Screening and characterization of multi-copy suppressors for the Dna2 mutants in Saccharomyces cerevisiae

초록

Saccharomyces cerevisiae Dna2 protein involved in Okazaki fragment processing is a multifunctional enzyme with two essential enzyme activities, endonuclease and helicase. The endonuclease activity is essential to remove RNA-DNA primers of Okazaki fragments, while the helicase facilitates the removal of secondary structures formed in the flap structure during Okazaki fragment processing. In an effort to identify genes interacting genetically with DNA2, multi-copy suppressors (gene dosage-dependent suppressors) were screened that rescue the lethality of Dna2 mutants devoid of helicase (dna2KE) or endonuclease (dna2DA) activities. As results, several genes with unknown functions were isolated as well as the previously identified suppressors such as RPA and Fen-1. A putative helicase, MPH1, was identied as a suppressor for dna2KE. The purified Mph1 protein possessed both ATPase and helicase activities, suggesting that it might replace the function of Dna2 helicase. The suppressors for dna2DA mutants are likely to be involved in DNA repair, transcriptional regulation, or translation processes. Surprisingly, all these suppressors also rescued the growth defect of other Dna2 mutants. The suppression mechanisms by these suppressors are currently under investigation.