Saccharomyces cerevisiae MPH1 Gene, Required for Homologous Recombination-mediated Mutation Avoidance, Encodes a 3′ to 5′ DNA Helicase

Abstract

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.DNA helicases are involved in essentially all the metabolic pathways that entail melting of the DNA double helix, such as DNA replication, homologous recombination (HR), 1 and DNA repair reactions (1, 2). These enzymes invariably possess the ability to hydrolyze a nucleoside triphosphate (most often ATP) when DNA (in general ssDNA) is present, and they are capable of utilizing the free energy derived from the nucleotide hydrolytic reaction to translocate on the DNA. Upon encountering a duplex region, the base pairing in the DNA is disrupted to result in the separation of DNA strands. Certain DNA helicase enzymes, e.g. those that belong to the RecQ helicase family, are specific for DNA structures that arise during homologous recombination and when replication forks stall (1, 2). Interestingly, the translocase activity of some helicases enables them to dislodge proteins from their path of movement, and this property of the helicase enzymes in question is believed to be germane for their biological roles (3-6).A number of DNA helicases, including the yeast Srs2 and Sgs1 proteins and the human BLM and WRN proteins, play roles in modulating homologous recombination efficiency and outcome, preventing replication fork demise, and the general maintenance of the genome (1, 2, 7). In humans, inactivation of the BLM and WRN helicases can lead to Bloom syndrome and Werner syndrome, which are marked by cancer predisposition. These findings aptly underscore the importance for delineating the functions of DNA helicases in homologous recombination and related processes.The Saccharomy...

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

“…Mph1 is an ATP-dependent helicase with 39-59 polarity (Prakash et al 2005), which was initially identified in a screen for mutants with increased spontaneous mutation rate as measured by the formation of canavanine resistant colonies (Entian et al 1999). Based on the epistatic relationship of MPH1 and RAD51 with respect to canavanine reversion rate and MMS damage sensitivity, Schü rer et al (2004) proposed that Mph1 functions in one of several HR pathways.…”

“…We used a DNA mobility shift assay with radiolabeled DNA substrates to investigate whether purified Mph1 protein (Prakash et al 2005) has affinity for various DNA structures, including the D-loop and the HJ that are generated during recombination. In these experiments, we incubated Mph1 with both the D-loop substrate containing a 59 tail and one of the remaining DNA substrates, dsDNA, ssDNA, DNA bubble, and the HJ.…”

“…4A), predicted to be deficient in ATPase and helicase activities, in yeast cells. Using the procedure that we originally devised for wild-type Mph1 (Prakash et al 2005), the mph1 D209N protein was purified to near homogeneity (Supplemental Fig. 4B).…”

“…The D209N mutation was introduced into Mph1 using the QuickChange Site-Directed Mutagenesis kit (Stratagene) and pYES2-Mph1 (2m, URA3, and GAL1-MPH1) (Prakash et al 2005) as the template. The protein expression plasmid pYES2-mph1 D209N was maintained in the protease-deficient strain BJ5464 (Supplemental Table 1), and protein expression was induced by galactose addition.…”

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…It is a 3 -5 DNA helicase (Bennett et al, 1998) interacting with topoisomerases Top3 and Top2 (Gangloff et al, 1994;Watt et al, 1995). Another ATP-dependent DNA helicase with 3 -5 polarity, playing a role in genome stability, is encoded by the MPH1 gene from Saccharomyces cerevisiae (Scheller et al, 2000;Prakash et al, 2005). We have previously reported that mph1 mutants show increased sensitivity to genotoxic agents and elevated spontaneous mutation rate, dependent on a functional TLS (Entian et al, 1999;Scheller et al, 2000).…”