The <Emphasis Type="Italic">dds20</Emphasis><Superscript>+</Superscript> Gene Controls a Novel Rad51<Superscript>Sp</Superscript>-Dependent Pathway of Recombinational Repair in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

Repair of DNA double-strand break (DSB) is an evolutionary conserved Rad51-mediated mechanism. In yeasts, Rad51 paralogs, Saccharomyces cerevisiae Rad55-Rad57 and Schizosaccharomyces pombe Rhp55-Rhp57 are mediators of the nucleoprotein Rad51 filament formation. As shown in this work, a novel Rad51^Sp-dependent pathway of DSB repair acts in S. pombe parallel to the pathway mediated by Rad51 paralogs. A new gene dds20 ⁺ that controls this pathway was identified. The overexpression of dds20 ⁺ partially suppresses defects of mutant rhp55Δ in DNA repair. Cells of dds20Δ manifest hypersensitivity to a variety of genotoxins. Epistatic analysis revealed that dds20 ⁺ is a gene of the recombinational repair group. The role of Dds20 in repair of spontaneous damages occurring in the process of replication and mating-type switching remains unclear. The results obtained suggest that Dds20 has functions beyond the mitotic S phase. The Dds20 protein physically interacts with Rhp51(Rad51^Sp). Dds20 is assumed to operate at early recombinational stages and to play a specific role in the Rad51 protein filament assembly differing from that of Rad51 paralogs.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 736–745.Original Russian Text Copyright © 2005 by Salakhova, Savchenko, Khasanov, Chepurnaya, Korolev, Bashkirov.     

Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe

Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved.     

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.     

Role of the Saccharomyces cerevisiae Rad51 paralogs in sister chromatid recombination

Rad51 requires a number of other proteins, including the Rad51 paralogs, for efficient recombination in vivo. Current evidence suggests that the yeast Rad51 paralogs, Rad55 and Rad57, are important in formation or stabilization of the Rad51 nucleoprotein filament. To gain further insights into the function of the Rad51 paralogs, reporters were designed to measure spontaneous or double-strand break (DSB)-induced sister or nonsister recombination. Spontaneous sister chromatid recombination (SCR) was reduced 6000-fold in the rad57 mutant, significantly more than in the rad51 mutant. Although the DSB-induced recombination defect of rad57 was suppressed by overexpression of Rad51, elevated temperature, or expression of both mating-type alleles, the rad57 defect in spontaneous SCR was not strongly suppressed by these same factors. In addition, the UV sensitivity of the rad57 mutant was not strongly suppressed by MAT heterozygosity, even though Rad51 foci were restored under these conditions. This lack of suppression suggests that Rad55 and Rad57 have different roles in the recombinational repair of stalled replication forks compared with DSB repair. Furthermore, these data suggest that most spontaneous SCR initiates from single-stranded gaps formed at stalled replication forks rather than DSBs.     

Cell phenotypes of a mutant in the gene encoding a Rad51 paralog in fission yeast

The discovery of three Rad51 paralogs in Saccharomyces cerevisiae (Rad55, Rad57, and Dmc1), four in Schizosaccharomyces pombe (Rhp55, Rhp57, Rlp 1, and Dmc 1), and six in human (Rad51 B, Rad51 C, Rad51 D, Xrcc2, Xrcc3, and Dmcl) indicate the functional diversity and specialization of RecA-like proteins in the line from the lower to higher organisms. This paper reports characterization of a number of mitotic and meiotic phenotypes of the cells mutant in rlpl gene, encoding a paralog of Rad5 1, in fission yeasts. No evident role of Rlp I protein in the repair of spontaneous lesions emerging during mating type switching was found. Rlpl does not interact physically with Dmcl. An elevated expression of rhp51 has a dominant negative effect on the cell survivability of rlpl mutant exposed to a DNA-damaging agent. We assume that Rlp 1 acts at the stages of recombination connected with disassembling of the nucleoprotein filament formed by Rhp51 protein.     

A postsynaptic role for Rhp55/57 that is responsible for cell death in Deltarqh1 mutants following replication arrest in Schizosaccharomyces pombe

Following replication arrest, multiple cellular responses are triggered to maintain genomic integrity. In fission yeast, the RecQ helicase, Rqh1, plays a critical role in this process. This is demonstrated in Deltarqh1 cells that, following treatment with hydroxyurea (HU), undergo an aberrant mitosis leading to cell death. Previous data suggest that Rqh1 functions with homologous recombination (HR) in recovery from replication arrest. We have found that loss of the HR genes rhp55(+) or rhp57(+), but not rhp51(+) or rhp54(+), suppresses the HU sensitivity of Deltarqh1 cells. Much of this suppression requires Rhp51 and Rhp54. In addition, this suppression is partially dependent on swi5(+). In budding yeast, overexpressing Rad51 (the Rhp51 homolog) minimized the need for Rad55/57 (Rhp55/57) in nucleoprotein filament formation. We overexpressed Rhp51 in Schizosaccharomyces pombe and found that it greatly reduced the requirement for Rhp55/57 in recovery from DNA damage. However, overexpressing Rhp51 did not change the Deltarhp55 suppression of the HU sensitivity of Deltarqh1, supporting an Rhp55/57 function during HR independent of nucleoprotein filament formation. These results are consistent with Rqh1 playing a role late in HR following replication arrest and provide evidence for a postsynaptic function for Rhp55/57.     

The F-Box DNA helicase Fbh1 prevents Rhp51-dependent recombination without mediator proteins

A key step in homologous recombination is the loading of Rad51 onto single-stranded DNA to form a nucleoprotein filament that promotes homologous DNA pairing and strand exchange. Mediator proteins, such as Rad52 and Rad55-Rad57, are thought to aid filament assembly by overcoming an inhibitory effect of the single-stranded-DNA-binding protein replication protein A. Here we show that mediator proteins are also required to enable fission yeast Rad51 (called Rhp51) to function in the presence of the F-box DNA helicase Fbh1. In particular, we show that the critical function of Rad22 (an orthologue of Rad52) in promoting Rhp51-dependent recombination and DNA repair can be mostly circumvented by deleting fbh1. Similarly, the reduced growth/viability and DNA damage sensitivity of an fbh1(-) mutant are variously suppressed by deletion of any one of the mediators Rad22, Rhp55, and Swi5. From these data we propose that Rhp51 action is controlled through an interplay between Fbh1 and the mediator proteins. Colocalization of Fbh1 with Rhp51 damage-induced foci suggests that this interplay occurs at the sites of nucleoprotein filament assembly. Furthermore, analysis of different fbh1 mutant alleles suggests that both the F-box and helicase activities of Fbh1 contribute to controlling Rhp51.     

Physical interaction between recombinational proteins Rhp51 and Rad22 in Schizosaccharomyces pombe

In eukaryotes, Rad51 and Rad52 are two key components of homologous recombination and recombinational repair. These two proteins interact with each other. Here we investigated the role of interaction between Rhp51 and Rad22, the fission yeast homologs of Rad51 and Rad52, respectively, on the function of each protein. We identified a direct association between the two proteins and their self-interactions both in vivo and in vitro. We also determined the binding domains of each protein that mediate these interactions. To characterize the role of Rhp51-Rad22 interaction, we used random mutagenesis to identify the mutants Rhp51 and Rad22, which cannot interact each other. Interestingly, we found that mutant Rhp51 protein, which cannot interact with either Rad22 or Rti1 (G282D), lost its DNA repair ability. In contrast, mutant Rad22 proteins, which cannot specifically bind to Rhp51 (S379L and P381L), maintained their DNA repair ability. These results suggest that the interaction between Rhp51 and Rad22 is crucial for the recombinational repair function of Rhp51. However, the significance of this interaction on the function of Rad22 remains to be characterized further.     

Phosphorylation of Rad55 on serines 2, 8, and 14 is required for efficient homologous recombination in the recovery of stalled replication forks

DNA damage checkpoints coordinate the cellular response to genotoxic stress and arrest the cell cycle in response to DNA damage and replication fork stalling. Homologous recombination is a ubiquitous pathway for the repair of DNA double-stranded breaks and other checkpoint-inducing lesions. Moreover, homologous recombination is involved in postreplicative tolerance of DNA damage and the recovery of DNA replication after replication fork stalling. Here, we show that the phosphorylation on serines 2, 8, and 14 (S2,8,14) of the Rad55 protein is specifically required for survival as well as for normal growth under genome-wide genotoxic stress. Rad55 is a Rad51 paralog in Saccharomyces cerevisiae and functions in the assembly of the Rad51 filament, a central intermediate in recombinational DNA repair. Phosphorylation-defective rad55-S2,8,14A mutants display a very slow traversal of S phase under DNA-damaging conditions, which is likely due to the slower recovery of stalled replication forks or the slower repair of replication-associated DNA damage. These results suggest that Rad55-S2,8,14 phosphorylation activates recombinational repair, allowing for faster recovery after genotoxic stress.     

Cell phenotypes of a mutant in the gene encoding a Rad51 paralog in fission yeast

The discovery of three Rad51 paralogs in Saccharomyces cerevisiae (Rad55, Rad57, and Dmc1), four in Schizosaccharomyces pombe (Rhp55, Rhp57, Rlp1, and Dmc1), and six in human (Rad51B, Rad51C, Rad51D, Xrcc2, Xrcc3, and Dmc1) indicate the functional diversity and specialization of RecA-like proteins in the line from the lower to higher organisms. This paper reports characterization of a number of mitotic and meiotic phenotypes of the cells mutant in rlp1 gene, encoding a paralog of Rad51, in fission yeasts. No evident role of Rlp1 protein in the repair of spontaneous lesions emerging during mating type switching was found. Rlp1 does not interact physically with Dmc1. An elevated expression of rhp51 has a dominant negative effect on the cell survivability of rlp1Δ mutant exposed to a DNA-damaging agent. We assume that Rlp1 acts at the stages of recombination connected with disassembling of the nucleoprotein filament formed by Rhp51 protein.     

Fission yeast Swi5 protein, a novel DNA recombination mediator

The Schizosaccharomyces pombe Swi5 protein forms two distinct protein complexes, Swi5-Sfr1 and Swi5-Swi2, each of which plays an important role in the related but functionally distinct processes of homologous recombination and mating-type switching, respectively. The Swi5-Sfr1 mediator complex has been shown to associate with the two RecA-like recombinases, Rhp51 (spRad51) and Dmc1, and to stimulate in vitro DNA strand exchange reactions mediated by these proteins. Genetic analysis indicates that Swi5-Sfr1 works independently of another mediator complex, Rhp55-Rhp57, during Rhp51-dependent recombinational repair. In addition, mutations affecting the two mediators generate distinct repair spectra of HO endonuclease-induced DNA double strand breaks, suggesting that these recombination mediators differently regulate recombination outcomes in an independent manner.     

Dds20 operates in cds1-independent mechanism of tolerance to UV-induced DNA damage in Schizosaccharomyces pombe cells

Repair of DNA double-stranded breaks caused by ionizing radiation or cellular metabolization, homologous recombination, is an evolutionary conserved process controlled by RAD52 group genes. Genes of recombinational repair also play a leading role in the response to DNA damage caused by UV light. Cells with deletion in gene dds20 of recombinational repair were shown to manifest hypersensitivity to the action of UV light at lowered incubation temperature. Epistatic analysis revealed that dds20+ is not a member of the NER and UVER gene groups responsible for the repair of DNA damage induced by UV light. The Dds protein has functions in the Cds1-independent mechanism of UV damage tolerance of DNA.     

New insights into the mechanism of homologous recombination in yeast

Genome stability is of primary importance for the survival and proper functioning of all organisms. Double-strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. Repair of DSBs by homologous recombination provides an efficient and fruitful pathway to restore chromosomal integrity. Exciting new work in yeast has lately provided insights into this complex process. Many of the proteins involved in recombination have been isolated and the details of the repair mechanism are now being unraveled at the molecular level. In this review, we focus on recent studies which dissect the recombinational repair of a single broken chromosome. After DSB formation, a decision is made regarding the mechanism of repair (recombination or non-homologous end-joining). This decision is under genetic control. Once committed to the recombination pathway, the broken chromosomal ends are resected by a still unclear mechanism in which the DNA damage checkpoint protein Rad24 participates. At this stage several proteins are recruited to the broken ends, including Rad51p, Rad52p, Rad55p, Rad57p, and possibly Rad54p. A genomic search for homology ensues, followed by strand invasion, promoted by the Rad51 filament with the participation of Rad55p, Rad57p and Rad54p. DNA synthesis then takes place, restoring the resected ends. Crossing-over formation depends on the length of the homologous recombining sequences, and is usually counteracted by the activity of the mismatch repair system. Given the conservation of the repair mechanisms and genes throughout evolution, these studies have profound implications for other eukaryotic organisms.     

Suppression of the Double-Strand-Break-Repair Defect of the Saccharomyces cerevisiae rad57 Mutant

The Rad51 paralogs Rad55 and Rad57 form a heterodimer required to mediate the formation and/or stabilization of the Rad51 filament. To further characterize the function of Rad55-Rad57, we used a combination of rad57 partial suppressors to determine whether the DNA repair and recombination defects of the rad57 mutant could be completely suppressed. The combination of all suppressors, elevated temperature, srs2, rad51-I345T, and mating-type (MAT) heterozygosity resulted in almost complete suppression of the rad57 mutant defect in the recruitment of Rad51 to DNA-damaged sites, as well as survival in response to ionizing radiation and camptothecin. In a physical assay to monitor the kinetics of double-strand-break (DSB)-induced gene conversion, the rad57 mutant defect was effectively suppressed by srs2 and MAT heterozygosity, but these same suppressors failed to suppress the spontaneous recombination defect. Thus the Rad55-Rad57 heterodimer appears to have a unique function in spontaneous recombination that is not essential for DSB repair. Furthermore, we investigated the currently unknown mechanism of rad57 suppression by MAT heterozygosity and found that it is independent of DNL4.     

Rad54 protein possesses chromatin-remodeling activity stimulated by the Rad51-ssDNA nucleoprotein filament

In Saccharomyces cerevisiae, the Rad54 protein participates in the recombinational repair of double-strand DNA breaks together with the Rad51, Rad52, Rad55 and Rad57 proteins. In vitro, Rad54 interacts with Rad51 and stimulates DNA strand exchange promoted by Rad51 protein. Rad54 is a SWI2/SNF2-related protein that possesses double-stranded DNA-dependent ATPase activity and changes DNA topology in an ATP hydrolysis-dependent manner. Here we show that Rad54 catalyzes bidirectional nucleosome redistribution by sliding nucleosomes along DNA. Nucleosome redistribution is greatly stimulated by the Rad51 nucleoprotein filament but does not require the presence of homologous single-stranded DNA within the filament. On the basis of these data, we propose that Rad54 facilitates chromatin remodeling and, perhaps more generally, protein clearing at the homology search step of genetic recombination.     

Role for the fission yeast RecQ helicase in DNA repair in G2

Members of the RecQ helicase subfamily are mutated in several human genomic instability syndromes, such as Bloom, Werner, and Rothmund-Thomson syndromes. We show that Rqh1, the single Schizosaccharomyces pombe homologue, is a 3'-to-5' helicase and exists with Top3 in a high-molecular-weight complex. top3 deletion is inviable, and this is suppressed by concomitant loss of rqh1 helicase activity or loss of recombination functions. This is consistent with RecQ helicases in other systems. By using epistasis analysis of the UV radiation sensitivity and by analyzing the kinetics of Rhp51 (Rad51 homologue), Rqh1, and Top3 focus formation in response to UV in synchronized cells, we identify the first evidence of a function for Rqh1 and Top3 in the repair of UV-induced DNA damage in G(2). Our data provide evidence that Rqh1 functions after Rad51 focus formation during DNA repair. We also identify a function for Rqh1 upstream of recombination in an Rhp18-dependent (Rad18 homologue) pathway. The model that these data allow us to propose helps to reconcile different interpretations of RecQ family helicase function that have arisen between work based on the S. pombe system and models based on studies of Saccharomyces cerevisiae SGS1 suggesting that RecQ helicases act before Rad51.     

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.     

ATP-dependent and ATP-independent roles for the Rad54 chromatin remodeling enzyme during recombinational repair of a DNA double strand break

The efficient and accurate repair of DNA double strand breaks (DSBs) is critical to cell survival, and defects in this process can lead to genome instability and cancers. In eukaryotes, the Rad52 group of proteins dictates the repair of DSBs by the error-free process of homologous recombination (HR). A critical step in eukaryotic HR is the formation of the initial Rad51-single-stranded DNA presynaptic nucleoprotein filament. This presynaptic filament participates in a homology search process that leads to the formation of a DNA joint molecule and recombinational repair of the DSB. Recently, we showed that the Rad54 protein functions as a mediator of Rad51 binding to single-stranded DNA, and here, we find that this activity does not require ATP hydrolysis. We also identify a novel Rad54-dependent chromatin remodeling event that occurs in vivo during the DNA strand invasion step of HR. This ATP-dependent remodeling activity of Rad54 appears to control subsequent steps in the HR process.