The product of Saccharomyces cerevisiae WHIP/MGS1, a gene related to replication factor C genes, interacts functionally with DNA polymerase delta.

The Saccharomyces cerevisiae gene WHIP/ MGS1 encodes a protein related to the subunits of Replication Factor C (RFC). We found that the RFC-like motifs in Whip/Mgs1 are essential for its function. Furthermore, by screening for synthetic dosage lethality, we have shown that overexpression of MGS1 causes lethality in combination with mutations in genes that encode replication proteins such as DNA polymerase delta, RFC, PCNA and RPA. Moreover, loss of MGS1 function interferes with the ability of multicopy PCNA to suppress the replication defect of the rfc5-1 mutant. At permissive temperatures, deletion of MGS1 suppresses the hydroxyurea (HU) sensitivity of pol31 and pol32 mutants, which bear mutations in the smaller subunits of DNA polymerase delta, and at semipermissive and non-permissive temperatures mgs1delta partially alleviates the growth defects of the pol31 mutant. We also report that the growth defect and HU sensitivity of the pol31 mutant are suppressed by mms2delta and rad18delta mutations. We suggest that Mgs1 interacts with the DNA replication machinery to modulate the function of DNA polymerase delta during replication or replication-associated repair, and influences the choice of the pathway employed for replication fork reactivation. Possible roles of Mgs1, DNA polymerase delta, Rad18 and Mms2 in replication and replication fork restart are discussed.

The N-terminal region of Sgs1, which interacts with Top3, is required for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 disruptants.

The SGS1 gene of Saccharomyces (cerevisiae is a homologue of the genes affected in Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome. Disruption of the SGS1 gene is associated with high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea (HU), and with hyper-recombination phenotypes, including interchromosomal recombination between heteroalleles. SGS1 encodes a protein which has a helicase domain similar to that of Escherichia coli RecQ. A comparison of amino acid sequences among helicases of the RecQ family reveals that Sgs1,WRN, and BLM share a conserved region adjacent to the C-terminal part of the helicase domain (C-terminal conserved region). In addition, Sgs1 contains two highly charged acidic regions in its N-terminal region and the HRDC (helicase and RNaseD C-terminal) domain at its C-terminal end. These regions were also found in BLM and WRN, and in Rqh1 from Schizosaccharomyces pombe. In this study, we demonstrate that the C-terminal conserved region, as well as the helicase motifs, of Sgs1 are essential for complementation of MMS sensitivity and suppression of hyper-recombination in sgs1 mutants. In contrast, the highly charged acidic regions, the HRDC domain, and the C-terminal 252 amino acids were dispensable for the complementation of these phenotypes. Surprisingly, the N-terminal 45 amino acids of Sgs1 were absolutely required for the suppression of the above phenotypes. Introduction of missense mutations into the region encoding amino acids 4-13 abolished the ability of Sgsl to complement MMS sensitivity and suppress hyper-recombination in sgs1 mutants, and also prevented its interaction with Top3, indicating that interaction with Top3 via the N-terminal region of Sgs1 is involved in the complementation of MMS sensitivity and the suppression of hyper-recombination.

A novel protein interacts with the Werner's syndrome gene product physically and functionally.

Werner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging. The gene responsible for WS encodes a protein homologous to Escherichia coli RecQ. Here we describe a novel Werner helicase interacting protein (WHIP), which interacts with the N-terminal portion of Werner protein (WRN), containing the exonuclease domain. WHIP, which shows homology to replication factor C family proteins, is conserved from E. coli to human. Ectopically expressed WHIP and WRN co-localized in granular structures in the nucleus. The functional relationship between WHIP and WRN was indicated by genetic analysis of yeast cells. Disruptants of the SGS1 gene of Saccharomyces cerevisiae, which is the WRN homologue in yeast, show an accelerated aging phenotype and high sensitivity to methyl methanesulfonate as compared with wild-type cells. Disruption of the yeast WHIP (yWHIP) gene in wild-type cells and sgs1 disruptants resulted in slightly accelerated aging and enhancement of the premature aging phenotype of sgs1 disruptants, respectively. In contrast, disruption of the yWHIP gene partially alleviated the sensitivity to methyl methanesulfonate of sgs1 disruptants.

Involvement of SGS1 in DNA damage-induced heteroallelic recombination that requires RAD52 in Saccharomyces cerevisiae.

The SGS1 gene of Saccharomyces cerevisiae is homologous to the genes that are mutated in Bloom's syndrome and Werner's syndrome in humans. Disruption of SGS1 results in high sensitivity to methyl methanesulfonate (MMS), poor sporulation, and a hyper-recombination phenotype including recombination between heteroalleles. In this study, we found that SGS1 forms part of the RAD52 epistasis group when cells are exposed to MMS. Exposure to DNA-damaging agents causes a striking, Rad52-dependent, increase in heteroallelic recombination in wild-type cells, but not in sgs1 disruptants. However, in the absence of DNA damage, the frequency of heteroallelic recombination in sgs1 disruptants was several-fold higher than in wild-type cells, as described previously. These results imply a function for Sgs1: it acts to suppress spontaneous heteroallelic recombination, and to promote DNA damage-induced heteroallelic recombination.

Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions.

The SGS1 gene of Saccharomyces cerevisiae is a homologue for the Bloom's syndrome and Werner's syndrome genes. The disruption of the SGS1 gene resulted in very poor sporulation, and the majority of the cells were arrested at the mononucleated stage. The recombination frequency measured by a return-to-growth assay was reduced considerably in sgs1 disruptants. However, double-strand break formation, which is a key event in the initiation of meiotic DNA recombination, occurred; crossover and noncrossover products were observed in the disruptants, although the amounts of these products were slightly decreased compared with those in wild-type cells. The spores produced by sgs1 disruptants showed relatively high viability. The sgs1 spo13 double disruptants sporulated poorly, like the sgs1 disruptants, but spore viability was reduced much more than with either sgs1 or spo13 single disruptants. Disruption of the RED1 or RAD17 gene partially alleviated the poor-sporulation phenotype of sgs1 disruptants, indicating that portions of the population of sgs1 disruptants are blocked by the meiotic checkpoint. The poor sporulation of sgs1 disruptants was complemented with a mutated SGS1 gene encoding a protein lacking DNA helicase activity; however, the mutated gene could suppress neither the sensitivity of sgs1 disruptants to methyl methanesulfonate and hydroxyurea nor the mitotic hyperrecombination phenotype of sgs1 disruptants.

Elevation of sister chromatid exchange in Saccharomyces cerevisiae sgs1 disruptants and the relevance of the disruptants as a system to evaluate mutations in Bloom's syndrome gene.

The SGS1 of Saccharomyces cerevisiae is a homologue of the Bloom's syndrome and Werner's syndrome genes. The sgs1 disruptants show hyperrecombination, higher sensitivity to methyl methanesulfonate and hydroxyurea, and poor sporulation. In this study, we found that sister chromatid exchange was increased in sgs1 disruptants. We made mutated SGS1 genes coding a protein proved to lack DNA helicase activity (sgs1-hd), having equivalent missense mutations found in Bloom's syndrome patients (sgs1-BS1, sgs1-BS2). None of the mutated genes could suppress the higher sensitivity to methyl methanesulfonate and hydroxyurea and the increased frequency of interchromosomal recombination and sister chromatid exchange of sgs1 disruptants. On the other hand, all of the mutant genes were able to complement the poor sporulation phenotype of sgs1 disruptants, although the values were not as high as that of wild-type SGS1.

Different domains of Sgs1 are required for mitotic and meiotic functions.

The SGS1 of Saccharomyces cerevisiae is a homologue for human Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson's syndrome causative genes. Disruptants of SGS1 show high sensitivity to methyl methanesulfonate (MMS) and hydroxyurea, and hyper recombination phenotypes including interchromosomal homologous recombination in mitotic growth. In addition, sgs1 disruptants show poor sporulation and a reduced level of meiotic recombination as assayed by return-to-growth. We examined domains of Sgs1 required for mitotic and meiotic functions of Sgs1 by transfecting variously mutated SGS1 into sgs1 disruptants. The N-terminal 1-401 amino acid region was required for complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations of sgs1 disruptants in mitotic growth and for complementation of poor sporulation and of reduced meiotic recombination. Although the N-terminal 1-125 amino acid region was absolutely required for the complementation of MMS sensitivity and suppression of hyper heteroallelic recombinations in mitotic growth, it was dispensable for the meiotic functions. In contrast, the highly acidic region (400-596 amino acid) was dispensable for the mitotic functions but a deletion of this region affected the meiotic functions. The C-terminal 1271-1350 amino acid region containing a HRDC (helicase and RNaseD C-terminal) domain was dispensable for the mitotic and meiotic functions. Although DNA helicase activity of Sgs1 was not required for Sgs1 to complement the meiotic functions, a deletion of helicase motifs III-IV (842-1046 amino acid) abolished the complementing activity of Sgs1, indicating that a structurally intact helicase domain is necessary for Sgs1 to fulfill its meiotic functions.

The lipid-lowering profile in rodents. AZ-1355, a new dibenzoxazepine derivative.

The lipid-lowering profile of ethyl 10,11-dihydro-4-methoxydibenz[b,f]-(1,4)oxazepine-8-carboxylate (AZ-1355) has been evaluated using clofibrate as a reference compound. This compound is structurally unrelated to any other hypolipidemic agent. AZ-1355 was selected not only for its effect in reducing serum lipids, but also because it inhibits platelet aggregation in vivo and elevates the prostaglandin I2/thromboxane A2 ratio in vitro. It lowers serum total cholesterol in Triton-treated hyperlipidemic mice, and also lowers serum total cholesterol and triglyceride in dietary hyperlipidemic rats. In golden hamsters chosen for further evaluation, AZ-1355 reduced serum, liver and cardiac lipids, improved the beta/alpha-lipoprotein ratio and increased the HDL cholesterol. Thus, it is apparent that the lipid-lowering profile of AZ-1355 differs from that of clofibrate.