[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.

[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.

[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 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.

[The dds20+ gene controls a novel Rad51Sp-dependent pathway of recombinational repair in Schizosaccharomyces pombe].

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 RadS1 filament formation. As shown in this work, a novel RAD51Sp-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 rhp55delta in DNA repair. Cells of dds20delta 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 (Rad51Sp). 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.

[Recombinational repair in Schizosaccharomyces pombe: role in maintaining genomic integrity]. / Rekombinatsionnaia reparatsiia u Schizosaccharomyces pombe: rol' v podderzhanii tselostnosti genoma.

Recombinational repair was first detected in budding yeast Saccharomyces cerevisiae and was also studied in fission yeast Schizosaccharomyces pombe over the recent decade. The discovery of Sch. pombe homologs of the S. cerevisiae RAD52 genes made it possible not only to identify and to clone their vertebrate counterparts, but also to study in detail the role of DNA recombination in certain cell processes. For instance, recombinational repair was shown to play a greater role in maintaining genome integrity in fission yeast and in vertebrates compared with S. cerevisiae. The present state of the problem of recombinational double-strand break repair in fission yeast is considered with a focus on comparisons between Sch. pombe and higher eukaryotes. The role of double-strand break repair in maintaining genome stability is discussed.

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.

DNA repair protein Rad55 is a terminal substrate of the DNA damage checkpoints.

Checkpoints, which are integral to the cellular response to DNA damage, coordinate transient cell cycle arrest and the induced expression of DNA repair genes after genotoxic stress. DNA repair ensures cellular survival and genomic stability, utilizing a multipathway network. Here we report evidence that the two systems, DNA damage checkpoint control and DNA repair, are directly connected by demonstrating that the Rad55 double-strand break repair protein of the recombinational repair pathway is a terminal substrate of DNA damage and replication block checkpoints. Rad55p was specifically phosphorylated in response to DNA damage induced by the alkylating agent methyl methanesulfonate, dependent on an active DNA damage checkpoint. Rad55p modification was also observed after gamma ray and UV radiation. The rapid time course of phosphorylation and the recombination defects identified in checkpoint-deficient cells are consistent with a role of the DNA damage checkpoint in activating recombinational repair. Rad55p phosphorylation possibly affects the balance between different competing DNA repair pathways.

A new recombinational DNA repair gene from Schizosaccharomyces pombe with homology to Escherichia coli RecA.

A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved.

A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates.

Exoribonucleases are important enzymes for the turnover of cellular RNA species. We have isolated the first mammalian cDNA from mouse demonstrated to encode a 5'-3' exoribonuclease. The structural conservation of the predicted protein and complementation data in Saccharomyces cerevisiae suggest a role in cytoplasmic mRNA turnover and pre-rRNA processing similar to that of the major cytoplasmic exoribonuclease Xrn1p in yeast. Therefore, a key component of the mRNA decay system in S. cerevisiae has been conserved in evolution from yeasts to mammals. The purified mouse protein (mXRN1p) exhibited a novel substrate preference for G4 RNA tetraplex-containing substrates demonstrated in binding and hydrolysis experiments. mXRN1p is the first RNA turnover function that has been localized in the cytoplasm of mammalian cells. mXRN1p was distributed in small granules and was highly enriched in discrete, prominent foci. The specificity of mXRN1p suggests that RNAs containing G4 tetraplex structures may occur in vivo and may have a role in RNA turnover.

[Localization of RecA-like proteins in preparations of spread nuclei of mouse spermatocytes at meiotic prophase I]. / Lokalizatsiia RecA-podobnykh belkov na preparatakh rasplastannykh iader spermatotsitov I poriadka myshi.

Earlier, polyclonal antibodies to Escherichia coli RecA protein were used to identify immunologically related proteins in meiotic spermatocytes of different eukaryotes. At least one such protein proved to be a component of the synaptonemal complex (SC) [1]. Subsequent experiments on localization of RecA-like antigens in SCs of spermatocytes were performed by indirect immunocytochemical methods and electron microscopy, which showed that RecA-like protein(s) at early leptotene are largely associated with chromatin. During SC formation (at leptotene and zygotene), they are found in both lateral elements and the central space of SC. In some cases, RecA-like proteins are associated with SC substructures that resemble recombination nodules. When spermatocytes enter late diplotene, Rec-A-like proteins cease to be detected in SC structures.

Identification of functional domains in the Sep1 protein (= Kem1, Xrn1), which is required for transition through meiotic prophase in Saccharomyces cerevisiae.

The Sep1 (also known as Kem1, Xrn1, Rar5, DST2/Stpbeta) protein of Saccharomyces cerevisiae is an Mr 175,000 multifunctional exonuclease with suspected roles in RNA turnover and in the microtubular cytoskeleton as well as in DNA recombination and DNA replication. The most striking phenotype of SEP1 null mutations is quantitative arrest during meiotic prophase at the pachytene stage. We have constructed a set of N- and C-terminal as well as internal deletions of the large SEP1 gene. Analysis of these deletion mutations on plasmids in a host carrying a null allele (sep1 ) revealed that at least 270 amino acids from the C-terminus of the wild-type protein were dispensable for complementing the slow growth and benomyl hypersensitivity of a null mutant. In contrast, any deletion at the N-terminus abrogated complementing activity for these phenotypes. The sequences essential for function correspond remarkably well with the regions of Sep1 that are homologous to its Schizosaccharomyces pombe counterpart Exo2. In addition, these experiments showed that, despite the high intracellular levels of Sep1, over-expression of this protein above these levels is detrimental to the cell. We discuss the potential cellular roles of the Sep1 protein as a microtubule-nucleic acid interface protein linking its suspected function in the microtubular cytoskeleton with its role as a nucleic acid binding protein.

Use of monoclonal antibodies in the functional characterization of the Saccharomyces cerevisiae Sep1 protein.

The Saccharomyces cerevisiae strand-exchange protein 1 (Sep1 also known as Xrn1, Kem1, Rar5, Stp beta/DST2) has been demonstrated to mediate the formation of hybrid DNA from model substrates of linear double-stranded and circular single-stranded DNA in vitro. To delineate the mechanism by which Sep1 acts in the strand-exchange reaction, we analyzed mouse anti-Sep1 monoclonal antibodies for inhibition of the Sep1 in vitro activity. Of 12 class-G immunoglobulins tested, four were found to consistently inhibit the Sep1-mediated strand-exchange reaction. The inhibiting antibodies were tested for inhibition of a variety of Sep1-catalyzed DNA reactions including exonuclease activity on double-stranded and single-stranded DNA, renaturation of complementary single-stranded DNA and condensation of DNA into large aggregates. All four inhibiting antibodies had no effect on the exonuclease activity of Sep1. Three antibodies specifically blocked DNA aggregation. In addition, one antibody inhibited renaturation of complementary single-stranded DNA. This inhibition pattern underlines the importance of condensation of DNA into large aggregates in conjunction with double-stranded DNA exonuclease activity for the in vitro homologous pairing activity of Sep1. The implications of these data for the interpretation of proteins which promote homologous pairing of DNA are discussed, in particular in light of the reannealing activity of the p53 human tumor-suppressor protein.

A role of Sep1 (= Kem1, Xrn1) as a microtubule-associated protein in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells lacking the SEP1 (also known as XRN1, KEM1, DST2, RAR5) gene function exhibit a number of phenotypes in cellular processes related to microtubule function. Mutant cells show increased sensitivity to the microtubule-destabilizing drug benomyl, increased chromosome loss, a karyogamy defect, impaired spindle pole body separation, and defective nuclear migration towards the bud neck. Analysis of the arrest morphology and of the survival during arrest strongly suggests a structural defect accounting for the benomyl hypersensitivity, rather than a regulatory defect in a checkpoint. Biochemical analysis of the purified Sep1 protein demonstrates its ability to promote the polymerization of procine brain and authentic S.cerevisiae tubulin into flexible microtubules in vitro. Furthermore, Sep1 co-sediments with these microtubules in sucrose cushion centrifugation. Genetic analysis of double mutant strains containing a mutation in SEP1 and in one of the genes coding for alpha- or beta-tubulin further suggests interaction between Sep1 and microtubules. Taken together these three lines of evidence constitute compelling evidence for a role of Sep1 as an accessory protein in microtubule function in the yeast S.cerevisiae.

[Antibodies against Escherichia coli RecA protein reveal two nuclear proteins in bovine spermatocytes which interact with synaptonemal complex structures of meiotic chromosomes of various eukaryotic organisms]. / Antitela k RecA-belku Escherichia coli vyiavliaiut dva iadernykh belka v spermatotsitakh byka, kotorye vzaimodeistvuiut so strukturami sinaptonemnogo kompleksa meioticheskikh khromosom riada éukarioticheskikh organizmov.

Three RecA-like proteins were detected in bovine meiotic cells using antibodies against Escherichia coli RecA protein. After isolation and purification of these RecA-like proteins their molecular weights appeared to be equal to 37, 70 and 130 kD. The 37 kD protein accompanies all the stages of spermatogenesis up to the stage of mature spermatozoa. The 70 kD protein is detectable only in nuclei of cells at the stage of prophase I of meiotic division. These RecA-like proteins are involved in the formation of structural elements of the synaptonemal complex (SC) and are detected in the SC composition in meiotic cells not only of mammals but also of plants and insects, which suggests the evolutionary conservative character of these proteins.

Imprecise excision of plasmid pE194 from the chromosomes of Bacillus subtilis pE194 insertion strains.

Plasmid pE194 has been shown to be rescued by integration after cultivation of infected Bacillus subtilis recE4 cells at a restrictive high temperature. The plasmid is also spontaneously excised from the chromosome at a low frequency by precise or imprecise excision (J. Hofemeister, M. Israeli-Reches, and D. Dubnau, Mol. Gen. Genet. 189:58-68, 1983). We have investigated nine excision plasmids, carrying insert DNA 1 to 6 kbp in length, either in a complete pE194 or in a partially deleted pE194 copy. Type 1 (additive) excision plasmids have the left- and right-junction DNAs preserved as 13-bp direct repeats (5'-GGGGAGAAAACAT-3') corresponding to the region between positions 864 and 876 in pE194. In type 2 (substitutive) excision plasmids, a conserved 13-bp sequence remains only at the right junction while the left junction has been deleted during the excision process. The type 3 excision plasmid carries at each junction the tetranucleotide 5'-TCCC-3', present in pE194 between positions 1995 and 1998. Although we isolated the excision plasmids from different integration mutants, the insert DNAs of eight independently isolated plasmids showed striking sequence homology, suggesting that they originated from one distinct region of the B. subtilis chromosome. Thus, we postulate that imprecise excision of pE194 occurs most frequently after its translocation from the original insertion site into a preferred excision site within the host chromosome. The imprecise excision from this site occurs at excision breakpoints outside the pE194-chromosome junctions in a chromosomal region which remains to be investigated further.

Homologous recombination between plasmid and chromosomal DNA in Bacillus subtilis requires approximately 70 bp of homology.

To determine the minimal DNA sequence homology required for recombination in Bacillus subtilis, we developed a system capable of distinguishing between homologous and illegitimate recombination events during plasmid integration into the chromosome. In this system the recombination frequencies were measured between ts pE194 derivatives carrying segments of the chromosomal beta-gluconase gene (bglS) of various lengths and the bacterial chromosome, using selection for erythromycin resistance at the non-permissive temperature. Homologous recombination events, resulting in disruption of the bglS gene, were easily detected by a colorimetric assay for beta-gluconase activity. A linear dependence of recombination frequency on homology length was observed over an interval of 77 bp. It was found that approximately 70 bp of homology is required for detectable homologous recombination. Homologous recombination was not detected when only 25 bp of homology between plasmid and chromosome were provided. The data indicate that homology requirements for recombination in B. subtilis differ from those in Escherichia coli.

[Determination of the minimal length DNA homologous region required for plasmid integration into the Bacillus subtilis chromosome via homologous recombination]. / Opredelenie minimal'noi dliny gomologichnogo uchastka DNK, neobkhodimoi dlia integratsii plazmid v khromosomu Bacillus subtilis posredstvom gomologichnoi rekombinatsii.

With a view to determine a minimal sequence length of homology necessary for RecE-dependent homologous recombination in Bacillus subtilis cells, we developed a system, based on interaction between plasmid replicon and bacterial chromosome. Recombination frequencies were measured between ts plasmid pE194 derivatives carrying chromosomal beta-glucuronidase gene (bglS) fragments of various length, and a bacterial chromosome. The homologous recombination events resulted in bglS gene disruption. Approx. 70 bp of homology were found to be necessary for detectable homologous recombination. Homologous recombination was not detected when homology was equal 25 bp. These data indicate that homology requirement for recombination in B. subtilis differs from that in Escherichia coli.