The Role of Chromatin Assembly Factors in Induced Mutagenesis at Low Levels of DNA Damage.

The problem of low-dose irradiation has been discussed in the scientific literature for several decades, but it is impossible to come to a generally accepted conclusion about the presence of any specific features of low-dose irradiation in contrast to acute irradiation. We were interested in the effect of low doses of UV radiation on the physiological processes, including repair processes in cells of the yeast Saccharomyces cerevisiae, in contrast to high doses of radiation. Cells utilize excision repair and DNA damage tolerance pathways without significant delay of the cell cycle to address low levels of DNA damage (such as spontaneous base lesions). For genotoxic agents, there is a dose threshold below which checkpoint activation is minimal despite the measurable activity of the DNA repair pathways. Here we report that at ultra-low levels of DNA damage, the role of the error-free branch of post-replicative repair in protection against induced mutagenesis is key. However, with an increase in the levels of DNA damage, the role of the error-free repair branch is rapidly decreasing. We demonstrate that with an increase in the amount of DNA damage from ultra-small to high, asf1Δ-specific mutagenesis decreases catastrophically. A similar dependence is observed for mutants of gene-encoding subunits of the NuB4 complex. Elevated levels of dNTPs caused by the inactivation of the SML1 gene are responsible for high spontaneous reparative mutagenesis. The Rad53 kinase plays a key role in reparative UV mutagenesis at high doses, as well as in spontaneous repair mutagenesis at ultra-low DNA damage levels.

Genetic Analysis of the Hsm3 Protein Function in Yeast Saccharomyces cerevisiae NuB4 Complex.

In the nuclear compartment of yeast, NuB4 core complex consists of three proteins, Hat1, Hat2, and Hif1, and interacts with a number of other factors. In particular, it was shown that NuB4 complex physically interacts with Hsm3p. Early we demonstrated that the gene HSM3 participates in the control of replicative and reparative spontaneous mutagenesis, and that hsm3Δ mutants increase the frequency of mutations induced by different mutagens. It was previously believed that the HSM3 gene controlled only some minor repair processes in the cell, but later it was suggested that it had a chaperone function with its participation in proteasome assembly. In this work, we analyzed the properties of three hsm3Δ, hif1Δ, and hat1Δ mutants. The results obtained showed that the Hsm3 protein may be a functional subunit of NuB4 complex. It has been shown that hsm3- and hif1-dependent UV-induced mutagenesis is completely suppressed by inactivation of the Polη polymerase. We showed a significant role of Polη for hsm3-dependent mutagenesis at non-bipyrimidine sites (NBP sites). The efficiency of expression of RNR (RiboNucleotid Reducase) genes after UV irradiation in hsm3Δ and hif1Δ mutants was several times lower than in wild-type cells. Thus, we have presented evidence that significant increase in the dNTP levels suppress hsm3- and hif1-dependent mutagenesis and Polη is responsible for hsm3- and hif1-dependent mutagenesis.

Genetic analysis reveals different roles of Schizosaccharomyces pombe sfr1/dds20 in meiotic and mitotic DNA recombination and repair.

DNA double-strand break (DSB) repair mediated by the Rad51 pathway of homologous recombination is conserved in eukaryotes. In yeast, Rad51 paralogs, Saccharomyces cerevisiae Rad55-Rad57 and Schizosaccharomyces pombe Rhp55-Rhp57, are mediators of Rad51 nucleoprotein formation. The recently discovered S. pombe Sfr1/Dds20 protein has been shown to interact with Rad51 and to operate in the Rad51-dependent DSB repair pathway in parallel to the paralog-mediated pathway. Here we show that Sfr1 is a nuclear protein and acts downstream of Rad50 in DSB processing. sfr1Delta is epistatic to rad18 (-) and rad60 (-), and Sfr1 is a high-copy suppressor of the replication and repair defects of a rad60 mutant. Sfr1 functions in a Cds1-independent UV damage tolerance mechanism. In contrast to mitotic recombination, meiotic recombination is significantly reduced in sfr1Delta strains. Our data indicate that Sfr1 acts in DSB repair mainly outside of S-phase, and is required for wild-type levels of meiotic recombination. We suggest that Sfr1 acts early in recombination and has a specific role in Rad51 filament assembly, distinct from that of the Rad51 paralogs.

Transferase and hydrolytic activities of the laminarinase from Rhodothermus marinus and its M133A, M133C, and M133W mutants.

Comparative studies of the transglycosylation and hydrolytic activities have been performed on the Rhodothermus marinus beta-1,3-glucanase (laminarinase) and its M133A, M133C, and M133W mutants. The M133C mutant demonstrated near 20% greater rate of transglycosylation activity in comparison with the M133A and M133W mutants that was measured by NMR quantitation of nascent beta(1-4) and beta(1-6) linkages. To obtain kinetic probes for the wild-type enzyme and Met-133 mutants, p-nitrophenyl beta-laminarin oligosaccharides of degree of polymerisation 2-8 were synthesized enzymatically. Catalytic efficiency values, k (cat)/K (m), of the laminarinase catalysed hydrolysis of these oligosaccharides suggested possibility of four negative and at least three positive binding subsites in the active site. Comparison of action patterns of the wild-type and M133C mutant in the hydrolysis of the p-nitrophenyl-beta-D-oligosac- charides indicated that the increased transglycosylation activity of the M133C mutant did not result from altered subsite affinities. The stereospecificity of the transglycosylation reaction also was unchanged in all mutants; the major transglycosylation products in hydrolysis of p-nitrophenyl laminaribioside were beta-glucopyranosyl-beta-1,3-D-glucopy- ranosyl-beta-1,3-D-glucopyranose and beta-glucopyranosyl-beta-1, 3-D-glucopyranosyl-beta-1,3-D-glucpyranosyl-beta-1,3-D- glucopyranoxside.

HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis.

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.

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.