Lcp5 acts as a multi-copy suppressor gene of a dna2 mutant by stimulating its nuclease activity

초록

Dna2 is a multifunctional enzyme that has both DNA helicase and endonuclease activities in the same polypeptide. Its endonuclease processes the long flap DNA generated by displacement DNA synthesis by polymerase delta and helicase activity plays a role to facilitate the removal of the flap DNA by its intrinsic endonuclease activity. In this study, we created a mutant yeast strain with the dna2 gene deleted from its chromosome. The mutant strain expresses the wild type Dna2 on one plasmid and the helicase-defective mutant Dna2K1080E on another plasmid. The mutant strain was used in screening for multi-copy suppressors of the dna2 mutant lacking the helicase activity. From this screening, the LCP5 gene was identified as a suppressor and the Lcp5 protein was expressed in E. coli and purified. Using an ELISA assay, we demonstrated that Dna2 interacts with Lcp5. Using a gel mobility shift assay, we showed that Dna2 and Lcp5 form a ternary complex with DNA. Furthermore, purified Lcp5 protein enhanced the nuclease activity of the Dna2 protein. These results suggest that Lcp5 may functionally interact with Dna2 in vivo.