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.     

Functional attributes of the Saccharomyces cerevisiae meiotic recombinase Dmc1

The role of Dmc1 as a meiosis-specific general recombinase was first demonstrated in Saccharomyces cerevisiae. Progress in understanding the biochemical mechanism of ScDmc1 has been hampered by its tendency to form inactive aggregates. We have found that the inclusion of ATP during protein purification prevents Dmc1 aggregation. ScDmc1 so prepared is capable of forming D-loops and responsive to its accessory factors Rad54 and Rdh54. Negative staining electron microscopy and iterative helical real-space reconstruction revealed that the ScDmc1-ssDNA nucleoprotein filament harbors 6.5 protomers per turn with a pitch of ～106 Å. The ScDmc1 purification procedure and companion molecular analyses should facilitate future studies on this recombinase.     

Schizosaccharomyces pombe Rdh54 (TID1) acts with Rhp54 (RAD54) to repair meiotic double-strand breaks

We report the characterization of rdh54+, the second fission yeast Schizosaccharomyces pombe Rad54 homolog. rdh54+ shares sequence and functional homology to budding yeast RDH54/TID1. Rdh54p is present during meiosis with appropriate timing for a meiotic recombination factor. It interacts with Rhp51 and the meiotic Rhp51 homolog Dmc1 in yeast two-hybrid assays. Deletion of rdh54+ has no effect on DNA damage repair during the haploid vegetative cell cycle. In meiosis, however, rdh54Delta shows decreased spore viability and homologous recombination with a concomitant increase in sister chromatid exchange. The rdh54Delta single mutant repairs meiotic breaks with similar timing to wild type, suggesting redundancy of meiotic recombination factors. Consistent with this, the rdh54Delta rhp54Delta double mutant fails to repair meiotic double strand breaks. Live cell analysis shows that rdh54Delta rhp54Delta asci do not arrest, but undergo both meiotic divisions with near normal timing, suggesting that failure to repair double strand breaks in S. pombe meiosis does not result in checkpoint arrest.     

ATP-dependent chromatin remodeling by the Saccharomyces cerevisiae homologous recombination factor Rdh54

Saccharomyces cerevisiae RDH54 is a key member of the evolutionarily conserved RAD52 epistasis group of genes needed for homologous recombination and DNA double strand break repair. The RDH54-encoded protein possesses a DNA translocase activity and functions together with the Rad51 recombinase in the D-loop reaction. By chromatin immunoprecipitation (ChIP), we show that Rdh54 is recruited, in a manner that is dependent on Rad51 and Rad52, to a site-specific DNA double strand break induced by the HO endonuclease. Because of its relatedness to Swi2/Snf2 chromatin remodelers, we have asked whether highly purified Rdh54 possesses chromatin-remodeling activity. Importantly, our results show that Rdh54 can mobilize a mononucleosome along DNA and render nucleosomal DNA accessible to a restriction enzyme, indicative of a chromatin-remodeling function. Moreover, Rdh54 co-operates with Rad51 in the utilization of naked or chromatinized DNA as template for D-loop formation. We also provide evidence for a strict dependence of the chromatin-remodeling attributes of Rdh54 on its ATPase activity and N-terminal domain. Interestingly, an N-terminal deletion mutant (rdh54Delta102) is unable to promote Rad51-mediated D-loop formation with a chromatinized template, while retaining substantial activity with naked DNA. These features of Rdh54 suggest a role of this protein factor in chromatin rearrangement during DNA recombination and repair.     

The Hop2 and Mnd1 proteins act in concert with Rad51 and Dmc1 in meiotic recombination

During the first meiotic division, homologous chromosomes (homologs) have to separate to opposite poles of the cell to ensure the right complement in the progeny. Homologous recombination provides a mechanism for a genome-wide homology search and physical linkage among the homologs before their orderly segregation. Rad51 and Dmc1 recombinases are the major players in these processes. Disruption of meiosis-specific HOP2 or MND1 genes leads to severe defects in homologous synapsis and an early-stage recombination failure resulting in sterility. Here we show that mouse Hop2 can efficiently form D-loops, the first recombination intermediates, but this activity is abrogated upon association with Mnd1. Furthermore, the Hop2-Mnd1 heterodimer physically interacts with Rad51 and Dmc1 recombinases and stimulates their activity up to 35-fold. Our data reveal an interplay among Hop2, Mnd1 and Rad51 and Dmc1 in the formation of the first recombination intermediates during meiosis.     

Exploring the multiple facets of the meiotic recombinase Dmc1

Meiotic recombination in eukaryotic cells requires two homologs of E. coli RecA protein, Rad51 and Dmc1. Until recently, the role of Dmc1 in meiotic recombination was mostly attributed to genetic studies as purified Dmc1 was found to be a much weaker recombinase than Rad51 in the test tube. Now, Sehorn and colleagues1 have reported that, like Rad51, human Dmc1 is an efficient recombinase in vitro. Dmc1 forms helical nucleoprotein filaments--the signature of classical recombinases such as Rad51. These observations reveal a high level of similitude between the Dmc1 and the Rad51 family of recombination enzymes in higher eukaryotes.     

Functional interactions between SPO11 and REC102 during initiation of meiotic recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, formation of the DNA double-strand breaks (DSBs) that initiate meiotic recombination requires the products of at least 10 genes. Spo11p is thought to be the catalytic subunit of the DNA cleaving activity, but the roles of the other proteins, and the interactions among them, are not well understood. This study demonstrates genetic and physical interactions between the products of SPO11 and another early meiotic gene required for DSB formation, REC102. We found that epitope-tagged versions of SPO11 and REC102 that by themselves were capable of supporting normal or nearly normal levels of meiotic recombination conferred a severe synthetic cold-sensitive phenotype when combined in the same cells. DSB formation, meiotic gene conversion, and spore viability were drastically reduced in the doubly tagged strain at a nonpermissive temperature. This conditional defect could be partially rescued by expression of untagged SPO11, but not by expression of untagged REC102, indicating that tagged REC102 is fully dominant for this synthetic phenotype. Both tagged and wild-type Spo11p co-immunoprecipitated with tagged Rec102p from meiotic cell extracts, indicating that these proteins are present in a common complex in vivo. Tagged Rec102p localized to the nucleus in whole cells and to chromatin on spread meiotic chromosomes. Our results are consistent with the idea that a multiprotein complex that includes Spo11p and Rec102p promotes meiotic DSB formation.     

Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination

During meiosis programmed DNA double-strand breaks (DSBs) are repaired by homologous recombination using the sister chromatid or the homologous chromosome (homolog) as a template. This repair results in crossover (CO) and non-crossover (NCO) recombinants. Only CO formation between homologs provides the physical linkages guiding correct chromosome segregation, which are essential to produce healthy gametes. The factors that determine the CO/NCO decision are still poorly understood. Using Schizosaccharomyces pombe as a model we show that the Rad51/Dmc1-paralog complexes Rad55-Rad57 and Rdl1-Rlp1-Sws1 together with Swi5-Sfr1 play a major role in antagonizing both the FANCM-family DNA helicase/translocase Fml1 and the RecQ-type DNA helicase Rqh1 to limit hybrid DNA formation and promote Mus81-Eme1-dependent COs. A common attribute of these protein complexes is an ability to stabilize the Rad51/Dmc1 nucleoprotein filament, and we propose that it is this property that imposes constraints on which enzymes gain access to the recombination intermediate, thereby controlling the manner in which it is processed and resolved.     

Yeast recombination factor Rdh54 functionally interacts with the Rad51 recombinase and catalyzes Rad51 removal from DNA

The Saccharomyces cerevisiae RDH54-encoded product, a member of the Swi2/Snf2 protein family, is needed for mitotic and meiotic interhomologue recombination and DNA repair. Previous biochemical studies employing Rdh54 purified from yeast cells have shown DNA-dependent ATP hydrolysis and DNA supercoiling by this protein, indicative of a DNA translocase function. Importantly, Rdh54 physically interacts with the Rad51 recombinase and promotes D-loop formation by the latter. Unfortunately, the low yield of Rdh54 from the yeast expression system has greatly hampered the progress on defining the functional interactions of this Swi2/Snf2-like factor with Rad51. Here we describe an E. coli expression system and purification scheme that together provide milligram quantities of nearly homogeneous Rdh54. Using this material, we demonstrate that Rdh54-mediated DNA supercoiling leads to transient DNA strand opening. Furthermore, at the expense of ATP hydrolysis, Rdh54 removes Rad51 from DNA. We furnish evidence that the Rad51 binding domain resides within the N terminus of Rdh54. Accordingly, N-terminal truncation mutants of Rdh54 that fail to bind Rad51 are also impaired for functional interactions with the latter. Interestingly, the rdh54 K352R mutation that ablates ATPase activity engenders a DNA repair defect even more severe than that seen in the rdh54Delta mutant. These results provide molecular information concerning the role of Rdh54 in homologous recombination and DNA repair, and they also demonstrate the functional significance of Rdh54.Rad51 complex formation. The Rdh54 expression and purification procedures described here should facilitate the functional dissection of this DNA recombination/repair factor.     

Mechanisms of distinctive mismatch tolerance between Rad51 and Dmc1 in homologous recombination

Homologous recombination (HR) is a primary DNA double-strand breaks (DSBs) repair mechanism. The recombinases Rad51 and Dmc1 are highly conserved in the RecA family; Rad51 is mainly responsible for DNA repair in somatic cells during mitosis while Dmc1 only works during meiosis in germ cells. This spatiotemporal difference is probably due to their distinctive mismatch tolerance during HR: Rad51 does not permit HR in the presence of mismatches, whereas Dmc1 can tolerate certain mismatches. Here, the cryo-EM structures of Rad51–DNA and Dmc1–DNA complexes revealed that the major conformational differences between these two proteins are located in their Loop2 regions, which contain invading single-stranded DNA (ssDNA) binding residues and double-stranded DNA (dsDNA) complementary strand binding residues, stabilizing ssDNA and dsDNA in presynaptic and postsynaptic complexes, respectively. By combining molecular dynamic simulation and single-molecule FRET assays, we identified that V273 and D274 in the Loop2 region of human RAD51 (hRAD51), corresponding to P274 and G275 of human DMC1 (hDMC1), are the key residues regulating mismatch tolerance during strand exchange in HR. This HR accuracy control mechanism provides mechanistic insights into the specific roles of Rad51 and Dmc1 in DNA double-strand break repair and may shed light on the regulatory mechanism of genetic recombination in mitosis and meiosis.     

Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny

The development of meiotic division and associated genetic recombination paved the way for evolutionary changes. However, the secondary and tertiary structure and functional domains of many of the proteins involved in genetic recombination have not been studied in detail. We used the human Dmc1 gene product along with secondary and tertiary domain structures of Escherichia coli RecA protein to help determine the molecular structure and function of maize Dmc1, which is required for synaptonemal complex formation and cell cycle progression. The maize recombinase Dmc1 gene was cloned and characterized, using rice Dmc1 cDNA as an orthologue. The deduced amino acid sequence was used for elaborating its 3-D structure, and functional analysis was made with the CDD software, showing significant identity of the Dmc1 gene product in Zea mays with that of Homo sapiens. Based on these results, the domains and motives of WalkerA and WalkerB as ATP binding sites, a multimer site (BRC) interface, the putative ssDNA binding L1 and L2 loops, the putative dsDNA binding helix-hairpin-helix, a polymerization motif, the subunit rotation motif, and a small N-terminal domain were proposed for maize recombinase Dmc1.     

Double duty for Exo1 during meiotic recombination

Comment on: Zakharyevich K, et al. Mol Cell 2010; 40:1001-15.     

The meiotic bouquet promotes homolog interactions and restricts ectopic recombination in Schizosaccharomyces pombe

Chromosome architecture undergoes extensive, programmed changes as cells enter meiosis. A highly conserved change is the clustering of telomeres at the nuclear periphery to form the "bouquet" configuration. In the fission yeast Schizosaccharomyces pombe the bouquet and associated nuclear movement facilitate initial interactions between homologs. We show that Bqt2, a meiosis-specific protein required for bouquet formation, is required for wild-type levels of homolog pairing and meiotic allelic recombination. Both gene conversion and crossing over are reduced and exhibit negative interference in bqt2Delta mutants, reflecting reduced homolog pairing. While both the bouquet and nuclear movement promote pairing, only the bouquet restricts ectopic recombination (that between dispersed repetitive DNA). We discuss mechanisms by which the bouquet may prevent deleterious translocations by restricting ectopic recombination.     

Characterization of the Roles of the Saccharomyces Cerevisiae Rad54 Gene and a Homologue of Rad54, Rdh54/Tid1, in Mitosis and Meiosis

The RAD54 gene, which encodes a protein in the SWI2/SNF2 family, plays an important role in recombination and DNA repair in Saccharomyces cerevisiae. The yeast genome project revealed a homologue of RAD54, RDH54/TID1. Properties of the rdh54/tid1 mutant and the rad54 rdh54/tid1 double mutant are shown for mitosis and meiosis. The rad54 mutant is sensitive to the alkylating agent, methyl methanesulfonate (MMS), and is defective in interchromosomal and intrachromosomal gene conversion. The rdh54/tid1 single mutant, on the other hand, does not show any significant deficiency in mitosis. However, the rad54 rdh54/tid1 mutant is more sensitive to MMS and more defective in interchromosomal gene conversion than is the rad54 mutant, but shows the same frequency of intrachromosomal gene conversion as the rad54 mutant. These results suggest that RDH54/TID1 is involved in a minor pathway of mitotic recombination in the absence of RAD54. In meiosis, both single mutants produce viable spores at slightly reduced frequency. However, only the rdh54/tid1 mutant, but not the rad54 mutant, shows significant defects in recombination: retardation of the repair of meiosis-specific double-strand breaks (DSBs) and delayed formation of physical recombinants. Furthermore, the rad54 rdh54/tid1 double mutant is completely defective in meiosis, accumulating DSBs with more recessed ends than the wild type and producing fewer physical recombinants than the wild type. These results suggest that one of the differences between the late stages of mitotic recombination and meiotic recombination might be specified by differential dependency on the Rad54 and Rdh54/Tid1 proteins.     

Multiple mechanisms of meiotic recombination

In this issue, reveal that different meiotic recombination mechanisms predominate in fission yeast and budding yeast. Budding yeast usually form crossover recombinants through double Holliday junctions, whereas fission yeast unexpectedly appear to form crossover recombinants through single junctions.     

Targeted stimulation of meiotic recombination

Meiotic recombination in Saccharomyces cerevisiae is initiated by programmed DNA double-strand breaks (DSBs), a process that requires the Spo11 protein. DSBs usually occur in intergenic regions that display open chromatin accessibility, but other determinants that control their frequencies and non-random chromosomal distribution remain obscure. We report that a Spo11 construct bearing the Gal4 DNA binding domain not only rescues spo11Delta spore inviability and catalyzes DSB formation at natural sites but also strongly stimulates DSB formation near Gal4 binding sites. At GAL2, a naturally DSB-cold locus, Gal4BD-Spo11 creates a recombinational hotspot that depends on all the other DSB gene functions, showing that the targeting of Spo11 to a specific site is sufficient to stimulate meiotic recombination that is under normal physiological control.     

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.