Sgs1 Helicase Activity Is Required for Mitotic but Apparently Not for Meiotic Functions

Abstract

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 poorsporulation 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.Proteins having DNA helicase activity play important roles in many processes involving DNA, such as replication, repair, and recombination. The product of the Escherichia coli recQ gene, which has DNA helicase activity, is a member of the RecF pathway of recombination. The recF mutants lack conjugal recombination proficiency and UV resistance in the background of recBCD (lacking active endonuclease V) and sbcBC (lacking active exonuclease I), and recQ deletion mutants in the background of recBC sbcBC display UV and methyl methanesulfonate (MMS) sensitivity (22,30).We (38) and Puranam and Blackshear (32) cloned cDNAs encoding a RecQ homologue of human cells, DNA helicase Q1 and RECQL, respectively. Since these are the same gene, we tentatively designated this gene RECQL1. We also cloned a gene of Saccharomyces cerevisiae encoding a protein having DNA helicase motifs with high homology to those of E. coli RecQ and human ATPase RECQL1. This gene soon was found to be identical to the SGS1 (slow growth suppressor 1) gene.A mutant allele of the SGS1 gene was identified as a suppressor of the slow-growth phenotype of top3 mutants (11). Two-hybrid experiments indicated that the yeast Sgs1 protein interacts with DNA topoisomerase III (Top3) (11) as well as DNA topoisomerase II (50). The protein encoded by the SGS1 gene has seven conserved helicase motifs, and Sgs1 was shown to actually have DNA helicase activity (3, 23). Deletion mutants of the SGS1 gene showed a reduction in the fidelity of chromosome segregation during mitosis and meiosis (50, 51); mitotic hyperrecombination phenotypes in interchromosomal homo...

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

“…The yeast strains used in this study are as follows : MR101 , ds1 (MR101 in sgs1 ∆::AUR1/sgs1 ∆::AUR1). Construction of ds1 mutants was described previously (Miyajima et al, 2000).…”

“…sgs1 mutations were shown to be synthetic lethal in combination with mutations in the SRS2 gene (Lee et al, 1999). Finally, sgs1 mutants also show defects in sporulation and meiotic recombination (Watt et al, 1995;Gangloff et al, 1999;Miyajima et al, 2000).…”

“…We recently demonstrated that 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 neither complement the sensitivity of sgs1 disruptants to MMS and HU nor suppress the mitotic hyper recombination phenotypes of sgs1 disruptants (Miyajima et al, 2000;Onoda et al, 2000a). Thus, it is likely that Sgs1 plays different roles in meiosis and in mitotic growth.…”

“…Deletion mutants of the SGS1 gene showed a reduction in the fidelity of chromosome segregation during mitosis and meiosis (Watt et al, 1995;, premature aging (Sinclair et al, 1997a;Sinclair and Guarente, 1997b), and mitotic hyper recombination phenotypes including interchromosomal homologous recombination, intrachromosomal excision recombination, ectopic recombination (Watt et al, 1996), unequal sister-chromatid recombination (Onoda et al, 2000a), and illegitimate recombination (Yamagata et al, 1998). The sgs1 mutants showed higher sensitivity to methyl methanesulfonate (MMS) (Frei and Gasser, 2000;Miyajima et al, 2000;Onoda et al, 2000a) and hydroxyurea (HU) (Miyajima et al, 2000;Onoda et al, 2000a) than wild-type cells. Although sgs1 haploid cells did not show apparent sensitivity to ionizing radiation or UV light (Watt et al, 1996), recently it was demonstrated that sgs1 diploid cells showed moderate sensitivity to X-ray (Gangloff et al, 2000) and UV irradiation (Gangloff et al, 2000;Onoda et al, 2000b).…”

ArecQGene Homolog from the Basidiomycetous MushroomLentinula edodes: Sequence Analysis and Expression

“…The sgs1 defective mutation causes S. cerevisiae a slight delay in growth and sensitivity to the DNA-damaging compound methylmethanesulfonate (MMS). 39) We examined whether the Le.recQ gene is able to complement these phenotypes of the sgs1 mutant. S. cerevisiae 966NS-1 carrying the Le.recQ cDNA expression plasmid pYES2-Le.recQ, i.…”