reaction between homologous DNA molecules. A three-dimensional reconstruction of a RecA filament on double-stranded DNA has been previously determined from electron micrographs, and the reconstruction displays a clear axial polarity. The RecA-mediated strand-exchange reaction between a double-stranded DNA and a homologous single-stranded DNA that is complexed with a RecA helical polymer proceeds with a known polarity. Using image analysis of electron micrographs, we have determined the relation between the structural polarity of RecA filaments and the 3' and 5' polarity of single-stranded DNA. Thus, the structural polarity of RecA filaments can now be related to the direction in which the RecA-mediated strand-exchange reaction advances along the complexed single-stranded DNA. 相似文献
12.
Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells
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We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced approximately 30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 microm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions. 相似文献
13.
Lantsov VA 《Molekuliarnaia biologiia》2007,41(3):467-477
Bacterial RecA protein is a prototype of ATP-dependent homologous recombinases found ubiquitously from bacteriophages up to human beings. When RecA filament is forming on single-stranded DNA in the presence of ATP, it initiates the strand exchange reaction with homologous double-stranded DNA. Among three phases of the reaction (the search for homology, the three-stranded structure annealing in conjunction with the switch of pairing, and the strand displacement) the first one is the most enigmatic and least studied. As commonly recognized, this phase is directed by a special (stretched) filament structure and does not required any additional consumption of energy in ATP hydrolysis. The novel approaches in the study of strand exchange reaction, using short oligonucleotides as DNA substrates and sensitive methods for a real-time monitoring of the reaction suggest that all three phases of the reaction depend on the ATP hydrolysis. 相似文献
14.
In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding. 相似文献
15.
Postreplication repair in E. coli: strand exchange reactions of gapped DNA by RecA protein 总被引:12,自引:0,他引:12
Stephen C. West Era Cassuto Paul Howard-Flanders 《Molecular & general genetics : MGG》1982,187(2):209-217
Summary We have used a sensitive gel electrophoresis assay to detect the products of Escherichia coli RecA protein catalysed strand exchange reactions between gapped and duplex DNA molecules. We identify structures that correspond to joint molecules formed by homologous pairing, and show that joint molecules are converted by RecA protein into heteroduplex monomers by reciprocal strand exchanges. However, strand exchanges only occur when there is a 3-terminus complementary to the single stranded DNA in the gap. In the absence of a complementary free end, the two DNA molecules pair and short heteroduplex regions are formed by localised interwinding. 相似文献
16.
Electron microscopic visualization of the RecA protein-mediated pairing and branch migration phases of DNA strand exchange 总被引:7,自引:0,他引:7
J C Register G Christiansen J Griffith 《The Journal of biological chemistry》1987,262(26):12812-12820
The RecA protein of Escherichia coli will drive the pairing and exchange of strands between homologous DNA molecules in a reaction stimulated by single-stranded binding protein. Here, reactions utilizing three homologous DNA pairs which can undergo both paranemic and plectonemic joining were examined by electron microscopy: supertwisted double-stranded (ds) DNA and linear single-stranded (ss) DNA, linear dsDNA and circular ssDNA, and linear dsDNA and colinear ssDNA. Several major observations were: (i) with RecA protein bound to the DNA, plectonemic joints were ultrastructurally indistinguishable from paranemic joints; (ii) complexes which appeared to be joined both paranemically and plectonemically were present in these reactions in roughly equal numbers; and (iii) in complexes undergoing strand exchange, both DNA partners were often enveloped within a RecA protein filament consisting of hundreds of RecA protein monomers and several kilobases of DNA. These observations suggest that, following RecA protein-ssDNA filament formation, strand exchange proceeds by a pathway that can be divided structurally into three phases: pairing, envelopment/exchange, and release of the products. 相似文献
17.
The function of the secondary DNA-binding site of RecA protein during DNA strand exchange. 总被引:4,自引:1,他引:3
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RecA protein features two distinct DNA-binding sites. During DNA strand exchange, the primary site binds to single-stranded DNA (ssDNA), forming the helical RecA nucleoprotein filament. The weaker secondary site binds double-stranded DNA (dsDNA) during the homology search process. Here we demonstrate that this site has a second important function. It binds the ssDNA strand that is displaced from homologous duplex DNA during DNA strand exchange, stabilizing the initial heteroduplex DNA product. Although the high affinity of the secondary site for ssDNA is essential for DNA strand exchange, it renders DNA strand exchange sensitive to an excess of ssDNA which competes with dsDNA for binding. We further demonstrate that single-stranded DNA-binding protein can sequester ssDNA, preventing its binding to the secondary site and thereby assisting at two levels: it averts the inhibition caused by an excess of ssDNA and prevents the reversal of DNA strand exchange by removing the displaced strand from the secondary site. 相似文献
18.
19.
Processes fundamental to all models of genetic recombination include the homologous pairing and subsequent exchange of DNA strands. Biochemical analysis of these events has been conducted primarily on the recA protein of Escherichia coli, although proteins which can promote such reactions have been purified from many sources, both prokaryotic and eukaryotic. The activities of these homologous pairing and DNA strand exchange proteins are either ATP-dependent, as predicted based on the recA protein paradigm, or, more unexpectedly, ATP-independent. This review examines the reactions promoted by both classes of proteins and highlights their similarities and differences. The mechanistic implications of the apparent existence of 2 classes of strand exchange protein are discussed. 相似文献
20.
The archaeal RadA protein is a homologue of the Escherichia coli RecA and Saccharomyces cerevisiae Rad51 proteins and possesses the same biochemical activities. Here, using in vitro selection, we show that the Sulfolobus solfataricus RadA protein displays the same preference as its homologues for binding to DNA sequences that are rich in G residues, and under-represented in A and C residues. The RadA protein also displays enhanced pairing activity with these in vitro-selected sequences. These parallels between the archaeal, eukaryal and bacterial proteins further extend the universal characteristics of DNA strand exchange proteins. 相似文献