Cerevisiae Rad51 Research Articles

Rad51 determines pathway usage in post-replication repair.

Stalled replication forks can be processed by several distinct mechanisms collectively called post-replication repair which includes homologous recombination, fork regression, and translesion DNA synthesis. However, the regulation of the usage between these pathways is not fully understood. The Rad51 protein plays a pivotal role in maintaining genomic stability through its roles in HR and in protecting stalled replication forks from degradation. We report the isolation of separation-of-function mutations in Saccharomyces cerevisiae Rad51 that retain their recombination function but display a defect in fork protection leading to a shift in post-replication repair pathway usage from HR to alternate pathways including mutagenic translesion synthesis. Rad51-E135D and Rad51-K305N show normal in vivo and in vitro recombination despite changes in their DNA binding profiles, in particular to dsDNA, with a resulting effect on their ATPase activities. The mutants lead to a defect in Rad51 recruitment to stalled forks in vivo as well as a defect in the protection of dsDNA from degradation by Dna2-Sgs1 and Exo1 in vitro . A high-resolution cryo-electron microscopy structure of the Rad51-ssDNA filament at 2.4 Å resolution provides a structural basis for a mechanistic understanding of the mutant phenotypes. Together, the evidence suggests a model in which Rad51 binding to duplex DNA is critical to control pathway usage at stalled replication forks.

Characterization of the RAD52 Gene in the Budding Yeast Naumovozyma castellii.

Several sources of DNA damage compromise the integrity and stability of the genome of every organism. Specifically, DNA double-strand breaks (DSBs) can have lethal consequences for the cell. To repair this type of DNA damage, the cells employ homology-directed repair pathways or non-homologous end joining. Homology-directed repair requires the activity of the RAD52 epistasis group of genes. Rad52 is the main recombination protein in the budding yeast Saccharomyces cerevisiae, and rad52Δ mutants have been characterized to show severe defects in DSB repair and other recombination events. Here, we identified the RAD52 gene in the budding yeast Naumovozyma castellii. Our analysis showed that the primary amino acid sequence of N. castellii Rad52 shared 70% similarity with S. cerevisiae Rad52. To characterize the gene function, we developed rad52Δ mutant strains by targeted gene replacement transformation. We found that N. castellii rad52Δ mutants showed lowered growth capacity, a moderately altered cell morphology and increased sensitivity to genotoxic agents. The decreased viability of the N. castellii rad52Δ mutants in the presence of genotoxic agents indicates that the role of the Rad52 protein in the repair of DNA damage is conserved in this species.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

The Role of the Rad55-Rad57 Complex in DNA Repair.

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55–Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55–Rad57 in HR.

The Rad51 paralog complex Rad55-Rad57 acts as a molecular chaperone during homologous recombination

Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.

Abstract 2394: Rad52 residues associated with colonic adenocarcinoma dictate its cooperative assembly on ssDNA during homologous recombination

Abstract DNA repair is essential for maintaining genomic integrity. Homologous recombination (HR) repairs double- stranded DNA breaks (DSBs) and defects lead to the accumulation of mutations, which results in genomic instability and a plethora of breast, ovarian, colonic and skin cancers. HR is initiated when a DSB is recognized and resected by exonucleases to yield single-stranded DNA (ssDNA), which is then coated and protected by replication protein A (RPA). Next, Rad51 recombinases access the ssDNA preoccupied by RPA. The Rad51 filament is then able to undergo strand-invasion and drive the subsequent stages of HR. The four DNA Binding domains in RPA (DBDs A, B, C and D) enable very tight RPA-ssDNA binding. Therefore, exchange of RPA for Rad51 is assisted by a group of proteins called mediators during this pre-synaptic phase of HR. RAD52 is one such pro-recombinogenic mediator and functions to promote RPA-Rad51 exchange. However, the mechanistic details underlying how Rad52 mediates RPA-Rad51 exchange on ssDNA are not fully resolved. Mutations in mediator proteins such as RAD52 and BRCA2 are prevalent in many cancers. For example, the COSMIC database shows there have been 139 sampled cases of mutations of Rad52 in solid tumors. To understand how Rad52 fulfills its mediator functions, it is important to first understand how Rad52 associates with DNA, and how this affects RPA-Rad51 exchange on ssDNA. To this extent, we have systematically characterized the DNA binding properties of Saccharomyces cerevisiae Rad52 and investigated the effects of Rad52 on RPA-DNA binding in different DNA contexts. Our results show that. Rad52 exists as a heptamer and binds to ssDNA with a Kd of 40.2 ± 3.3 nM which is similar to RPA binding to ssDNA with a Kd of 29.2 ± 4 nM. Using Protein Induced Fluorescent Enhancement (PIFE), we show that Rad52 binds cooperatively towards the 5′ end of DNA. This cooperativity arises from DNA binding to two distinct sites in Rad52: outer- and inner-binding site. In humans, mutations in the amino acids located in the inner binding site has been found in adenocarcinoma. We have perturbed and characterized a specific inner site mutation, R37A, that is associated with multiple cases of colonic adenocarcinoma, as well as other amino acids in the inner and outer binding sites. This has allowed us to develop a model of dynamic ssDNA-Rad52 binding. Finally, we show that this cooperativity in Rad52 selectively affects the DNA binding of RPA-DBD-D on a 5′ overhang DNA substrate. A detailed mechanistic insight into Rad52-ssDNA binding will aid in a better understanding of HR, which can contribute to the development of small-molecule inhibitor drugs that can be used as effective chemotherapeutic agents against cancers in the future. Citation Format: Emma A. Tillison, Nilisha Pokhrel, Jaigeeth Deveryshetty, Edwin Antony. Rad52 residues associated with colonic adenocarcinoma dictate its cooperative assembly on ssDNA during homologous recombination [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2394.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.

Saccharomyces cerevisiae Mus81-Mms4 and Rad52 can cooperate in the resolution of recombination intermediates.

Mus81 is a well-conserved DNA structure-specific endonuclease which belongs to the XPF/Rad1 family of proteins that are involved in DNA nucleotide excision repair. Mus81 forms a heterodimer with a non-catalytic subunit, Mms4, in Saccharomyces cerevisiae (Eme1/EME1 in Schizosaccharomyces pombe and mammals). Recent evidence shows that Mus81 functions redundantly with Sgs1, a member of the ubiquitous RecQ family of DNA helicases, to process toxic recombinant intermediates. In budding yeast, homologous recombination is regulated by the Rad52 epistasis group of proteins, including Rad52, which stimulates the main steps of DNA sequence-homology searching. Mus81 was proven to act in the Rad52-dependent pathway. Here, we demonstrate that Rad52 and Mus81-Mms4 possesses a functional interaction; the presence of Rad52 significantly enhances the endonuclease activity of Mus81-Mms4 on a broad range of its preferred synthetic substrates. Furthermore, this functional interaction is demonstrated to be species specific. We fragmented Rad52 and found that the N-terminal fragment from the 86th to 169th amino acid residue, which belongs to DNA-binding and self-association domains, can stimulate Mus81-Mms4 endonuclease. These results strongly support the notion that Rad52 and Mus81-Mms4 collaborate and work jointly in processing of homologous recombination intermediates.

Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52.

Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays. Moreover, the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different (plasmid, PCR and ssDNA) donor templates were confirmed. We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7. In addition, we also applied the fusion strategy for modifying the IGF2 gene of porcine PK15 cells, which further demonstrated a 2.2-fold increase in HDR frequency. In conclusion, our data suggests that Rad52-Cas9 fusion is a good option for enhancing CRISPR/Cas9-mediated HDR, which may be of use in future studies involving precise genome editing.

Promotion of presynaptic filament assembly by the ensemble of S. cerevisiae Rad51 paralogues with Rad52

The conserved budding yeast Rad51 paralogues, including Rad55, Rad57, Csm2 and Psy3 are indispensable for homologous recombination (HR)-mediated chromosome damage repair. Rad55 and Rad57 are associated in a heterodimer, while Csm2 and Psy3 form the Shu complex with Shu1 and Shu2. Here we show that Rad55 bridges an interaction between Csm2 with Rad51 and Rad52 and, using a fully reconstituted system, demonstrate that the Shu complex synergizes with Rad55–Rad57 and Rad52 to promote nucleation of Rad51 on single-stranded DNA pre-occupied by replication protein A (RPA). The csm2–F46A allele is unable to interact with Rad55, ablating the ability of the Shu complex to enhance Rad51 presynaptic filament assembly in vitro and impairing HR in vivo. Our results reveal that Rad55–Rad57, the Shu complex and Rad52 act as a functional ensemble to promote Rad51-filament assembly, which has important implications for understanding the role of the human RAD51 paralogues in Fanconi anaemia and cancer predisposition.

Protein dynamics during presynaptic-complex assembly on individual single-stranded DNA molecules.

Homologous recombination is a conserved pathway for repairing double–stranded breaks, which are processed to yield single–stranded DNA overhangs that serve as platforms for presynaptic complex assembly. Here we use single–molecule imaging to reveal the interplay between Saccharomyce cerevisiae RPA, Rad52, and Rad51 during presynaptic complex assembly. We show that Rad52 binds RPA–ssDNA and suppresses RPA turnover, highlighting an unanticipated regulatory influence on protein dynamics. Rad51 binding extends the ssDNA, and Rad52–RPA clusters remain interspersed along the presynaptic complex. These clusters promote additional binding of RPA and Rad52. Together, our work illustrates the spatial and temporal progression of RPA and Rad52 association with the presynaptic complex, and reveals a novel RPA–Rad52–Rad51–ssDNA intermediate, which has implications for understanding how the activities of Rad52 and RPA are coordinated with Rad51 during the later stages recombination.

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.

An elegans Solution for Crossover Formation

An elegans Solution for Crossover Formation

Characterization of the Interaction between the Saccharomyces cerevisiae Rad51 Recombinase and the DNA Translocase Rdh54

The Saccharomyces cerevisiae Rdh54 protein is a member of the Swi2/Snf2 family of DNA translocases required for meiotic and mitotic recombination and DNA repair. Rdh54 interacts with the general recombinases Rad51 and Dmc1 and promotes D-loop formation with either recombinase. Rdh54 also mediates the removal of Rad51 from undamaged chromatin in mitotic cells, which prevents formation of nonrecombinogenic complexes that can otherwise become toxic for cell growth. To determine which of the mitotic roles of Rdh54 are dependent on Rad51 complex formation, we finely mapped the Rad51 interaction domain in Rdh54, generated N-terminal truncation variants, and characterized their attributes biochemically and in cells. Here, we provide evidence suggesting that the N-terminal region of Rdh54 is not necessary for the response to the DNA-damaging agent methyl methanesulfonate. However, truncation variants missing 75-200 residues at the N terminus are sensitive to Rad51 overexpression. Interestingly, a hybrid protein containing the N-terminal region of Rad54, responsible for Rad51 interaction, fused to the Swi2/Snf2 core of Rdh54 is able to effectively complement the sensitivity to both methyl methanesulfonate and excess Rad51 in rdh54 null cells. Altogether, these results reveal a distinction between damage sensitivity and Rad51 removal with regard to Rdh54 interaction with Rad51.

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.

The Intra-S Phase Checkpoint Targets Dna2 to Prevent Stalled Replication Forks from Reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ∼10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.

Rad51 paralogues Rad55–Rad57 balance the antirecombinase Srs2 in Rad51 filament formation

Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements, and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyzes the signature reactions of recombination, homology search and DNA strand invasion 1,2. Eukaryotes also possess Rad51 paralogs, whose exact role in recombination remains to be defined 3. Here we show that the budding yeast Rad51 paralogs, the Rad55-Rad57 heterodimer, counteract the anti-recombination activity of the Srs2 helicase. Rad55-Rad57 associate with the Rad51-ssDNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51/Rad55-Rad57 co-filament resists disruption by the Srs2 anti-recombinase by blocking Srs2 translocation involving a direct protein interaction between Rad55-Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogs in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 anti-recombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55-Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogs, which are known to be involved in cancer predisposition and human disease.

Enhancement of gene targeting in human cells by intranuclear permeation of the Saccharomyces cerevisiae Rad52 protein

The introduction of exogenous DNA in human somatic cells results in a frequency of random integration at least 100-fold higher than gene targeting (GT), posing a seemingly insurmountable limitation for gene therapy applications. We previously reported that, in human cells, the stable over-expression of the Saccharomyces cerevisiae Rad52 gene (yRAD52), which plays the major role in yeast homologous recombination (HR), caused an up to 37-fold increase in the frequency of GT, indicating that yRAD52 interacts with the double-strand break repair pathway(s) of human cells favoring homologous integration. In the present study, we tested the effect of the yRad52 protein by delivering it directly to the human cells. To this purpose, we fused the yRAD52 cDNA to the arginine-rich domain of the TAT protein of HIV (tat11) that is known to permeate the cell membranes. We observed that a recombinant yRad52tat11 fusion protein produced in Escherichia coli, which maintains its ability to bind single-stranded DNA (ssDNA), enters the cells and the nuclei, where it is able to increase both intrachromosomal recombination and GT up to 63- and 50-fold, respectively. Moreover, the non-homologous plasmid DNA integration decreased by 4-fold. yRAD52tat11 proteins carrying point mutations in the ssDNA binding domain caused a lower or nil increase in recombination proficiency. Thus, the yRad52tat11 could be instrumental to increase GT in human cells and a ‘protein delivery approach’ offers a new tool for developing novel strategies for genome modification and gene therapy applications.

2P127 Real-time observation of DNA Loop formation by Saccharomyces cerevisiae Rad54(The 48th Annual Meeting of the Biophysical Society of Japan)

2P127 Real-time observation of DNA Loop formation by Saccharomyces cerevisiae Rad54(The 48th Annual Meeting of the Biophysical Society of Japan)

Regulation of Meiotic Recombination via Mek1-Mediated Rad54 Phosphorylation

A preference for homologs over sister chromatids in homologous recombination is a fundamental difference in meiotic versus mitotic cells. In budding yeast, the bias for interhomolog recombination in meiosis requires the Dmc1 recombinase and the meiosis-specific kinase Mek1, which suppresses engagement of sister chromatids by the mitotic recombinase Rad51. Here, a combination of proteomic, biochemical, and genetic approaches has identified an additional role for Mek1 in inhibiting the activity of the Rad51 recombinase through phosphorylation of its binding partner, Rad54. Rad54 phosphorylation of threonine 132 attenuates complex formation with Rad51, and a negative charge at this position reduces Rad51 function in vitro and in vivo. Thus, Mek1 phosphorylation provides a dynamic means of controlling recombination partner choice in meiosis in two ways: (1) it reduces Rad51 activity through inhibition of Rad51/Rad54 complex formation, and (2) it suppresses Rad51-mediated strand invasion of sister chromatids via a Rad54-independent mechanism.