The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing

doi:10.1074/jbc.M808894200

The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing*

^‡Center for DNA Replication and Genome Instability, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea and the ^§Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

1 To whom correspondence should be addressed. Tel.: 82-42-350-2637; Fax: 82-42-350-2610; E-mail: yeonsooseo{at}kaist.ac.kr.

Abstract

Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3′ to 5′ DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated MPH1 as a multicopy suppressor of the dna2K1080E helicase-negative lethal mutant. Purified Mph1 stimulated the endonuclease activities of both Fen1 and Dna2, which act faithfully in the processing of Okazaki fragments. This stimulation required neither ATP hydrolysis nor the helicase activity of Mph1. Multicopy expression of MPH1 also suppressed the temperature-sensitive growth defects in cells expressing dna2Δ405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Δ405N mutant protein. The stimulation of Fen1 by Mph1 was limited to flap-structured substrates; Mph1 hardly stimulated the 5′ to 3′ exonuclease activity of Fen1. Mph1 binds to flap-structured substrate more efficiently than to nicked duplex structures, suggesting that the stimulatory effect of Mph1 is exerted through its binding to DNA substrates. In addition, we found that Mph1 reversed the inhibitory effects of replication protein A on Fen1 activity. Our biochemical and genetic data indicate that the in vivo suppression of Dna2 defects observed with both dna2K1080E and dna2Δ405N mutants occur via stimulation of Fen1 activity. These findings suggest that Mph1 plays an important, although not essential, role in processing of Okazaki fragments by facilitating the formation of ligatable nicks.

Footnotes

↵2 The abbreviations used are: pol, polymerase; RPA, replication protein A; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; ATPγS, adenosine 5′-O-(thiotriphosphate); DTT, dithiothreitol; BSA, bovine serum albumin; nt, nucleotide(s); SD, synthetic-dropout medium.
↵3 C. H. Lee and Y. S. Seo, unpublished data.
↵4 C. H. Lee, unpublished observation.
↵5 Y.-S. Seo, unpublished observation.
↵* This work was supported by Korea Science and Engineering Foundation Grants funded by the Ministry of Education, Science and Technology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3.
- Received November 24, 2008.
- Revision received January 28, 2009.
The American Society for Biochemistry and Molecular Biology, Inc.