Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3' to 5' DNA helicase.
The MPH1 (mutator pHenotype 1) gene of Saccharomyces cerevisiae was identified on the basis of elevated spontaneous mutation rates of haploid cells deleted for this gene. Further studies showed that MPH1 functions to channel DNA lesions into an error-free DNA repair pathway. The Mph1 protein contains the seven conserved motifs of the superfamily 2 (SF2) family of nucleic acid unwinding enzymes. Genetic analyses have found epistasis of the mph1 deletion with mutations in the RAD52 gene group that mediates homologous recombination and DNA repair by homologous recombination. To begin dissecting the biochemical functions of the MPH1-encoded product, we have expressed it in yeast cells and purified it to near homogeneity. We show that Mph1 has a robust ATPase function that requires single-stranded DNA for activation. Consistent with its homology to members of the SF2 helicase family, we find a DNA helicase activity in Mph1. We present data to demonstrate that the Mph1 DNA helicase activity is fueled by ATP hydrolysis and has a 3' to 5' polarity with respect to the DNA strand on which this protein translocates. The DNA helicase activity of Mph1 is enhanced by the heterotrimeric single-stranded DNA binding protein replication protein A. These results, thus, establish Mph1 as an ATP-dependent DNA helicase, and the availability of purified Mph1 should facilitate efforts at deciphering the role of this protein in homologous recombination and mutation avoidance.
Prakash
Rohit
R
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Krejci
Lumir
L
Van Komen
Stephen
S
Anke Schürer
Kirsten
K
Kramer
Wilfried
W
Sung
Patrick
P
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000235:genetics*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D000939:Epitopes / Q000737:chemistry; D006868:Hydrolysis; D008274:Magnesium / Q000737:chemistry; D009154:Mutation; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D021381:Protein Transport; D020365:RNA Helicases / Q000235:genetics / Q000502:physiology*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D004277:DNA, Single-Stranded; D000939:Epitopes; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D011189:Potassium Chloride; D004247:DNA; D000251:Adenosine Triphosphatases; C491240:MPH1 protein, S cerevisiae; D004265:DNA Helicases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases; D008274:Magnesium