Rad51 Uses One Mechanism to Drive DNA Strand Exchange in Both Directions

Rad51 protein forms nucleoprotein filaments on single-stranded DNA (ssDNA) and then pairs that DNA with the complementary strand of incoming duplex DNA. In apparent contrast with published results, we demonstrate that Rad51 protein promotes an extensive pairing of long homologous DNAs in the absence of replication protein A. This pairing exists only within the Rad51 filament; it was previously undetected because it is lost upon deproteinization. We further demonstrate that RPA has a critical postsynaptic role in DNA strand exchange, stabilizing the DNA pairing initiated by Rad51 protein. Stabilization of the Rad51-generated DNA pairing intermediates can be can occur either by binding the displaced strand with RPA or by degrading the same DNA strand using exonuclease VII. The optimal conditions for Rad51-mediated DNA strand exchange used here minimize the secondary structure in singlestranded DNA, minimizing the established presynaptic role of RPA in facilitating Rad51 filament formation. We verify that RPA has little effect on Rad51 filament formation under these conditions, assigning the dramatic stimulation of strand exchange nevertheless afforded by RPA to its postsynaptic function of removing the displaced DNA strand from Rad51 filaments.Homologous genetic recombination is an integral feature of DNA metabolism in all organisms. Functions include the repair of replication forks halted at DNA barriers such as DNA lesions and breaks and the repair of double-strand breaks in DNA arising from nonreplication sources. Central steps in these processes are carried out by recombinases such as the bacterial RecA protein or the eukaryotic Rad51 protein. These proteins bind first to single-stranded DNA within a gap or a DNA terminal extension, forming an extended nucleoprotein filament. The filament then initiates a search for homologous double-stranded DNA (dsDNA) 1 and pairs the bound singlestranded DNA with the complementary strand of the incoming duplex in a process known as DNA strand exchange. Synapsis is the point at which homologous alignment of the two DNAs is achieved, and reactions are often divided into presynaptic (nucleoprotein filament formation) and postsynaptic (extension of the paired DNA segment) phases. The Rad51 protein of Saccharomyces cerevisiae promotes a very efficient DNA strand exchange reaction under the right reaction conditions (1, 2).Single-stranded DNA-binding proteins, generally referred to as SSBs, have a multitude of roles in DNA metabolism. As part of their role in genetic recombination, SSBs stimulate recombinase-mediated in vitro DNA strand exchange reactions. The SSB of S. cerevisiae is replication protein A, or RPA. RPA has been implicated in the recombination pathway genetically (3-7) and through its physical interaction with recombination protein Rad52 (8). Although RPA stimulates most in vitro DNA strand exchange reactions promoted by the Rad51 protein, RPA is not required for Rad51-mediated DNA strand exchange in reactions with oligonucleotides (2, 9). When much longer DNA deri...

Section: Be Stabilized By Nucleolytic Degradation Of the Displacedmentioning

confidence: 86%

The Rad51-dependent Pairing of Long DNA Substrates Is Stabilized by Replication Protein A

Eggler

Inman

Cox

2002

“…Originally we assumed that JM2 must use the complementary overhang and thus form via the strand replacement model (13). There were, however, two problems with this interpretation.…”

Section: Discussionmentioning

confidence: 99%

“…6A, 5). This model takes advantage of the unique ability of Rad51 to promote strand exchange in both the 3Ј to 5Ј and 5Ј to 3Ј direction (13). However, whereas Namsaraev and Berg have reported that strand transfer initiates only from the complementary overhang (16), in this model the second dsL substrate must initiate via the end where the complementary strand is recessed.…”

Section: Table I Electron Microscopy Analysis Of Jm1 From Heterologoumentioning

confidence: 99%

See 1 more Smart Citation

Structure of Reaction Intermediates Formed duringSaccharomyces cerevisiae Rad51-catalyzed Strand Transfer

Holmes¹,

Scandellari²,

Benjamin³

et al. 2002

The process by which the Saccharomyces cerevisiae strand transfer protein, Rad51, seeks out homologous sequences in vivo can be modeled by an in vitro reaction between a single-stranded DNA circle and a doublestranded linear DNA. In addition to the substrates and products, electrophoresis of reaction mixtures resolves two groups of low mobility bands. Here we show that the low mobility bands formed during strand transfer by Rad51 (or Escherichia coli RecA) represent joint molecules (JM) between the two substrates. One group, which we name JM1, is an obligatory reaction intermediate in which the complementary strand from the duplex substrate has been partially transferred to the single-stranded circle. Our assignment is based on pulse-chase and restriction enzyme digestion experiments and verified by electron microscopy. The slower moving group of bands, designated JM2, is formed by an unexpected reaction between JM1 and a second doublestranded linear substrate. Strand transfer of the second duplex initiates noncanonically from the end where the complementary strand is recessed. Thus JM2 is formed by two strand transfer reactions with the same singlestranded circular substrate but with opposite polarities. Finally, we show that the multiple sharp bands in JM1 and JM2 are the result of substrate sequences that pause strand transfer.During the initial steps of DNA double-strand break repair, a strand transfer protein such as Saccharomyces cerevisiae Rad51 forms a filament along the 3Ј single-stranded tails generated from broken DNA ends (1). The resulting nucleoprotein filament then searches the genome for sequences homologous to the tail that can serve as templates for the repair synthesis needed to restore the continuity of the chromosome (2). Rad51 is called a strand transfer protein because it catalyzes the invasion of the single-stranded tail into the intact template duplex and transfers base pairing interactions from the identical strand of the template duplex to the tail. The 3Ј end of the transferred tail can then serve as a primer for repair synthesis (3). The mechanisms of the homology search and subsequent strand transfer are not well understood. Indeed, it is difficult to imagine a process that achieves both the speed and fidelity with which a sequence homolog is found in vivo.The homology search process and the broader mechanism of strand transfer proteins have been studied using an in vitro strand transfer reaction that recapitulates the first steps of homologous recombination (4) (Fig. 1A). Whereas early work focused on the Escherichia coli RecA protein (5), the Saccharomyces cerevisiae Rad51 protein has been favored for studies of recombination in eukaryotes (6, 7).In the model strand transfer reaction, Rad51 coats a singlestranded circular (ssC) 1 DNA to form a nucleoprotein filament that associates with a homologous double-stranded linear (dsL) DNA. Once the nucleoprotein filament has aligned the homologous partners in a paranemic joint, which involves no net intertwining of the substrate DNAs, st...

“…ATP hydrolysis-dependent bypass of heterologous DNA has been argued to require the generation of rotory torque and/or recycling of the RecA protomers during the strand exchange reaction (30,33,34). RAD51 displays a dramatically reduced capacity to bypass heterologous DNA during strand exchange (26,(35)(36)(37). Taken together, these observations suggest that altered ATP processing might account for the disparities between hRAD51 and bacterial RecA activities.…”

mentioning

confidence: 99%

Biochemical Characterization of the Human RAD51 Protein

Tombline

Fishel²

2002

The prototypical bacterial RecA protein promotes recombination/repair by catalyzing strand exchange between homologous DNAs. While the mechanism of strand exchange remains enigmatic, ATP-induced cooperativity between RecA protomers is critical for its function. A human RecA homolog, human RAD51 protein (hRAD51), facilitates eukaryotic recombination/repair, although its ability to hydrolyze ATP and/or promote strand exchange appears distinct from the bacterial RecA. We have quantitatively examined the hRAD51 ATPase. The catalytic efficiency (k cat /K m ) of the hRAD51 ATPase was ϳ50-fold lower than the RecA ATPase. Altering the ratio of DNA/hRAD51 and including salts that stimulate DNA strand exchange (ammonium sulfate and spermidine) were found to affect the catalytic efficiency of hRAD51. The average site size of hRAD51 was determined to be ϳ3 nt (bp) for both single-stranded and double-stranded DNA. Importantly, hRAD51 lacks the magnitude of ATP-induced cooperativity that is a hallmark of RecA. Together, these results suggest that hRAD51 may be unable to coordinate ATP hydrolysis between neighboring protomers.