The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processing Journal Article
| Authors: | Kang, Y. H.; Kang, M. J.; Kim, J. H.; Lee, C. H.; Cho, I. T.; Hurwitz, J.; Seo, Y. S. |
| Article Title: | 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,weisolated 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 Mph1was 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. |
| Keywords: | controlled study; protein expression; unclassified drug; genetics; mutation; nonhuman; molecular genetics; dna replication; protein function; protein analysis; phenotype; metabolism; membrane proteins; enzyme activation; dead box protein; dead-box rna helicases; amines; dna; molecular sequence data; protein purification; saccharomyces cerevisiae; nucleotide sequence; nucleic acids; membrane protein; base sequence; in-vivo; helicase; mutant proteins; saccharomyces cerevisiae proteins; okazaki fragments; amino acids; organic acids; adenosine triphosphate; flap endonuclease 1; okazaki fragment; saccharomyces cerevisiae protein; dna metabolism; endonuclease; exonuclease; hydrolysis; temperature sensitivity; dna helicases; inhibitory effect; n-terminal; atp hydrolysis; dna helicase; dna substrates; duplex structures; endonuclease activities; exonuclease activity; genetic data; helicase activities; in-cell; replication protein a; stimulatory effects; structured substrate; temperature-sensitive growth; gene encoding; substrates; dna2 endonuclease; fungal protein; mutator phenotype 1 protein; acyltransferase; dna2 protein, s cerevisiae; fen1 protein, s cerevisiae; mph1 protein, s cerevisiae; dna nick translation; fungus growth; lethal mutant; acetyltransferases |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 284 |
| Issue: | 16 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2009-04-17 |
| Start Page: | 10376 |
| End Page: | 10386 |
| Language: | English |
| DOI: | 10.1074/jbc.M808894200 |
| PUBMED: | 19181670 |
| PROVIDER: | scopus |
| PMCID: | PMC2667725 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 5" - "Export Date: 30 November 2010" - "CODEN: JBCHA" - "Source: Scopus" |
