Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair.
A mutant allele of SGS1 of Saccharomyces cerevisiae was identified as a suppressor of the slow-growth phenotype of top3 mutants. We previously reported the involvement of Top3 via the interaction with the N-terminal region of Sgs1 in the complementation of methylmethanesulfonate (MMS) sensitivity and the suppression of hyper recombination of a sgs1 mutant. In this study, we found that several amino acids residues in the N-terminal region of Sgs1 between residues 4 and 33 were responsible for binding to Top3 and essential for complementing the sensitivity to MMS of sgsl cells. Two-hybrid assays suggested that the region of Top3 responsible for the binding to Sgs1 was bipartite, with portion in the N- and C-terminal domains. Although disruption of the SGS1 gene suppressed the semi-lethality of the top3 mutant of strain MR, the sgsl-top3 double mutant grew more slowly and was more sensitive to MMS than the sgsl single mutant, indicating that Top3 plays some role independently of Sgs1. The DNA topoisomerase activity of Top3 was required for the Top3 function to repair DNA damages induced by MMS, as shown by the fact that the TOP3 gene carrying a mutation (Phe for Tyr) at the amino acid residue essential for its activity (residue 356) failed to restore the MMS sensitivity of sgs1-top3 to the level of that of the sgs1 single mutant. Epistatic analysis using the sgs1-top3 double mutant, rad52 mutant and sgs1-top3-rad52 triple mutant indicated that TOP3 belongs to the RAD52 recombinational repair pathway.
Onodera
Ryoko
R
Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Seki
Masayuki
M
Ui
Ayako
A
Satoh
Yurie
Y
Miyajima
Atsuko
A
Onoda
Fumitoshi
F
Enomoto
Takemi
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D004265:DNA Helicases / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004271:DNA, Fungal / Q000502:physiology; D017353:Gene Deletion; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D053484:RecQ Helicases; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; D053484:RecQ Helicases; D004264:DNA Topoisomerases, Type I