Genetic and biochemical characterization of a novel Saccharomyces cerevisiae gene, SRL5

초록

In an attempt to identify novel genes involved in DNA metabolism, we analyzed the proteins that were eluted from the single-stranded DNA cellulose column by the addition of denatured salmon sperm DNA. The eluted proteins were identified with the use of Mass spectrometry. The majority were RPA, the eukaryotic single-stranded DNA binding protein (SSB), and Rim1, the mitochondrial SSB. Minor proteins included histones and some replication or repair proteins such as RFC, ligase, CDC45, msh2 and msh3 proteins. In addition, some uncharacterized proteins were also identified such as YKR015C and YDL156W. YDL156W gene is a putative protein of unknown function, containing three WD domains (WD-40 repeat). We found that the deletion of this gene suppress the lethality of mutations of the essential S phase checkpoint genes, RAD53 and LCD1. On the basis of this observation we refer to this gene as SRL5 (Suppressor of Rad53 null Lethality, or Suppressor of Rad53 and Lcd1). We also found that the srl5D mutant cells were resistant to hydroxyurea and evidenced higher dNTP levels than were observed in the wild-type cells. The mutant cells also showed a significantly faster growth rate and higher dNTP levels at low temperature (16oC) than were observed in the wild-type cells, whereas we detected no differences in the growth rate at 30oC. TAP affinity purification showed that Srl5 interacts with RPA and hitones. The function of Srl5 was discussed on the basis of these observations.