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

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.

[1]  P. Burgers.  Polymerase Dynamics at the Eukaryotic DNA Replication Fork* , 2009, Journal of Biological Chemistry.

[2]  R. Bambara,et al.  Pif1 Helicase Directs Eukaryotic Okazaki Fragments toward the Two-nuclease Cleavage Pathway for Primer Removal* , 2008, Journal of Biological Chemistry.

[3]  P. Burgers,et al.  Title Partial reconstitution of DNA large loop repair with purifiedproteins from Saccharomyces cerevisiae , 2018 .

[4]  K. Myung,et al.  Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination , 2008, The Journal of cell biology.

[5]  B. Van Houten,et al.  DNA tandem lesion repair by strand displacement synthesis and nucleotide excision repair. , 2008, Biochemistry.

[6]  Andrzej Stasiak,et al.  The Fanconi anemia protein FANCM can promote branch migration of Holliday junctions and replication forks. , 2008, Molecular cell.

[7]  Ellen Fanning,et al.  A dynamic model for replication protein A (RPA) function in DNA processing pathways , 2006, Nucleic acids research.

[8]  J. Hurwitz,et al.  Isolation of human Dna2 endonuclease and characterization of its enzymatic properties , 2006, Nucleic acids research.

[9]  Yeon-Soo Seo,et al.  In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki fragment processing , 2005, Nucleic acids research.

[10]  P. Sung,et al.  Saccharomyces cerevisiae MPH1 Gene, Required for Homologous Recombination-mediated Mutation Avoidance, Encodes a 3′ to 5′ DNA Helicase* , 2005, Journal of Biological Chemistry.

[11]  Jay Z. Parrish,et al.  Novel function of the flap endonuclease 1 complex in processing stalled DNA replication forks , 2005, EMBO reports.

[12]  K. Komori,et al.  Cooperation of the N-terminal Helicase and C-terminal Endonuclease Activities of Archaeal Hef Protein in Processing Stalled Replication Forks* , 2004, Journal of Biological Chemistry.

[13]  T. Ushimaru,et al.  Fission Yeast Dna2 Is Required for Generation of the Telomeric Single-Strand Overhang , 2004, Molecular and Cellular Biology.

[14]  R. Bambara,et al.  Flap endonuclease 1: a central component of DNA metabolism. , 2004, Annual review of biochemistry.

[15]  Janaki Veeraraghavan,et al.  Saccharomyces cerevisiae Flap Endonuclease 1 Uses Flap Equilibration To Maintain Triplet Repeat Stability , 2004, Molecular and Cellular Biology.

[16]  Christian J. Rudolph,et al.  Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair. , 2004, Genetics.

[17]  K. Bae,et al.  Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro. , 2003, Nucleic acids research.

[18]  D. Gordenin,et al.  Okazaki Fragment Maturation in Yeast , 2003, The Journal of Biological Chemistry.

[19]  R. Bambara,et al.  The Protein Components and Mechanism of Eukaryotic Okazaki Fragment Maturation , 2003, Critical reviews in biochemistry and molecular biology.

[20]  D. W. Kim,et al.  Coupling of DNA Helicase and Endonuclease Activities of Yeast Dna2 Facilitates Okazaki Fragment Processing* , 2002, The Journal of Biological Chemistry.

[21]  L. Hoopes,et al.  Dynamic Localization of an Okazaki Fragment Processing Protein Suggests a Novel Role in Telomere Replication , 2002, Molecular and Cellular Biology.

[22]  U. Hübscher,et al.  Replication of the lagging strand: a concert of at least 23 polypeptides. , 2001, Molecules and cells.

[23]  G. Dianov,et al.  Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity , 2001, The EMBO journal.

[24]  Jung-Ae Kim,et al.  RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes , 2001, Nature.

[25]  Jung-Ae Kim,et al.  Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease. , 2001, Nucleic acids research.

[26]  M. Dixon,et al.  Inhibition of FEN-1 processing by DNA secondary structure at trinucleotide repeats. , 1999, Molecular cell.

[27]  A. Hinnen,et al.  Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach , 1999, Molecular and General Genetics MGG.

[28]  R. Wellinger,et al.  Accumulation of Single-Stranded DNA and Destabilization of Telomeric Repeats in Yeast Mutant Strains Carrying a Deletion of RAD27 , 1999, Molecular and Cellular Biology.

[29]  G. Marsischky,et al.  Eukaryotic DNA mismatch repair. , 1999, Current opinion in genetics & development.

[30]  Kyoung-Hwa Lee,et al.  Dna2 of Saccharomyces cerevisiae Possesses a Single-stranded DNA-specific Endonuclease Activity That Is Able to Act on Double-stranded DNA in the Presence of ATP* , 1998, The Journal of Biological Chemistry.

[31]  Robert E. Johnson,et al.  Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication , 1998, Current Genetics.

[32]  Judith L Campbell,et al.  A yeast replicative helicase, Dna2 helicase, interacts with yeast FEN-1 nuclease in carrying out its essential function , 1997, Molecular and cellular biology.

[33]  R. Bambara,et al.  Enzymes and Reactions at the Eukaryotic DNA Replication Fork* , 1997, The Journal of Biological Chemistry.

[34]  M. O’Donnell,et al.  Reconstitution of human replication factor C from its five subunits in baculovirus-infected insect cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Judith L. Campbell,et al.  DNA2 Encodes a DNA Helicase Essential for Replication of Eukaryotic Chromosomes (*) , 1995, The Journal of Biological Chemistry.

[36]  Judith L Campbell,et al.  A yeast gene required for DNA replication encodes a protein with homology to DNA helicases. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[37]  Robert E. Johnson,et al.  Requirement of the yeast RTH1 5' to 3' exonuclease for the stability of simple repetitive DNA. , 1995, Science.

[38]  C. Pittenger,et al.  Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene , 1995, Journal of bacteriology.

[39]  J. Turchi,et al.  Enzymatic completion of mammalian lagging-strand DNA replication. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[40]  M. Lieber,et al.  The characterization of a mammalian DNA structure‐specific endonuclease. , 1994, The EMBO journal.

[41]  J. Turchi,et al.  The calf 5'- to 3'-exonuclease is also an endonuclease with both activities dependent on primers annealed upstream of the point of cleavage. , 1994, The Journal of biological chemistry.

[42]  P. Burgers,et al.  The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[43]  P. Burgers.  Mammalian cyclin/PCNA (DNA polymerase delta auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III. , 1988, Nucleic acids research.

[44]  J. Campbell,et al.  Isolation of yeast DNA replication mutants in permeabilized cells. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[45]  R. Benbow,et al.  DNA primase activity associated with DNA polymerase alpha from Xenopus laevis ovaries. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[46]  R. Conaway,et al.  A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos. , 1982, Proceedings of the National Academy of Sciences of the United States of America.