Saccharomyces cerevisiae Dmc1 protein promotes renaturation of single-strand DNA (ssDNA) and assimilation of ssDNA into homologous super-coiled duplex DNA

Dmc1 and Rad51 are eukaryotic RecA homologues that are involved in meiotic recombination. The expression of Dmc1 is limited to meiosis, whereas Rad51 is expressed in mitosis and meiosis. Dmc1 and Rad51 have unique and overlapping functions during meiotic recombination. Here we report the purification of the Dmc1 protein from the budding yeast Saccharomyces cerevisiae and present basic characterization of its biochemical activity. The protein has a weak DNA-dependent ATPase activity and binds both single-strand DNA (ssDNA) and double-strand DNA. Electrophoretic mobility shift assays suggest that DNA binding by Dmc1 is cooperative. Dmc1 renatures linearized plasmid DNA with first order reaction kinetics and without requiring added nucleotide cofactor. In addition, Dmc1 catalyzes strand assimilation of ssDNA oligonucleotides into homologous supercoiled duplex DNA in a reaction promoted by ATP or the non-hydrolyzable ATP analogue AMP-PNP.     

Fission yeast rad51 and dmc1, two efficient DNA recombinases forming helical nucleoprotein filaments

Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.     

Meiotic Recombination: An Affair of Two Recombinases

In E. coli, homologous recombination is catalyzed by the RecA recombinase. Two RecA-like factors, Rad51 and Dmc1, are found in eukaryotes. Whereas Rad51 is needed for homologous recombination reactions in both mitotic and meiotic cells, the role of Dmc1 is restricted to meiosis. Recent work has shown that, like RecA and Rad51, Dmc1 mediates the homologous DNA pairing strand exchange reaction via a filamentous intermediate assembled on single-stranded DNA. Emerging evidence suggests that the tumor suppressor BRCA2 functions in the assembly of nucleoprotein filaments of Rad51 and Dmc1. The manner in which Rad51 and Dmc1 functionally cooperate in meiotic recombination remains to be determined.     

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.     

Stimulation of fission yeast and mouse Hop2-Mnd1 of the Dmc1 and Rad51 recombinases

Genetic analysis of fission yeast suggests a role for the spHop2–Mnd1 proteins in the Rad51 and Dmc1-dependent meiotic recombination pathways. In order to gain biochemical insights into this process, we purified Schizosaccharomyces pombe Hop2-Mnd1 to homogeneity. spHop2 and spMnd1 interact by co-immunoprecipitation and two-hybrid analysis. Electron microscopy reveals that S. pombe Hop2–Mnd1 binds single-strand DNA ends of 3′-tailed DNA. Interestingly, spHop2-Mnd1 promotes the renaturation of complementary single-strand DNA and catalyses strand exchange reactions with short oligonucleotides. Importantly, we show that spHop2-Mnd1 stimulates spDmc1-dependent strand exchange and strand invasion. Ca²⁺ alleviate the requirement for the order of addition of the proteins on DNA. We also demonstrate that while spHop2-Mnd1 affects spDmc1 specifically, mHop2 or mHop2-Mnd1 stimulates both the hRad51 and hDmc1 recombinases in strand exchange assays. Thus, our results suggest a crucial role for S. pombe and mouse Hop2-Mnd1 in homologous pairing and strand exchange and reveal evolutionary divergence in their specificity for the Dmc1 and Rad51 recombinases.     

DNA binding and pairing activity of OsDmc1, a recombinase from rice

A cloned cDNA corresponding to OsDMC1 from rice anther tissue was expressed in Escherichia coli. The OsDmc1 protein was largely present in the inclusion bodies of the cell lysatE., which was solubilized by 8.0 M urea containing buffeR., purified to homogeneity by Ni-CAM agarose column chromatography, followed by renaturation to its native state through stepwise dialysis against reduced concentrations of urea. The purified protein cross-reacted with anti-yeast Dmc1 antibodies. The binding efficiency observed with circular single-stranded DNA (ssDNA) was similar to that with circular double-stranded DNA (dsDNA). The binding to either DNA showed no ATP dependencE., but required 5–10 mM Mg²⁺ in the presence of ATP. Even though the protein binding to dsDNA was as efficient as it was to ssDNA, the former induced no DNA dependent ATPasE., whereas the binding to ssDNA stimulated a significant level of DNA dependent ATPase activity. OsDmc1–ssDNA complex, with its ATPase proficiency, also mediated renaturation of homologous complementary strands as well as assimilation of single strands into homologous supercoiled duplexes leading to D-loop formation. The D-loop formation was lowered by excess of OsDmc1 protein. This D-loop formation activity was promoted by non-hydrolyzable ATP analog, AMP-PNP and was not observed in absence of ATP or presence of ADP/ATP--S. These properties reflected the classical hallmarks of a recombinase and represented the first biochemical characterization of a plant Dmc1 protein.     

Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination.     

The recombinases Rad51 and Dmc1 play distinct roles in DNA break repair and recombination partner choice in the meiosis of Tetrahymena

Repair of programmed DNA double-strand breaks (DSBs) by meiotic recombination relies on the generation of flanking 3' single-stranded DNA overhangs and their interaction with a homologous double-stranded DNA template. In various common model organisms, the ubiquitous strand exchange protein Rad51 and its meiosis-specific homologue Dmc1 have been implicated in the joint promotion of DNA-strand exchange at meiotic recombination sites. However, the division of labor between these two recombinases is still a puzzle. Using RNAi and gene-disruption experiments, we have studied their roles in meiotic recombination and chromosome pairing in the ciliated protist Tetrahymena as an evolutionarily distant meiotic model. Cytological and electrophoresis-based assays for DSBs revealed that, without Rad51p, DSBs were not repaired. However, in the absence of Dmc1p, efficient Rad51p-dependent repair took place, but crossing over was suppressed. Immunostaining and protein tagging demonstrated that only Dmc1p formed strong DSB-dependent foci on meiotic chromatin, whereas the distribution of Rad51p was diffuse within nuclei. This suggests that meiotic nucleoprotein filaments consist primarily of Dmc1p. Moreover, a proximity ligation assay confirmed that little if any Rad51p forms mixed nucleoprotein filaments with Dmc1p. Dmc1p focus formation was independent of the presence of Rad51p. The absence of Dmc1p did not result in compensatory assembly of Rad51p repair foci, and even artificial DNA damage by UV failed to induce Rad51p foci in meiotic nuclei, while it did so in somatic nuclei within one and the same cell. The observed interhomologue repair deficit in dmc1Δ meiosis is consistent with a requirement for Dmc1p in promoting the homologue as the preferred recombination partner. We propose that relatively short and/or transient Rad51p nucleoprotein filaments are sufficient for intrachromosomal recombination, whereas long nucleoprotein filaments consisting primarily of Dmc1p are required for interhomolog recombination.     

Meiotic Crossover Control by Concerted Action of Rad51-Dmc1 in Homolog Template Bias and Robust Homeostatic Regulation

During meiosis, repair of programmed DNA double-strand breaks (DSBs) by recombination promotes pairing of homologous chromosomes and their connection by crossovers. Two DNA strand-exchange proteins, Rad51 and Dmc1, are required for meiotic recombination in many organisms. Studies in budding yeast imply that Rad51 acts to regulate Dmc1''s strand exchange activity, while its own exchange activity is inhibited. However, in a dmc1 mutant, elimination of inhibitory factor, Hed1, activates Rad51''s strand exchange activity and results in high levels of recombination without participation of Dmc1. Here we show that Rad51-mediated meiotic recombination is not subject to regulatory processes associated with high-fidelity chromosome segregation. These include homolog bias, a process that directs strand exchange between homologs rather than sister chromatids. Furthermore, activation of Rad51 does not effectively substitute for Dmc1''s chromosome pairing activity, nor does it ensure formation of the obligate crossovers required for accurate homolog segregation. We further show that Dmc1''s dominance in promoting strand exchange between homologs involves repression of Rad51''s strand-exchange activity. This function of Dmc1 is independent of Hed1, but requires the meiotic kinase, Mek1. Hed1 makes a relatively minor contribution to homolog bias, but nonetheless this is important for normal morphogenesis of synaptonemal complexes and efficient crossing-over especially when DSB numbers are decreased. Super-resolution microscopy shows that Dmc1 also acts to organize discrete complexes of a Mek1 partner protein, Red1, into clusters along lateral elements of synaptonemal complexes; this activity may also contribute to homolog bias. Finally, we show that when interhomolog bias is defective, recombination is buffered by two feedback processes, one that increases the fraction of events that yields crossovers, and a second that we propose involves additional DSB formation in response to defective homolog interactions. Thus, robust crossover homeostasis is conferred by integrated regulation at initiation, strand-exchange and maturation steps of meiotic recombination.     

Regulation of Hed1 and Rad54 binding during maturation of the meiosis‐specific presynaptic complex

Most eukaryotes have two Rad51/RecA family recombinases, Rad51, which promotes recombination during mitotic double‐strand break (DSB) repair, and the meiosis‐specific recombinase Dmc1. During meiosis, the strand exchange activity of Rad51 is downregulated through interactions with the meiosis‐specific protein Hed1, which helps ensure that strand exchange is driven by Dmc1 instead of Rad51. Hed1 acts by preventing Rad51 from interacting with Rad54, a cofactor required for promoting strand exchange during homologous recombination. However, we have a poor quantitative understanding of the regulatory interplay between these proteins. Here, we use real‐time single‐molecule imaging to probe how the Hed1‐ and Rad54‐mediated regulatory network contributes to the identity of mitotic and meiotic presynaptic complexes. Based on our findings, we define a model in which kinetic competition between Hed1 and Rad54 helps define the functional identity of the presynaptic complex as cells undergo the transition from mitotic to meiotic repair.     

Homologous recombination properties of OsRad51, a recombinase from rice

cDNA corresponding to OsRad51 protein was isolated from cDNA library of rice flowers (Oryza sativa, Indica cultivar group) and cloned in to pET28a expression vector. The protein was over expressed in E. coli BL21 (DE3) and purified. Purified OsRad51 could bind single and double stranded DNA, however it showed higher affinity for single stranded DNA. Transmission Electron Microscopy (TEM) studies of OsRad51-DNA complexes showed that this protein formed ring like structures and bound DNA forming filaments. OsRad51 protein promoted renaturation of complementary single strands in to duplex DNA molecules and also showed ATPase activity, which was stimulated by single strand DNA. Fluorescence resonance energy transfer (FRET) assays revealed that OsRad51 promoted homology dependent renaturation as well as strand exchange reactions. Renaturation activity was ATP dependent; however strand exchange activity was ATP independent. This is the first report on in vitro characterization of Rad51 protein from crop plants.     

Roles of RecA homologues Rad51 and Dmc1 during meiotic recombination

RecA protein is involved in homology search and strand exchange processes during recombination. Mitotic cells in eukaryotes express one RecA, Rad51, which is essential for the repair of double-strand breaks (DSBs). Additionally, meiotic cells induce the second RecA, Dmc1. Both Rad51 and Dmc1 are necessary to generate a crossover between homologous chromosomes, which ensures the segregation of the chromosomes at meiotic division I. It is largely unknown how the two RecAs cooperate during meiotic recombination. In this review, recent advances on our knowledge about the roles of Rad51 and Dmc1 during meiosis are summarized and discussed.     

A comparative analysis of Dmc1 and Rad51 nucleoprotein filaments

The eukaryotic RecA homologs Rad51 and Dmc1 are essential for strand exchange between homologous chromosomes during meiosis. All members of the RecA family of recombinases polymerize on DNA to form helical nucleoprotein filaments, which is the active form of the protein. Here we compare the filament structures of the Rad51 and Dmc1 proteins from both human and budding yeast. Previous studies of Dmc1 filaments suggested that they might be structurally distinct from filaments of other members of the RecA family, including Rad51. The data presented here indicate that Rad51 and Dmc1 filaments are essentially identical with respect to several structural parameters, including persistence length, helical pitch, filament diameter, DNA base pairs per helical turn and helical handedness. These data, together with previous studies demonstrating similar in vitro recombinase activity for Dmc1 and Rad51, support the view that differences in the meiotic function of Rad51 and Dmc1 are more likely to result from the influence of distinct sets of accessory proteins than from intrinsic differences in filament structure.     

AtMND1 is required for homologous pairing during meiosis in Arabidopsis

Background  

Pairing of homologous chromosomes at meiosis is an important requirement for recombination and balanced chromosome segregation among the products of meiotic division. Recombination is initiated by double strand breaks (DSBs) made by Spo11 followed by interaction of DSB sites with a homologous chromosome. This interaction requires the strand exchange proteins Rad51 and Dmc1 that bind to single stranded regions created by resection of ends at the site of DSBs and promote interactions with uncut DNA on the homologous partner. Recombination is also considered to be dependent on factors that stabilize interactions between homologous chromosomes. In budding yeast Hop2 and Mnd1 act as a complex to promote homologous pairing and recombination in conjunction with Rad51 and Dmc1.     

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity.     

Recruitment of RecA homologs Dmc1p and Rad51p to the double-strand break repair site initiated by meiosis-specific endonuclease VDE (PI-SceI)

During meiosis, VDE (PI-SceI), a homing endonuclease in Saccharomyces cerevisiae, introduces a double-strand break (DSB) at its recognition sequence and induces homologous recombinational repair, called homing. Meiosis-specific RecA homolog Dmc1p, as well as mitotic RecA homolog Rad51p, acts in the process of meiotic recombination, being required for strand invasion and exchange. In this study, recruitment of Dmc1p and Rad51p to the VDE-induced DSB repair site is investigated by chromatin immunoprecipitation assay. It is revealed that Dmc1p and Rad51p are loaded to the repair site in an independent manner. Association of Rad51p requires other DSB repair proteins of Rad52p, Rad55p, and Rad57p, while loading of Dmc1p is facilitated by the different protein, Sae3p. Absence of Tid1p, which can bind both RecA homologs, appears specifically to cause an abnormal distribution of Dmc1p. Lack of Hop2, Mnd1p, and Sae1p does not impair recruitment of both RecA homologs. These findings reveal the discrete functions of each strand invasion protein in VDE-initiated homing, confirm the similarity between VDE-initiated homing and Spo11p-initiated meiotic recombination, and demonstrate the availability of VDE-initiated homing for the study of meiotic recombination.     

A RecA homologue in Ustilago maydis that is distinct and evolutionarily distant from Rad51 actively promotes DNA pairing reactions in the absence of auxiliary factors

Two RecA homologues have been identified to date in Ustilago maydis. One is orthologous to Rad51 while the other, Rec2, is structurally quite divergent and evolutionarily distant. DNA repair and recombination proficiency in U. maydis requires both Rec2 and Rad51. Here we have examined biochemical activities of Rec2 protein purified after overexpression of the cloned gene. Rec2 requires DNA as a cofactor to hydrolyze ATP and depends on ATP to promote homologous pairing and DNA strand exchange. ATPgammaS was found to substitute for ATP in all pairing reactions examined. With superhelical DNA and a homologous single-stranded oligonucleotide as substrates, Rec2 actively promoted formation and dissociation of D-loops. When an RNA oligonucleotide was substituted it was found that R-loops could also be formed and utilized as primer/template for limited DNA synthesis. In DNA strand exchange reactions using oligonucleotides, we found that Rec2 exhibited a pairing bias that is opposite that of RecA. Single-stranded oligonucleotides were activated for DNA strand exchange when attached as tails protruding from a duplex sequence due to enhanced binding of Rec2. The results indicate that Rec2 is competent, and in certain ways even better than Rad51, in the ability to provide the fundamental DNA pairing activity necessary for recombinational repair. We propose that the emerging paradigm for homologous recombination featuring Rad51 as the essential catalytic component for strand exchange may not be universal in eukaryotes.     

Heterology tolerance and recognition of mismatched base pairs by human Rad51 protein

Human Rad51 (hRad51) promoted homology recognition and subsequent strand exchange are the key steps in human homologous recombination mediated repair of DNA double-strand breaks. However, it is still not clear how hRad51 deals with sequence heterology between the two homologous chromosomes in eukaryotic cells, which would lead to mismatched base pairs after strand exchange. Excessive tolerance of sequence heterology may compromise the fidelity of repair of DNA double-strand breaks. In this study, fluorescence resonance energy transfer (FRET) was used to monitor the heterology tolerance of human Rad51 mediated strand exchange reactions, in real time, by introducing either G-T or I-C mismatched base pairs between the two homologous DNA strands. The strand exchange reactions were much more sensitive to G-T than to I-C base pairs. These results imply that the recognition of homology and the tolerance of heterology by hRad51 may depend on the local structural motif adopted by the base pairs participating in strand exchange. AnhRad51 mutant protein (hRad51K133R), deficient in ATP hydrolysis, showed greater heterology tolerance to both types of mismatch base pairing, suggesting that ATPase activity may be important for maintenance of high fidelity homologous recombination DNA repair.     

Down-Regulation of Rad51 Activity during Meiosis in Yeast Prevents Competition with Dmc1 for Repair of Double-Strand Breaks

Interhomolog recombination plays a critical role in promoting proper meiotic chromosome segregation but a mechanistic understanding of this process is far from complete. In vegetative cells, Rad51 is a highly conserved recombinase that exhibits a preference for repairing double strand breaks (DSBs) using sister chromatids, in contrast to the conserved, meiosis-specific recombinase, Dmc1, which preferentially repairs programmed DSBs using homologs. Despite the different preferences for repair templates, both Rad51 and Dmc1 are required for interhomolog recombination during meiosis. This paradox has recently been explained by the finding that Rad51 protein, but not its strand exchange activity, promotes Dmc1 function in budding yeast. Rad51 activity is inhibited in dmc1Δ mutants, where the failure to repair meiotic DSBs triggers the meiotic recombination checkpoint, resulting in prophase arrest. The question remains whether inhibition of Rad51 activity is important during wild-type meiosis, or whether inactivation of Rad51 occurs only as a result of the absence of DMC1 or checkpoint activation. This work shows that strains in which mechanisms that down-regulate Rad51 activity are removed exhibit reduced numbers of interhomolog crossovers and noncrossovers. A hypomorphic mutant, dmc1-T159A, makes less stable presynaptic filaments but is still able to mediate strand exchange and interact with accessory factors. Combining dmc1-T159A with up-regulated Rad51 activity reduces interhomolog recombination and spore viability, while increasing intersister joint molecule formation. These results support the idea that down-regulation of Rad51 activity is important during meiosis to prevent Rad51 from competing with Dmc1 for repair of meiotic DSBs.     

The meiosis-specific recombinase hDmc1 forms ring structures and interacts with hRad51

Eukaryotic cells encode two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, which are required for meiotic recombination. Rad51, like E.coli RecA, forms helical nucleoprotein filaments that promote joint molecule and heteroduplex DNA formation. Electron microscopy reveals that the human meiosis-specific recombinase Dmc1 forms ring structures that bind single-stranded (ss) and double-stranded (ds) DNA. The protein binds preferentially to ssDNA tails and gaps in duplex DNA. hDmc1-ssDNA complexes exhibit an irregular, often compacted structure, and promote strand-transfer reactions with homologous duplex DNA. hDmc1 binds duplex DNA with reduced affinity to form nucleoprotein complexes. In contrast to helical RecA/Rad51 filaments, however, Dmc1 filaments are composed of a linear array of stacked protein rings. Consistent with the requirement for two recombinases in meiotic recombination, hDmc1 interacts directly with hRad51.