Analysis of mutations in the mat1 region of Schizosaccharomyces pombe strain with the deletion of gene rhp55+

DNA double-strand breaks may occur both under the action of various exogenous factors and in the course of cell metabolism processes, in particular, upon mating type switching in yeast. Genes belonging to the epistatic group RAD52 are known to repair such DNA damage. Molecular defects in mating type switching occurring after the deletion of gene rhp55+ encoding the paralog of recombinational protein Rhp51, which is a functional homolog of Escherichia coli RecA, were studied in fission yeast. Analysis of stable nonswitching segregants in h90 rhp55 mutants with unchanged configuration of the mating type switching locus but with a drastically decreased level of double-strand DNA break formation at the mat1 :1 locus demonstrated changes in DNA sequences within the region responsible for the generation of the breaks. These changes might have resulted from incorrect gene conversion upon repair of double-strand DNA breaks in Schizosaccharomyces pombe rhp55 mutants.     

The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51 ⁺ and rhp55 ⁺. We constructed homothallic strains of genotypes h ⁹⁰ rhp51 and h ⁹⁰ rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h ⁹⁰ cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h ^? and h ⁺. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h ⁺ segregants had the h ^+N or h ⁹⁰ configuration of the mat region, whereas h ^?, the h ⁹⁰ configuration. Segregants h ^+N contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51 ⁺ and rhp55 ⁺ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

The role of recombinational repair proteins in mating type switching in fission yeast cells

DNA double-strand breaks (DSBs) occur after exposing cells to ionizing radiation or under the action of various antitumor antibiotics. They can be also generated in the course cell processes, such as meiosis and mating type switching in yeast. The most preferential mechanism for the correction of DNA DSB in yeasts is recombinational repair controlled by RAD52 group genes. The role of recombinational repair in mating type switching of fission yeast cells was examined on the example of genes of this group, rhp51+ and rhp51+. We constructed homothallic strains of genotypes h90 rhp51 and h90 rhp55, and found that mutant cells yielded colonies with the mottled phenotype. In addition, h90 cells with deletions in these genes were shown to segregate heterothallic iodine-negative colonies h- and h+. The genome region, responsible for the switching process in these segregants, was analyzed by DNA hybridization. As shown in this analysis, h+ segregants had the h+N or h90 configuration of the mat region, whereas h-, the h90 configuration. Segregants h+ contained DNA duplication in the mat region. DNA rearrangements were not detected at the mating type locus, but the level of DNA DSB formation was drastically decreased in these segregants. Thus, our results show that genes rhp51+ and rhp55+ are involved not only in the repair of induced DNA DSB, but also in the mechanism of mating type switching in fission yeast.     

A Novel Function of the DNA Repair Gene rhp6 in Mating-Type Silencing by Chromatin Remodeling in Fission Yeast

Recent studies have indicated that the DNA replication machinery is coupled to silencing of mating-type loci in the budding yeast Saccharomyces cerevisiae, and a similar silencing mechanism may operate in the distantly related yeast Schizosaccharomyces pombe. Regarding gene regulation, an important function of DNA replication may be in coupling of faithful chromatin assembly to reestablishment of the parental states of gene expression in daughter cells. We have been interested in isolating mutants that are defective in this hypothesized coupling. An S. pombe mutant fortuitously isolated from a screen for temperature-sensitive growth and silencing phenotype exhibited a novel defect in silencing that was dependent on the switching competence of the mating-type loci, a property that differentiates this mutant from other silencing mutants of S. pombe as well as of S. cerevisiae. This unique mutant phenotype defined a locus which we named sng1 (for silencing not governed). Chromatin analysis revealed a switching-dependent unfolding of the donor loci mat2P and mat3M in the sng1⁻ mutant, as indicated by increased accessibility to the in vivo-expressed Escherichia coli dam methylase. Unexpectedly, cloning and sequencing identified the gene as the previously isolated DNA repair gene rhp6. RAD6, an rhp6 homolog in S. cerevisiae, is required for postreplication DNA repair and ubiquitination of histones H2A and H2B. This study implicates the Rad6/rhp6 protein in gene regulation and, more importantly, suggests that a transient window of opportunity exists to ensure the remodeling of chromatin structure during chromosome replication and recombination. We propose that the effects of the sng1⁻/rhp6⁻ mutation on silencing are indirect consequences of changes in chromatin structure.     

The involvement of Srs2 in post-replication repair and homologous recombination in fission yeast

Homologous recombination is important for the repair of double-strand breaks and daughter strand gaps, and also helps restart stalled and collapsed replication forks. However, sometimes recombination is inappropriate and can have deleterious consequences. To temper recombination, cells have employed DNA helicases that unwind joint DNA molecules and/or dissociate recombinases from DNA. Budding yeast Srs2 is one such helicase. It can act by dissociating Rad51 nucleoprotein filaments, and is required for channelling DNA lesions to the post-replication repair (PRR) pathway. Here we have investigated the role of Srs2 in controlling recombination in fission yeast. Similar to budding yeast, deletion of fission yeast srs2 results in hypersensitivity to a range of DNA damaging agents, rhp51-dependent hyper-recombination and synthetic sickness when combined with rqh1^– that is suppressed by deleting rhp51, rhp55 or rhp57. Epistasis analysis indicates that Srs2 and the structure-specific endonuclease Mus81–Eme1 function in a sub-pathway of PRR for the tolerance/repair of UV-induced damage. However, unlike in Saccharomyces cerevisiae, Srs2 is not required for channelling lesions to the PRR pathway in Schizosaccharomyces pombe. In addition to acting as an antirecombinase, we also show that Srs2 can aid the recombinational repair of camptothecin-induced collapsed replication forks, independently of PRR.     

DNA damage induced mating type switching in Saccharomyces cerevisiae.

Haploid cells of the yeast Saccharomyces cerevisiae are able to undergo a differentiation-like process: they can switch their mating type between the a and the alpha state. The molecular mechanism of this interconversion of mating types is intrachromosomal gene conversion. It has been shown in a variety of studies that mating type switching in heterothallic strains can be induced by DNA damaging agents, and that different DNA damaging agents differ in the length of incubation after treatment required for induction. Because X-rays induce switching immediately after irradiation and because the DNA double-strand break repair pathway is required for switching, the event initiating heterothallic mating type switching is likely to be a DNA double-strand break. Therefore the assay for heterothallic mating type switching may screen for the induction of DNA double-strand breaks. Several aspects indicating a relationship of mating type switching to mechanisms associated with carcinogenesis are discussed.     

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.     

Mating type-like conversion promoted by the 2 micrograms circle site-specific recombinase: implications for the double-strand-gap repair model.

Double-strand breaks in DNA are known to promote recombination in Saccharomyces cerevisiae. Yeast mating type switching, which is a highly efficient gene conversion event, is apparently initiated by a site-specific double-strand break. The 2 micrograms circle site-specific recombinase, FLP, has been shown to make double-strand breaks in its substrate DNA. By using a hybrid 2 micrograms circle::Tn5 plasmid, a portion of which resembles, in its DNA organization, the active (MAT) and the silent (HML) yeast mating type loci, it is shown that FLP mediates a conversion event analogous to mating type switching. Whereas the FLP site-specific recombination is not dependent on the RAD52 gene product, the FLP-induced conversion is abolished in a rad52 background. The FLP-promoted conversion in vivo can be faithfully reproduced by making a double-stranded gap in vitro in the vicinity of the FLP site and allowing the gap to be repaired in vivo.     

The role of novel genes rrp1+ and rrp2+ in the repair of DNA damage in Schizosaccharomyces pombe

We identified two predicted proteins in Schizosaccharomyces pombe, Rrp1 (SPAC17A2.12) and Rrp2 (SPBC23E6.02) that share 34% and 36% similarity to Saccharomyces cerevisiae Ris1p, respectively. Ris1p is a DNA-dependent ATP-ase involved in gene silencing and DNA repair. Rrp1 and Rrp2 also share similarity with S. cerevisiae Rad5 and S. pombe Rad8, containing SNF2-N, RING finger and Helicase-C domains. To investigate the function of the Rrp proteins, we studied the DNA damage sensitivities and genetic interactions of null mutants with known DNA repair mutants. Single Δrrp1 and Δrrp2 mutants were not sensitive to CPT, 4NQO, CDPP, MMS, HU, UV or IR. The double mutants Δrrp1 Δrhp51 and Δrrp2 Δrhp51 plus the triple Δrrp1 Δrrp2 Δrhp51 mutant did not display significant additional sensitivity. However, the double mutants Δrrp1 Δrhp57 and Δrrp2 Δrhp57 were significantly more sensitive to MMS, CPT, HU and IR than the Δrhp57 single mutant. The checkpoint response in these strains was functional. In S. pombe, Rhp55/57 acts in parallel with a second mediator complex, Swi5/Sfr1, to facilitate Rhp51-dependent DNA repair. Δrrp1 Δsfr1 and Δrrp2 Δsfr1 double mutants did not show significant additional sensitivity, suggesting a function for Rrp proteins in the Swi5/Sfr1 pathway of DSB repair. Consistent with this, Δrrp1 Δrhp57 and Δrrp2 Δrhp57 mutants, but not Δrrp1 Δsfr1 or Δrrp2 Δsfr1 double mutants, exhibited slow growth and aberrations in cell and nuclear morphology that are typical of Δrhp51.     

Interconversion of Yeast Mating Types II. Restoration of Mating Ability to Sterile Mutants in Homothallic and Heterothallic Strains

The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HMa and hmα loci are blocs of unexpressed α regulatory information, and HMα and hma loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.     

The repair of double-strand breaks in the nuclear DNA of Saccharomyces cerevisiae and its genetic control

Summary With the use of neutral sucrose sedimentation techniques, the size of unirradiated nuclear DNA and the repair of double-strand breaks induced in it by ionizing radiation have been determined in both wild-type and homozygous rad52 diploids of the yeast Saccharomyces cerevisiae. The number average molecular weight of unirradiated DNA in these experiments is 3.0×10⁸±0.3 Daltons. Double-strand breaks are induced with a frequency of 0.58×10^-10 per Daltonkrad in the range of 25 to 100 krad. Since repair at low doses is observed in wild-type but not homozygous rad52 strains, the corresponding rad52 gene product is concluded to have a role in the repair process. Cycloheximide was also observed to inhibit repair to a limited extent indicating a requirement for protein synthesis. Based on the sensitivity of various mutants and the induction frequency of double-strand breaks, it is concluded that there are 1 to 2 double-strand breaks per lethal event in diploid cells incapable of repairing these breaks.     

Rad22 protein, a rad52 homologue in Schizosaccharomyces pombe, binds to DNA double-strand breaks

DNA double-strand breaks can be introduced by exogenous agents or during normal cellular processes. Genes belonging to the RAD52 epistasis group are known to repair these breaks in budding yeast. Among these genes, RAD52 plays a central role in homologous recombination and DNA double-strand break repair. Despite its importance, its mechanism of action is not yet clear. It is known, however, that the human homologue of Rad52 is capable of binding to DNA ends in vitro. Herein, we show that Rad22 protein, a Rad52 homologue in the fission yeast Schizosaccharomyces pombe, can similarly bind to DNA ends at double-strand breaks. This end-binding ability was demonstrated in vitro by electron microscopy and by protection from exonuclease attack. We also showed that Rad22 specifically binds near double-strand break associated with mating type switching in vivo by chromatin immunoprecipitation analysis. This is the first evidence that a recombinational protein directly binds to DNA double-strand breaks in vivo.     

Control of cell type in yeast by the mating type locus: The α1-α2 hypothesis

We have extended the genetic analysis of four mutants carrying defective MATα alleles in order to determine how the mating type locus controls yeast cell types: a, a, and $\text{a}\text{α}$. First, we have mapped the defect in the mutant VC73 to the mating type locus by diploid and tetraploid segregation analysis. Second, we have determined that the mutations in these strains define two complementation groups, MATα1 and MATα2. The MATα1 gene is proposed to be a positive regulator of α mating functions. The MATα2 gene product is proposed to have two roles, as a negative regulator of a-specific mating functions and as a regulator of $\text{a}\text{α}$ cell functions (required for sporulation, for inhibition of mating and other processes). This view of MATα leads to the prediction that matα1^?matα2^? mutants should have the mating ability of an a cell and that matα1^?matα2^?/MATα strains should mate as α and be unable to sporulate. Such double mutants have been constructed and behave as predicted. We therefore propose that a-specific mating functions in MATa cells are constitutively expressed due to the absence of the MATα2 gene product and that α-specific mating functions are not expressed due to the absence of the MATα1 gene product.     

Fission yeast Swi5/Sfr1 and Rhp55/Rhp57 differentially regulate Rhp51-dependent recombination outcomes

Several accessory proteins referred to as mediators are required for the full activity of the Rad51 (Rhp51 in fission yeast) recombinase. In this study, we analyzed in vivo functions of the recently discovered Swi5/Sfr1 complex from fission yeast. In normally growing cells, the Swi5-GFP protein localizes to the nucleus, where it forms a diffuse nuclear staining pattern with a few distinct foci. These spontaneous foci do not form in swi2Delta mutants. Upon UV irradiation, Swi5 focus formation is induced in swi2Delta mutants, a response that depends on Sfr1 function, and Sfr1 also forms foci that colocalize with damage-induced Rhp51 foci. The number of UV-induced Rhp51 foci is partially reduced in swi5Delta and rhp57Delta mutants and completely abolished in an swi5Delta rhp57Delta double mutant. An assay for products generated by HO endonuclease-induced DNA double-strand breaks (DSBs) reveals that Rhp51 and Rhp57, but not Swi5/Sfr1, are essential for crossover production. These results suggest that Swi5/Sfr1 functions as an Rhp51 mediator but processes DSBs in a manner different from that of the Rhp55/57 mediator.     

Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage

We have identified two fission yeast homologs of budding yeast Rad4 and human xeroderma pigmentosum complementation group C (XP-C) correcting protein, designated Rhp4A and Rhp4B. Here we show that the rhp4 genes encode NER factors that are required for UV-induced DNA damage repair in fission yeast. The rhp4A-deficient cells but not the rhp4B-deficient cells are sensitive to UV irradiation. However, the disruption of both rhp4A and rhp4B resulted in UV sensitivity that was greater than that of the rhp4A-deficient cells, revealing that Rhp4B plays a role in DNA repair on its own. Fission yeast has two pathways to repair photolesions on DNA, namely, nucleotide excision repair (NER) and UV-damaged DNA endonuclease-dependent excision repair (UVER). Studies with the NER-deficient rad13 and the UVER-deficient (Delta)uvde mutants showed the two rhp4 genes are involved in NER and not UVER. Assessment of the ability of the various mutants to remove cyclobutane pyrimidine dimers (CPDs) from the rbp2 gene locus indicated that Rhp4A is involved in the preferential repair of lesions on the transcribed DNA strand and plays the major role in fission yeast NER. Rhp4B in contrast acts as an accessory protein in non-transcribed strand (NTS) repair.     

Mus81, Rhp51(Rad51), and Rqh1 Form an Epistatic Pathway Required for the S-Phase DNA Damage Checkpoint

The S-phase DNA damage checkpoint slows the rate of DNA synthesis in response to damage during replication. In the fission yeast Schizosaccharomyces pombe, Cds1, the S-phase-specific checkpoint effector kinase, is required for checkpoint signaling and replication slowing; upon treatment with the alkylating agent methyl methane sulfonate, cds1Δ mutants display a complete checkpoint defect. We have identified proteins downstream of Cds1 required for checkpoint-dependant slowing, including the structure-specific endonuclease Mus81 and the helicase Rqh1, which are implicated in replication fork stability and the negative regulation of recombination. Removing Rhp51, the Rad51 recombinase homologue, suppresses the slowing defect of rqh1Δ mutants, but not that of mus81Δ mutant, defining an epistatic pathway in which mus81 is epistatic to rhp51 and rhp51 is epistatic to rqh1. We propose that restraining recombination is required for the slowing of replication in response to DNA damage.     

Identification of the DNA damage-responsive elements of therhp51 + gene,arecA andRAD51 homolog from the fission yeastSchizosaccharomyces pombe

TheSchizosaccharomyces pombe rhp51 ⁺ gene encodes a recombinational repair protein that shares significant sequence identities with the bacterial RecA and theSaccharomyces cerevisiae RAD51 protein. Levels ofrhp51 ⁺ mRNA increase following several types of DNA damage or inhibition of DNA synthesis. Anrhp51::ura4 fusion gene was used to identify the cis-acting promoter elements involved in regulatingrhp51 ⁺ expression in response to DNA damage. Two elements, designated DRE1 and DRE2 (fordamage-responsiveelement), match a decamer consensus URS (upstream repressing sequence) found in the promoters of many other DNA repair and metabolism genes fromS. cerevisiae. However, our results show that DRE1 and DRE2 each function as a UAS (upstream activating sequence) rather than a URS and are also required for DNA-damage inducibility of the gene. A 20-bp fragment located downstream of both DRE1 and DRE2 is responsible for URS function. The DRE1 and DRE2 elements cross-competed for binding to two proteins of 45 and 59 kDa. DNase I footprint analysis suggests that DRE1 and DRE2 bind to the same DNA-binding proteins. These results suggest that the DRE-binding proteins may play an important role in the DNA-damage inducibility ofrhp51 ⁺ expression.     

Functional overlap between the structure-specific nucleases Yen1 and Mus81-Mms4 for DNA-damage repair in S. cerevisiae

In eukaryotic cells, multiple DNA repair mechanisms respond to a wide variety of DNA lesions. Homologous recombination-dependent repair provides a pathway for dealing with DNA double-strand breaks and replication fork demise. A key step in this process is the resolution of recombination intermediates such as Holliday junctions (HJs). Recently, nucleases from yeast (Yen1) and human cells (GEN1) were identified that can resolve HJ intermediates, in a manner analogous to the E. coli HJ resolvase RuvC. Here, we have analyzed the role of Yen1 in DNA repair in S. cerevisiae, and show that while yen1Δ mutants are repair-proficient, yen1Δ mus81Δ double mutants are exquisitely sensitive to a variety of DNA-damaging agents that disturb replication fork progression. This phenotype is dependent upon RAD52, indicating that toxic recombination intermediates accumulate in the absence of Yen1 and Mus81. After MMS treatment, yen1Δ mus81Δ double mutants arrest with a G2 DNA content and unsegregated chromosomes. These findings indicate that Yen1 can act upon recombination/repair intermediates that arise in MUS81-defective cells following replication fork damage.