Structure of REC2, a recombinational repair gene of Ustilago maydis, and its function in homologous recombination between plasmid and chromosomal sequences.

Mutation in the REC2 gene of Ustilago maydis leads to defects in DNA repair, recombination, and meiosis. Analysis of the primary sequence of the Rec2 protein reveals a region with significant homology to bacterial RecA protein and to the yeast recombination proteins Dmc1, Rad51, and Rad57. This homologous region in the U. maydis Rec2 protein was found to be functionally sensitive to mutation, lending support to the hypothesis that Rec2 has a functional RecA-like domain essential for activity in recombination and repair. Homologous recombination between plasmid and chromosomal DNA sequences is reduced substantially in the rec2 mutant following transformation. The frequency can be restored to a level approaching, but not exceeding, that observed in the wild-type strain if transformation is performed with cells containing multiple copies of REC2.     

Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Rad1–Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1–Rad10 endonuclease cleaves 3′ branches of DNA and aberrant 3′ DNA ends that are refractory to other 3′ processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1–Rad10 complex in DSB repair in yeast.     

Functions of the Snf2/Swi2 family Rad54 motor protein in homologous recombination

Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair of DNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54 is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicase activity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potent and processive motor protein that translocates on dsDNA, potentially executing several functions in recombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination that performs the key reactions of homology search and DNA strand invasion. Here, we will review the role of the Rad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast and human enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologous recombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.     

An essential role of DmRad51/SpnA in DNA repair and meiotic checkpoint control

Rad51 is a conserved protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells and during meiosis in germ cells. Yeast Rad51 mutants are viable but show meiosis defects. In the mouse, RAD51 deletions cause early embryonic death, suggesting that in higher eukaryotes Rad51 is required for viability. Here we report the identification of SpnA as the Drosophila Rad51 gene, whose sequence among the five known Drosophila Rad51-like genes is most closely related to the Rad51 homologs of human and yeast. DmRad51/spnA null mutants are viable but oogenesis is disrupted by the activation of a meiotic recombination checkpoint. We show that the meiotic phenotypes result from an inability to effectively repair DSBs. Our study further demonstrates that in Drosophila the Rad51-dependent homologous recombination pathway is not essential for DNA repair in the soma, unless exposed to DNA damaging agents. We therefore propose that under normal conditions a second, Rad51-independent, repair pathway prevents the lethal effects of DNA damage.     

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.     

RAD51B plays an essential role during somatic and meiotic recombination in Physcomitrella

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.     

Identification and characterization of the rlp1+, the novel Rad51 paralog in the fission yeast Schizosaccharomyces pombe

A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1Delta mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1Delta mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1Delta mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse.     

RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

Rad51 is a key protein in homologous recombination performing homology search and DNA strand invasion. After DNA strand exchange Rad51 protein is stuck on the double-stranded heteroduplex DNA product of DNA strand invasion. This is a problem, because DNA polymerase requires access to the invading 3′-OH end to initiate DNA synthesis. Here we show that, the Saccharomyces cerevisiae dsDNA motor protein Rad54 solves this problem by dissociating yeast Rad51 protein bound to the heteroduplex DNA after DNA strand invasion. The reaction required species-specific interaction between both proteins and the ATPase activity of Rad54 protein. This mechanism rationalizes the in vivo requirement of Rad54 protein for the turnover of Rad51 foci and explains the observed dependence of the transition from homologous pairing to DNA synthesis on Rad54 protein in vegetative and meiotic yeast cells.     

Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint

Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made null mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non-homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal.     

Three-dimensional microscopy of the Rad51 recombination protein during meiotic prophase.

An open question in meiosis is whether the Rad51 recombination protein functions solely in meiotic recombination or whether it is also involved in the chromosome homology search. To address this question, we have performed three-dimensional high-resolution immunofluorescence microscopy to visualize native Rad51 structures in maize male meiocytes. Maize has two closely related RAD51 genes that are expressed at low levels in differentiated tissues and at higher levels in mitotic and meiotic tissues. Cells and nuclei were specially fixed and embedded in polyacrylamide to maintain both native chromosome structure and the three dimensionality of the specimens. Analysis of Rad51 in maize meiocytes revealed that when chromosomes condense during leptotene, Rad51 is diffuse within the nucleus. Rad51 foci form on the chromosomes at the beginning of zygotene and rise to approximately 500 per nucleus by mid-zygotene when chromosomes are pairing and synapsing. During chromosome pairing, we consistently found two contiguous Rad51 foci on paired chromosomes. These paired foci may identify the sites where DNA sequence homology is being compared. During pachytene, the number of Rad51 foci drops to seven to 22 per nucleus. This higher number corresponds approximately to the number of chiasmata in maize meiosis. These observations are consistent with a role for Rad51 in the homology search phase of chromosome pairing in addition to its known role in meiotic recombination.     

DNA repair by a Rad22-Mus81-dependent pathway that is independent of Rhp51

In budding yeast most Rad51-dependent and -independent recombination depends on Rad52. In contrast, its homologue in fission yeast, Rad22, was assumed to play a less critical role possibly due to functional redundancy with another Rad52-like protein Rti1. We show here that this is not the case. Rad22 like Rad52 plays a central role in recombination being required for both Rhp51-dependent and -independent events. Having established this we proceed to investigate the involvement of the Mus81–Eme1 endonuclease in these pathways. Mus81 plays a relatively minor role in the Rhp51-dependent repair of DNA damage induced by ultraviolet light. In contrast Mus81 has a key role in the Rad22-dependent (Rhp51-independent) repair of damage induced by camptothecin, hydroxyurea and methyl-methanesulfonate. Furthermore, spontaneous intrachromosomal recombination that gives rise to deletion recombinants is impaired in a mus81 mutant. From these data we propose that a Rad22–Mus81-dependent (Rhp51-independent) pathway is an important mechanism for the repair of DNA damage in fission yeast. Consistent with this we show that in vitro Rad22 can promote strand invasion to form a D-loop that can be cleaved by Mus81.     

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.     

Functional analysis of the Drosophila Rad51 gene (spn-A) in repair of DNA damage and meiotic chromosome segregation

Rad51 is a crucial enzyme in DNA repair, mediating the strand invasion and strand exchange steps of homologous recombination (HR). Mutations in the Drosophila Rad51 gene (spn-A) disrupt somatic as well as meiotic double-strand break (DSB) repair, similar to fungal Rad51 genes. However, the sterility of spn-A mutant females prevented a thorough analysis of the role of Rad51 in meiosis. In this study, we generated transgenic animals that express spn-A dsRNA under control of an inducible promoter, and examined the effects of inhibiting expression of spn-A on DNA repair, meiotic recombination and meiotic chromosome pairing and segregation. We found that depletion of spn-A mRNA had no effect on the viability of non-mutagen-treated transgenic animals but greatly reduced the survival of larvae that were exposed to the radiomimetic drug MMS, in agreement with the MMS and X-ray sensitivity of spn-A mutant animals. We also found that increases in dose of spn-A gene enhanced larval resistance to MMS exposure, suggesting that at high damage levels, Rad51 protein levels may be limiting for DNA repair. spn-A RNAi strongly stimulated X-X nondisjunction and decreased recombination along the X in female meiosis, consistent with a requirement of Rad51 in meiotic recombination. However, neither RNAi directed against the spn-A mRNA nor homozygosity for a spn-A null mutation had any effect on male fertility or on X-Y segregation in male meiosis, indicating that Rad51 likely plays no role in male meiotic chromosome pairing. Our results support a central role for Rad51 in HR in both somatic and meiotic DSB repair, but indicate that Rad51 in Drosophila is dispensable for meiotic chromosome pairing. Our results also provide the first demonstration that RNAi can be used to inhibit the functions of meiotic genes in Drosophila.     

Identification and characterization of rad51 of Pneumocystis

Rad51, a eukaryotic homolog of RecA, is an important protein involved in DNA recombination and repair. We have characterized rad51 of Pneumocystis carinii and Pneumocystis murina. rad51 is a single copy gene that encodes a 1.2 kb mRNA, which contains an open reading frame encoding 343 amino acids. Rad51 from Pneumocystis showed high homology to those from yeast. ATP binding motifs GEFRTGKS and LLIVD, similar to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, are conserved in Pneumocystis Rad51. The recombinant protein when expressed in E. coli showed DNA-dependent ATPase activity. Since Rad51 is a key enzyme in DNA repair and recombination, it potentially plays an important role in the recombination process leading to antigenic variation and thereby resistance to host immune responses in Pneumocystis.     

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.     

A Conserved Sequence Extending Motif III of the Motor Domain in the Snf2-Family DNA Translocase Rad54 Is Critical for ATPase Activity

Rad54 is a dsDNA-dependent ATPase that translocates on duplex DNA. Its ATPase function is essential for homologous recombination, a pathway critical for meiotic chromosome segregation, repair of complex DNA damage, and recovery of stalled or broken replication forks. In recombination, Rad54 cooperates with Rad51 protein and is required to dissociate Rad51 from heteroduplex DNA to allow access by DNA polymerases for recombination-associated DNA synthesis. Sequence analysis revealed that Rad54 contains a perfect match to the consensus PIP box sequence, a widely spread PCNA interaction motif. Indeed, Rad54 interacts directly with PCNA, but this interaction is not mediated by the Rad54 PIP box-like sequence. This sequence is located as an extension of motif III of the Rad54 motor domain and is essential for full Rad54 ATPase activity. Mutations in this motif render Rad54 non-functional in vivo and severely compromise its activities in vitro. Further analysis demonstrated that such mutations affect dsDNA binding, consistent with the location of this sequence motif on the surface of the cleft formed by two RecA-like domains, which likely forms the dsDNA binding site of Rad54. Our study identified a novel sequence motif critical for Rad54 function and showed that even perfect matches to the PIP box consensus may not necessarily identify PCNA interaction sites.     

The Arabidopsis homologue of Xrcc3 plays an essential role in meiosis

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified from yeast (Rad55, Rad57 and Dmc1) to vertebrates (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3 and Dmc1). These Rad51-like proteins are all members of the genetic recombination and DNA damage repair pathways. The sequenced genome of Arabidopsis thaliana encodes putative homologues of all six vertebrate Rad51-like proteins. We have identified and characterized an Arabidopsis mutant defective for one of these, AtXRCC3, the homologue of XRCC3. atxrcc3 plants are sterile, while they have normal vegetative development. Cytological observation shows that the atxrcc3 mutation does not affect homologous chromosome synapsis, but leads to chromosome fragmentation after pachytene, thus disrupting both male and female gametogenesis. This study shows an essential role for AtXrcc3 in meiosis in plants and possibly in other higher eukaryotes. Furthermore, atxrcc3 cells and plants are hypersensitive to DNA-damaging treatments, supporting the involvement of this Arabidopsis Rad51-like protein in recombinational repair.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.