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 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.
Kang
Young-Hoon
YH
Center for DNA Replication and Genome Instability, Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.
Kang
Min-Jung
MJ
Kim
Jeong-Hoon
JH
Lee
Chul-Hwan
CH
Cho
Il-Taeg
IT
Hurwitz
Jerard
J
Seo
Yeon-Soo
YS
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004789:Enzyme Activation; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; C022262:Okazaki fragments; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000123:Acetyltransferases; C107118:ELO2 protein, S cerevisiae; C491240:MPH1 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D053487:DEAD-box RNA Helicases