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The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing.

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 dna2Delta405N, which lacks the N-terminal 405 amino acids of Dna2. However, Mph1 did not stimulate the endonuclease activity of the Dna2Delta405N 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 dna2Delta405N 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.

SUBMITTER: Kang YH

PROVIDER: S-EPMC2667725 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing.

Kang Young-Hoon YH Kang Min-Jung MJ Kim Jeong-Hoon JH Lee Chul-Hwan CH Cho Il-Taeg IT Hurwitz Jerard J Seo Yeon-Soo YS

The Journal of biological chemistry 20090129 16

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 MP ...[more]

PMID: 19181670

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Project description:RNA-DNA hybrids are common in chromosomal DNA. Persistent RNA-DNA hybrids result in replication fork stress, DNA breaks, and neurological disorders in humans. During replication, Okazaki fragment synthesis relies on frequent RNA primer placement, providing one of the most prominent forms of covalent RNA-DNA strands in vivo The mechanism of Okazaki fragment maturation, which involves RNA removal and subsequent DNA replacement, in bacteria lacking RNase HI remains unclear. In this work, we reconstituted repair of a linear model Okazaki fragment in vitro using purified recombinant enzymes from Bacillus subtilis We showed that RNase HII and HIII are capable of incision on Okazaki fragments in vitro and that both enzymes show mild stimulation by single-stranded DNA binding protein (SSB). We also showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation in vitro Furthermore, we found that YpcP is a 5' to 3' nuclease that can act on a wide variety of RNA- and DNA-containing substrates and exhibits preference for degrading RNA in model Okazaki fragments. Together, our data showed that RNase HIII and DNA polymerase I provide the primary pathway for Okazaki fragment maturation, whereas YpcP also contributes to the removal of RNA from an Okazaki fragment in vitroIMPORTANCE All cells are required to resolve the different types of RNA-DNA hybrids that form in vivo When RNA-DNA hybrids persist, cells experience an increase in mutation rate and problems with DNA replication. Okazaki fragment synthesis on the lagging strand requires an RNA primer to begin synthesis of each fragment. The mechanism of RNA removal from Okazaki fragments remains unknown in bacteria that lack RNase HI. We examined Okazaki fragment processing in vitro and found that RNase HIII in conjunction with DNA polymerase I represent the most efficient repair pathway. We also assessed the contribution of YpcP and found that YpcP is a 5' to 3' exonuclease that prefers RNA substrates with activity on Okazaki and flap substrates in vitro.

Dataset Information

The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing.

Publications

The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing.

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