Structural and functional conservation of error-free DNA postreplication repair in Schizosaccharomyces pombe.
DNA postreplication repair (PRR) is a cellular process by which cells survive replication-blocking lesions without removing the lesion. In the budding yeast Saccharomyces cerevisiae, MMS2 plays a key role in the error-free PRR pathway: the mms2 null mutant displays an increased spontaneous mutation rate and sensitivity to a variety of DNA damaging agents. In contrast, its human homologs appear to play a different role. In order to address whether the MMS2-mediated PRR pathway is conserved in eukaryotes, we isolated a Schizosaccharomyces pombe cDNA homologous to MMS2, which we named spm2(+). Using spm2(+) as a bait in a yeast two-hybrid screen, we identified a fission yeast cDNA homologous to UBC13 from various species and named it spu13(+). Two-hybrid analysis confirmed physical interaction between Spm2 and Spu13, and between Spm2 and budding yeast Ubc13. Genetic analysis shows that both spm2(+) and spu13(+) are able to functionally complement the corresponding budding yeast mutants. Furthermore, deletion of either spm2(+), spu13(+) or both genes from fission yeast results in an increased sensitivity to DNA damaging agents, suggesting that spm2(+) and spu13(+) indeed function in PRR. The fact that the spm2(-) spu13(-) double mutant showed sensitivity similar to that of the single mutant indicates that these two gene products act at the same step. Hence, our data strongly support the hypothesis that the PRR function mediated by UBC13-MMS2 is conserved throughout eukaryotes.
Brown
Morgan
M
Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Canada.
Zhu
Yu
Y
Hemmingsen
Sean M
SM
Xiao
Wei
W
D000595:Amino Acid Sequence; D002470:Cell Survival; D003001:Cloning, Molecular; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D017346:Protein Serine-Threonine Kinases; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases
D017931:DNA Primers; D018076:DNA, Complementary; D005656:Fungal Proteins; C112520:MMS2 protein, S cerevisiae; C091267:SIP2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D015534:Trans-Activators; C513353:Mms2 protein, S pombe; C471610:UBC13 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D017346:Protein Serine-Threonine Kinases; D008025:Ligases