Presynaptic Filament Research Articles

Crystal Structure of RecA from Deinococcus radiodurans: Insights into the Structural Basis of Extreme Radioresistance

The resistance of Deinococcus radiodurans (Dr) to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks. Dr RecA, the key protein in the repair of dsDNA breaks by homologous recombination, promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the remarkable repair capacity of Dr and the novel mechanistic features of its RecA protein, we have determined its X-ray crystal structure in complex with ATPgammaS at 2.5A resolution. Like RecA from Escherichia coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67 A. Although the overall fold of Dr RecA is similar to E.coli RecA, there is a large reorientation of the C-terminal domain, which in E.coli RecA has a site for binding dsDNA. Compared to E.coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. Unique amino acid residues in Dr RecA cluster around a flexible beta-hairpin that has also been implicated in DNA binding.

Zinc finger nuclease-boosted gene targeting and synergistic transient regenerative gene therapy for long-term stem cell gene therapy

Stem cells have both self-renewing and homing/differentiative capabilities and thereby provide for life-long cell replacement in tissues and organs. They are therefore the ideal targets for most gene therapy protocols, i.e. both for long-term and transient gene expression protocols where they are either the long term carriers of the therapeutic gene (inherited/degenerative disease) or the mobilized/recruted targets of a transient regenerative process such as the formation of new blood vessels. Long-term gene therapy is thus amenable to synergistic combinations where ex vivo/in vivo genetic engineering of stem cells can be associated with in vivo transient topical expression of minigenes encoding various factors such as homing, regenerative and differentiative ones. Such a strategy is still hampered by many hurdles of which random integration of therapeutic DNA is a major safety concern. Emerging technologies are however aimed at efficient site-specific integration of therapeutic transgenes and at endogenous gene repair/modification. Promising site-specific integration vectors are at the pre-clinical stage and rely on an adeno-associated virus (AAV) rep platform or on phage phiC31 integrase. However, unlike these approaches, gene targeting is driven by homologous recombination and has thus target flexibility. It mediates DNA exchanges between chromosomal DNA and transfecting/transducing DNA, thereby providing the means to modify at will the sequence of target chromosomal DNA. Gene targeting stands thus as the ultimate process both for gene repair/alteration and targeted (i.e. site-specific) transgene integration. Such a process is however highly inefficient unless target chromosomal DNA is struck by a double-strand break (DSB). In addition, it is overwhelmed by random integration. In order to increase gene targeting frequency and eliminate random integration, we devised an approach that relies on the transfer into target cells of premade presynaptic filaments, i.e. the very stochiometric complexes between recombinase protein and single-stranded DNA (ssDNA) that mediate the key reaction from homologous recombination (homologous DNA pairing-strand exchange with double-stranded [ds] DNA). Upon publication of the enzymatic properties of recombinase RAD51, we invented chimeric presynaptic filaments with a dsDNA core, and shifted from gene conversion to 'true' gene targeting. Chimeric zinc finger nucleases are now available that create sequence-specific DSBs in target chromosomal DNA and stimulate gene targeting as expected. Such designed nucleases open exciting potentialities for standard gene targeting (non-viral) but also for emerging AAV gene targeting that has been shown to be boosted by DSB too. In these approaches, gene targeting frequency is raised to the random-integration level, i. e. ~1% of transfected/transduced cells. Our current approach is thus discussed in terms of synergistic combinations in which random-integration is blocked by the use of premade presynaptic complexes while homologous recombination is promoted both by premade presynaptic complexes and by sequence-specific DSB of target chromosomal DNA. Long-term gene therapy is thus amenable to sophisticated protocols in which stem-cell gene-targeting is combined with transient regenerative gene therapy, and might therefore apply to the treatment of the neurologic symptoms of the Lesch–Nyhan disease in which the target is our paragon model, the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene.

Effects of tumor-associated mutations on Rad54 functions.

Yeast RAD54 gene, a member of the RAD52 epistasis group, plays an important role in homologous recombination and DNA double strand break repair. Rad54 belongs to the Snf2/Swi2 protein family, and it possesses a robust DNA-dependent ATPase activity, uses free energy from ATP hydrolysis to supercoil DNA, and cooperates with the Rad51 recombinase in DNA joint formation. There are two RAD54-homologous genes in human cells, hRAD54 and RAD54B. Mutations in these human genes have been found in tumors. These tumor-associated mutations map to conserved regions of the hRad54 and hRad54B proteins. Here we introduced the equivalent mutations into the Saccharomyces cerevisiae RAD54 gene in an effort to examine the functional consequences of these gene changes. One mutant, rad54 G484R, showed sensitivity to DNA-damaging agents and reduced homologous recombination rates, indicating a loss of function. Even though the purified rad54 G484R mutant protein retained the ability to bind DNA and interact with Rad51, it was nearly devoid of ATPase activity and was similarly defective in DNA supercoiling and D-loop formation. Two other mutants, rad54 N616S and rad54 D442Y, were not sensitive to genotoxic agents and behaved like the wild type allele in homologous recombination assays. Consistent with the mild phenotype associated with the rad54 N616S allele, its encoded protein was similar to wild type Rad54 protein in biochemical attributes. Because dysfunctional homologous recombination gives rise to genome instability, our results are consistent with the premise that tumor-associated mutations in hRad54 and Rad54B could contribute to the tumor phenotype or enhance the genome instability seen in tumor cells.

Mutations in a Conserved Motif Inhibit Single-stranded DNA Binding and Recombination Mediator Activities of Bacteriophage T4 UvsY Protein

The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsY-ssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNA-dependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.

Recruitment of the Recombinational Repair Machinery to a DNA Double-Strand Break in Yeast

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires members of the RAD52 epistasis group. Here we use chromatin immunoprecipitation (ChIP) to examine the temporal order of recruitment of Rad51p, Rad52p, Rad54p, Rad55p, and RPA to a single, induced DSB in yeast. Our results suggest a sequential, interdependent assembly of Rad proteins adjacent to the DSB initiated by binding of Rad51p. ChIP time courses from various mutant strains and additional biochemical studies suggest that Rad52p, Rad55p, and Rad54p each help promote the formation and/or stabilization of the Rad51p nucleoprotein filament. We also find that all four Rad proteins associate with homologous donor sequences during strand invasion. These studies provide a near comprehensive view of the molecular events required for the in vivo assembly of a functional Rad51p presynaptic filament.

DNA helicase Srs2 disrupts the Rad51 presynaptic filament.

Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing.

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.

Rad54 Protein Exerts Diverse Modes of ATPase Activity on Duplex DNA Partially and Fully Covered with Rad51 Protein

Rad54 protein is a Snf2-like ATPase with a specialized function in the recombinational repair of DNA damage. Rad54 is thought to stimulate the search of homology via formation of a specific complex with the presynaptic Rad51 filament on single-stranded DNA. Herein, we address the interaction of Rad54 with Rad51 filaments on double-stranded (ds) DNA, an intermediate in DNA strand exchange with unclear functional significance. We show that Saccharomyces cerevisiae Rad54 exerts distinct modes of ATPase activity on partially and fully saturated filaments of Rad51 protein on dsDNA. The highest ATPase activity is observed on dsDNA containing short patches of yeast Rad51 filaments resulting in a 6-fold increase compared with protein-free DNA. This enhanced ATPase mode of yeast Rad54 can also be elicited by partial filaments of human Rad51 protein but to a lesser extent. In contrast, the interaction of Rad54 protein with duplex DNA fully covered with Rad51 is entirely species-specific. When yeast Rad51 fully covers dsDNA, Rad54 protein hydrolyzes ATP in a reduced mode at 60-80% of its rate on protein-free DNA. Instead, saturated filaments with human Rad51 fail to support the yeast Rad54 ATPase. We suggest that the interaction of Rad54 with dsDNA-Rad51 complexes is of functional importance in homologous recombination.

Rad52 Protein Has a Second Stimulatory Role in DNA Strand Exchange That Complements Replication Protein-A Function

Rad52 protein plays a central role in double strand break repair and homologous recombination in Saccharomyces cerevisiae. We have identified a new mechanism by which Rad52 protein stimulates Rad51 protein-promoted DNA strand exchange. This function of Rad52 protein is revealed when subsaturating amounts (relative to the single-stranded DNA concentration) of replication protein-A (RPA) are used. Under these conditions, Rad52 protein is needed for extensive DNA strand exchange. Interestingly, in this new role, Rad52 protein neither acts simply as a single strand DNA-binding protein per se nor, in contrast to its previously identified stimulatory roles, does it require physical interaction with RPA because it can be substituted by the Escherichia coli single strand DNA-binding protein. We propose that Rad52 protein acts by stabilizing the Rad51 presynaptic filament.

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair.

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3' ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative "bubble migration" mode of RDR could play a role in lesion bypass by the T4 replication machinery.

Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament

RAD54 is an important member of the RAD52 group of genes that carry out recombinational repair of DNA damage in the yeast Saccharomyces cerevisiae. Rad54 protein is a member of the Snf2/Swi2 protein family of DNA-dependent/stimulated ATPases, and its ATPase activity is crucial for Rad54 protein function. Rad54 protein and Rad54-K341R, a mutant protein defective in the Walker A box ATP-binding fold, were fused to glutathione-S-transferase (GST) and purified to near homogeneity. In vivo, GST-Rad54 protein carried out the functions required for methyl methanesulfonate sulfate (MMS), UV, and DSB repair. In vitro, GST-Rad54 protein exhibited dsDNA-specific ATPase activity. Rad54 protein stimulated Rad51/Rpa-mediated DNA strand exchange by specifically increasing the kinetics of joint molecule formation. This stimulation was accompanied by a concurrent increase in the formation of heteroduplex DNA. Our results suggest that Rad54 protein interacts specifically with established Rad51 nucleoprotein filaments before homology search on the duplex DNA and heteroduplex DNA formation. Rad54 protein did not stimulate DNA strand exchange by increasing presynaptic complex formation. We conclude that Rad54 protein acts during the synaptic phase of DNA strand exchange and after the formation of presynaptic Rad51 protein-ssDNA filaments.

Evidence for a four-strand exchange catalyzed by the RecA protein.

Strand exchange between two duplexes is usually initiated as a three-strand event that requires the presence of a single-stranded overhang or gap in one of the two molecules. Here we show that the RecA protein can catalyze a four-strand exchange. Specifically, it can recombine short hairpin substrates with homologous stems provided that one of the hairpins possesses a chimeric DNA/RNA backbone. This four-strand exchange reaction goes to completion in the presence of ATPgammaS and releases a stable heteroduplex upon removal of the RecA protein. Under identical conditions, strand exchange between two DNA hairpins is incomplete and generates a nascent heteroduplex that rapidly dissociates when the RecA protein is denatured. Since presynaptic filament formation does not appear to melt either type of hairpin, we propose that exchange occurs between homologously aligned duplexes that are extended and unwound within a RecA filament. The first reaction provides a mechanism for gene targeting by chimeric double-hairpin oligonucleotides while the second reaction explains the ability of the RecA protein to transiently align double-stranded DNA molecules.

The Mutant RecA Proteins, RecAR243Q and RecAK245N, Exhibit Defective DNA Binding in Homologous Pairing

In homologous pairing, the RecA protein sequentially binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), aligning the two DNA molecules within the helical nucleoprotein filament. To identify the DNA binding region, which stretches from the outside to the inside of the filament, we constructed two mutant RecA proteins, RecAR243Q and RecAK245N, with the amino acid substitutions of Arg243 to Gln and Lys245 to Asn, respectively. These amino acids are exposed to the solvent in the crystal structure of the RecA protein and are located in the central domain, which is believed to be the catalytic center of the homologous pairing activity. The mutations of Arg243 to Gln (RecAR243Q) and Lys245 to Asn (RecAK245N) impair the repair of UV-damaged DNAin vivoand cause defective homologous pairing of ssDNA and dsDNAin vitro.Although RecAR243Q is only slightly defective and RecAK245N is completely proficient in ssDNA binding to form the presynaptic filament, both mutant RecA proteins are defective in the formation of the three-component complex including ssDNA, dsDNA, and RecA protein. The ability to form dsDNA from complementary single strands is also defective in both RecAR243Q and RecAK245N. These results suggest that the region including Arg243 and Lys245 may be involved in the path of secondary DNA binding to the presynaptic filament.

Biochemical interactions within a ternary complex of the bacteriophage T4 recombination proteins uvsY and gp32 bound to single-stranded DNA.

The presynaptic phase of homologous recombination requires the formation of a filament of single-stranded DNA (ssDNA) coated with a recombinase enzyme. In bacteriophage T4, at least three proteins are required for the assembly of this presynaptic filament. In addition to the T4 recombinase, uvsX protein, the T4 ssDNA binding protein (gp32), and the uvsY recombination accessory protein are also required. Here we report on a detailed analysis of a tripartite filament containing ssDNA bound by stoichiometric quantities of both uvsY and gp32, which appears to be an important intermediate in the assembly of the T4 presynaptic filament. We demonstrate that uvsY and gp32 simultaneously co-occupy the ssDNA in a noncompetitive fashion. In addition, we show that protein-protein interactions between uvsY and gp32 are not required for the assembly of this ternary complex and do not affect the affinity of uvsY for the ssDNA lattice. Finally, we demonstrate that the interaction of gp32 with the ssDNA is destabilized within this complex, in a manner which is independent of gp32-uvsY interactions. The data suggest that the uvsY protein acts to remodel the gp32-ssDNA complex via uvsY-ssDNA interactions. The implications of these findings for the mechanism of presynapsis in the T4 recombination system are discussed.

Single-stranded DNA binding properties of the UvsX recombinase of bacteriophage T4: binding parameters and effects of nucleotides

Bacteriophage T4 provides an important model for the biochemistry and genetics of DNA metabolism. Phage-encoded proteins conduct all essential steps of T4 DNA replication, repair, and recombination. Central to these three processes is the T4 UvsX protein, a member of the filamentous, ATP-dependent class of general recombination enzymes typified by the Escherichia coli RecA protein. Like RecA, UvsX forms presynaptic filaments on single-stranded (ss) DNA, which are the obligatory nucleoprotein intermediates in recombination. Aspects of the T4 presynaptic filament are explored by quantitative characterization of the UvsX-ssDNA interaction using an etheno-derivitized single-stranded DNA molecule, ϵDNA, whose fluorescence is enhanced by UvsX binding. Studies with this model lattice show that UvsX exhibits a moderate level of cooperativity (ω = 100) when binding to ϵDNA with a binding-site size (n) equal to four nucleotide residues. Salt-stability studies of this complex reveal that the non-hydrolyzable ATP analog, ATPγS, induces a high-affinity binding mode that is distinguishable from complexes formed with ADP or in the absence of a nucleotide cofactor. With this new information, both functional relationships between the UvsX and RecA recombinases, and implications for UvsX interactions with the other proteins of the T4 presynaptic filament (UvsY and gp32) may be further explored.

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination. ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA.

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 microM duplex and 0.1-1.0 microM ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATPgammaS showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATPgammaS. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATPgammaS. Results in ATPgammaS using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATPgammaS, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range RecA-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.

The uvsY recombination protein of bacteriophage T4 forms hexamers in the presence and absence of single-stranded DNA.

A prerequisite to genetic recombination in the T4 bacteriophage is the formation of the presynaptic filament-a helical nucleoprotein filament containing stoichiometric amounts of the uvsX recombinase in complex with single-stranded DNA (ssDNA). Once formed, the filament is competent to catalyze homologous pairing and DNA strand exchange reactions. An important component in the formation of the presynaptic filament is the uvsY protein, which is required for optimal uvsX-ssDNA assembly in vitro, and essential for phage recombination in vivo. uvsY enhances uvsX activities by promoting filament formation and stabilizing filaments under conditions of low uvsX, high salt, and/or high gp32 (ssDNA-binding protein) concentrations. The molecular properties of uvsY include noncooperative binding to ssDNA and specific protein-protein interactions with both uvsX and gp32. Evidence suggests that all of these hetero-associations of the uvsY protein are important for presynaptic filament formation. However, there is currently no structural information available on the uvsY protein itself. In this study, we present the first characterization of the self-association of uvsY. Using hydrodynamic methods, we demonstrate that uvsY associates into a stable hexamer (s020,w = 6.0, M = 95 kDa) in solution and that this structure is competent to bind ssDNA. We further demonstrate that uvsY hexamers are capable of reversible association into higher aggregates in a manner dependent on both salt and protein concentration. The implications for presynaptic filament formation are discussed.

Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A.

The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. We find that Rad52 protein stimulates DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required.

Characterization of an amino-terminal fragment of the bacteriophage T4 uvsY recombination protein

The uvsY protein plays essential roles in homologous genetic recombination processes in the bacteriophage T4. In vitro, uvsY promotes the formation of presynaptic filaments containing stoichiometric amounts of the T4 uvsX recombinase bound to single-stranded DNA. uvsY protein has intrinsic binding activities towards ssDNA, uvsX, and gp32, the T4-encoded SSB, however, it has not been directly determined which of these activities are essential for uvsY's role in presynapsis. We have therefore sought to generate altered forms of uvsY deficient in uvsX- and/or gp32-binding, in order to assess whether these specific protein-protein interactions are essential for uvsY recombination functions. Limited chymotrypsinolysis of the 16 kDa uvsY protein generates two major fragments: an 11.4 kDa fragment containing the N-terminus of uvsY, and a 4.5 kDa C-terminal fragment. We have expressed and purified the large fragment as a fusion protein containing the N-terminal 101 amino acids of uvsY. We show that this truncated uvsY species, which we call uvsY nt, retains ssDNA-binding activity, but is devoid of both uvsX- and gp32-binding activities. Like native uvsY, uvsY nt stimulates the ssDNA-dependent ATPase activity of the uvsX protein, however, the synergistic effects observed between uvsY, uvsX, and gp32 are not observed with uvsY nt. In addition, uvsY nt weakly stimulates uvsX-catalyzed DNA strand exchange reactions. The latter result is surprising since it suggests that specific interactions with uvsX and/or gp32 are not absolutely essential for uvsY recombination functions. Taken together, the data are consistent with a model in which uvsY-ssDNA interactions alone are capable of promoting the assembly of functional uvsX-ssDNA complexes, while uvsY-protein interactions stabilize uvsX-ssDNA complexes.

The Gene 32 Single-Stranded DNA-Binding Protein Is Not Bound Stably to the Phage T4 Presynaptic Filament

A central reaction in homologous recombination is synapsis, which involves invasion of duplex DNA by a homologous single strand. A key intermediate in this process is the presynaptic filament, a protein–DNA complex composed of a “strand transferase” polymerized along the invading single strand. In this report, the organization and mechanism of assembly of the bacteriophage T4 presynaptic filament are explored. Three T4 proteins, encoded by the uvsX, uvsY and 32 genes, are involved in this process. It is demonstrated that a well-defined series of events involving multiple protein–DNA and protein–protein interactions is required to mediate a transition from an initial gene 32-DNA complex to a mature presynaptic filament in which the UvsX and UvsY proteins are in contact with the DNA and each other, while most or all of the gene 32 protein is removed from the complex.