Rad5-dependent DNA Repair Functions of the Saccharomyces cerevisiae FANCM Protein Homolog Mph1

Genome Instability Section, Genetics, and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA
Journal of Biological Chemistry (Impact Factor: 4.57). 06/2012; 287(32):26563-75. DOI: 10.1074/jbc.M112.369918
Source: PubMed


Interstrand cross-links (ICLs) covalently link complementary DNA strands, block DNA replication, and transcription and must be removed to allow cell survival. Several pathways, including the Fanconi anemia (FA) pathway, can faithfully repair ICLs and maintain genomic integrity; however, the precise mechanisms of most ICL repair processes remain enigmatic. In this study we genetically characterized a conserved yeast ICL repair pathway composed of the yeast homologs (Mph1, Chl1, Mhf1, Mhf2) of four FA proteins (FANCM, FANCJ, MHF1, MHF2). This pathway is epistatic with Rad5-mediated DNA damage bypass and distinct from the ICL repair pathways mediated by Rad18 and Pso2. In addition, consistent with the FANCM role in stabilizing ICL-stalled replication forks, we present evidence that Mph1 prevents ICL-stalled replication forks from collapsing into double-strand breaks. This unique repair function of Mph1 is specific for ICL damage and does not extend to other types of damage. These studies reveal the functional conservation of the FA pathway and validate the yeast model for future studies to further elucidate the mechanism of the FA pathway.

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Available from: Dana Branzei, Oct 11, 2015
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    • "In vivo tests were performed using epitope-tagged Mph1, Mhf1, and Smc5 expressed at endogenous levels from their chromosomal loci. Mhf1 coimmunoprecipitated with Mph1, consistent with their reported interaction (Fig. 1A, top; Daee et al. 2012). In addition, Mhf1 coimmunoprecipitated with Smc5, suggesting that scMHF also associates with Smc5 in vivo (Fig. 1A, bottom). "
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    ABSTRACT: Budding yeast Mph1 helicase and its orthologs drive multiple DNA transactions. Elucidating the mechanisms that regulate these motor proteins is central to understanding genome maintenance processes. Here, we show that the conserved histone fold MHF complex promotes Mph1-mediated repair of damaged replication forks but does not influence the outcome of DNA double-strand break repair. Mechanistically, scMHF relieves the inhibition imposed by the structural maintenance of chromosome protein Smc5 on Mph1 activities relevant to replication-associated repair through binding to Mph1 but not DNA. Thus, scMHF is a function-specific enhancer of Mph1 that enables flexible response to different genome repair situations. © 2015 Xue et al.; Published by Cold Spring Harbor Laboratory Press.
    Genes & development 05/2015; 29(10). DOI:10.1101/gad.259143.115 · 10.80 Impact Factor
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    • "This protein family is capable of fork reversal and promotes recombination at stalled replication forks (Blackford et al. 2012; Gari et al. 2008; Nandi and Whitby 2012; Prakash et al. 2005; Schurer et al. 2004; Sun et al. 2008; Zheng et al. 2011). Recent work suggests that budding yeast Mph1/FancM operates downstream of ScRad5 in repair of interstrand crosslinks (Daee et al. 2012). S. pombe has two Fml proteins: Fml1 and its paralogue, Fml2, which plays a minor role (Sun et al. 2008). "
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    ABSTRACT: Schizosaccharomyces pombe Rad8 is a conserved protein homologous to S. cerevisiae Rad5 and human HLTF that is required for error-free postreplication repair by contributing to polyubiquitylation of PCNA. It has three conserved domains: an E3 ubiquitin ligase motif, a SNF2-family helicase domain, and a family-specific HIRAN domain. Data from humans and budding yeast suggest that helicase activity contributes to replication fork regression and template switching for fork restart. We constructed specific mutations in the three conserved domains and found that both the E3 ligase and HIRAN domains are required for proper response to DNA damage caused by a variety of agents. In contrast, mutations in the helicase domain show no phenotypes in a wild-type background. To determine whether Rad8 functionally overlaps with other helicases, we compared the phenotypes of single and double mutants with a panel of 23 nonessential helicase mutants, which we categorized into five phenotypic groups. Synthetic phenotypes with rad8∆ were observed for mutants affecting recombination, and a rad8 helicase mutation affected the HU response of a subset of recombination mutants. Our data suggest that the S. pombe Rad8 ubiquitin ligase activity is important for response to a variety of damaging agents, while the helicase domain plays only a minor role in modulating recombination-based fork restart during specific forms of replication stress.
    G3-Genes Genomes Genetics 05/2014; 4(8). DOI:10.1534/g3.114.011346 · 3.20 Impact Factor
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    • "Physical and negative genetic interactions have been observed between Mph1 and Srs2 (Chiolo et al. 2005; St Onge et al. 2007). mph1 mutants share with both elg1 and srs2 genome instability phenotypes (Daee et al. 2012), suggesting that Mph1 may be sharing repair functions with both genes. DDC1: Ddc1 forms part of a PCNA-like molecule (called the 9-1-1 clamp). "
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    ABSTRACT: Elg1 and Srs2 are two proteins involved in maintaining genome stability in yeast. After DNA damage, the homotrimeric clamp PCNA, which provides stability and processivity to DNA polymerases and serves as a docking platform for DNA repair enzymes, undergoes modification by the ubiquitin-like molecule SUMO. PCNA SUMOylation helps recruit Srs2 and Elg1 to the replication fork. In the absence of Elg1, both SUMOylated PCNA and Srs2 accumulate at the chromatin fraction, indicating that Elg1 is required for removing SUMOylated PCNA and Srs2 from DNA. Despite this interaction, which suggests that the two proteins work together, double mutants elg1Δ srs2Δ have severely impaired growth as haploids and exhibit synergistic sensitivity to DNA damage and a synergistic increase in gene conversion. In addition, diploid elg1Δ srs2Δ double mutants are dead, which implies that an essential function in the cell requires at least one of the two gene products for survival. To gain information about this essential function, we have carried out a high copy number suppressor screen to search for genes that, when overexpressed, suppress the synthetic lethality between elg1Δ and srs2Δ. We report the identification of 36 such genes, which are enriched for functions related to DNA- and chromatin-binding, chromatin packaging and modification, and mRNA export from the nucleus.
    G3-Genes Genomes Genetics 05/2013; 3(5):917-926. DOI:10.1534/g3.113.005561 · 3.20 Impact Factor
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