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Mlh2 Is an Accessory Factor for DNA Mismatch Repair in Saccharomyces cerevisiae

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      Abstract

      In Saccharomyces cerevisiae, the essential mismatch repair (MMR) endonuclease Mlh1-Pms1 forms foci promoted by Msh2-Msh6 or Msh2-Msh3 in response to mispaired bases. Here we analyzed the Mlh1-Mlh2 complex, whose role in MMR has been unclear. Mlh1-Mlh2 formed foci that often colocalized with and had a longer lifetime than Mlh1-Pms1 foci. Mlh1-Mlh2 foci were similar to Mlh1-Pms1 foci: they required mispair recognition by Msh2-Msh6, increased in response to increased mispairs or downstream defects in MMR, and formed after induction of DNA damage by phleomycin but not double-stranded breaks by I- SceI. Mlh1-Mlh2 could be recruited to mispair-containing DNA in vitro by either Msh2-Msh6 or Msh2-Msh3. Deletion of MLH2 caused a synergistic increase in mutation rate in combination with deletion of MSH6 or reduced expression of Pms1. Phylogenetic analysis demonstrated that the S. cerevisiae Mlh2 protein and the mammalian PMS1 protein are homologs. These results support a hypothesis that Mlh1-Mlh2 is a non-essential accessory factor that acts to enhance the activity of Mlh1-Pms1.

      Author Summary

      Lynch syndrome (hereditary nonpolyposis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. In this syndrome, predisposition to cancer results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the human mismatch repair genes MLH1, MSH2, MSH6 or PMS2. In addition to these genes, various DNA replication factors and the excision factor EXO1 function in the repair of damaged DNA by the MMR pathway. In Saccharomyces cerevisiae, the MLH2 gene encodes a MutL homolog protein whose role in DNA mismatch repair has been unclear. Here, we used phylogenetic analysis to demonstrate that the S. cerevisiae Mlh2 protein and the mammalian Pms1 protein are homologs. A combination of genetics, biochemistry and imaging studies were used to demonstrate that the Mlh1-Mlh2 complex is recruited to mispair-containing DNA by the Msh2-Msh6 and Msh2-Msh3 mispair recognition complexes where it forms foci that colocalize with Mlh1-Pms1 foci (note that scPms1 is the homolog of hPms2) and augments the function of the Mlh1-Pms1 complex. Thus, this work establishes the Mlh1-Mlh2 complex as a non-essential accessory factor that functions in MMR.

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      Most cited references 76

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          MrBayes 3.2: Efficient Bayesian Phylogenetic Inference and Model Choice Across a Large Model Space

          Since its introduction in 2001, MrBayes has grown in popularity as a software package for Bayesian phylogenetic inference using Markov chain Monte Carlo (MCMC) methods. With this note, we announce the release of version 3.2, a major upgrade to the latest official release presented in 2003. The new version provides convergence diagnostics and allows multiple analyses to be run in parallel with convergence progress monitored on the fly. The introduction of new proposals and automatic optimization of tuning parameters has improved convergence for many problems. The new version also sports significantly faster likelihood calculations through streaming single-instruction-multiple-data extensions (SSE) and support of the BEAGLE library, allowing likelihood calculations to be delegated to graphics processing units (GPUs) on compatible hardware. Speedup factors range from around 2 with SSE code to more than 50 with BEAGLE for codon problems. Checkpointing across all models allows long runs to be completed even when an analysis is prematurely terminated. New models include relaxed clocks, dating, model averaging across time-reversible substitution models, and support for hard, negative, and partial (backbone) tree constraints. Inference of species trees from gene trees is supported by full incorporation of the Bayesian estimation of species trees (BEST) algorithms. Marginal model likelihoods for Bayes factor tests can be estimated accurately across the entire model space using the stepping stone method. The new version provides more output options than previously, including samples of ancestral states, site rates, site d N /d S rations, branch rates, and node dates. A wide range of statistics on tree parameters can also be output for visualization in FigTree and compatible software.
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            Author and article information

            Affiliations
            [1 ]Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America
            [2 ]German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581, Heidelberg, Germany
            [3 ]Department of Cellular and Molecular Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America
            [4 ]Moores-UCSD Cancer Center, University of California School of Medicine, San Diego, La Jolla, California, United States of America
            [5 ]Department of Medicine, University of California School of Medicine, San Diego, La Jolla, California, United States of America
            Duke University, United States of America
            Author notes

            The authors have declared that no competing interests exist.

            Conceived and designed the experiments: CSC HH CDP RDK. Performed the experiments: CSC HH AS NB KG CDP. Analyzed the data: CSC HH AS AD CDP RDK. Contributed reagents/materials/analysis tools: NB. Wrote the paper: CSC HH AS NB KG AD CDP RDK.

            Contributors
            Role: Editor
            Journal
            PLoS Genet
            PLoS Genet
            plos
            plosgen
            PLoS Genetics
            Public Library of Science (San Francisco, USA )
            1553-7390
            1553-7404
            May 2014
            8 May 2014
            : 10
            : 5
            24811092 4014439 PGENETICS-D-14-00059 10.1371/journal.pgen.1004327

            This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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            Pages: 14
            Funding
            This work was supported by NIH grants GM50006 (RDK), GM074215 (AD) and CA23100 (AD, RDK), a Damon Runyon Cancer Research Foundation Fellowship (CSC) and to the Harald zur Hausen Fellowship from the German Cancer Research Center, DKFZ (HH). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Research and Analysis Methods
            Model Organisms
            Yeast and Fungal Models
            Biology and Life Sciences
            Genetics
            Mutation

            Genetics

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