Mutations affecting a putative MutLalpha endonuclease motif impact multiple mismatch repair functions.

Mutations in DNA mismatch repair (MMR) lead to increased mutation rates and higher recombination between similar, but not identical sequences, as well as resistance to certain DNA methylating agents. Recently, a component of human MMR machinery, MutLalpha, has been shown to display a latent endonuclease activity. The endonuclease active site appears to include a conserved motif, DQHA(X)(2)E(X)(4)E, within the COOH-terminus of human PMS2. Substitution of the glutamic acid residue (E705) abolished the endonuclease activity and mismatch-dependent excision in vitro. Previously, we showed that the PMS2-E705K mutation and the corresponding mutation in Saccharomyces cerevisiae were both recessive loss of function alleles for mutation avoidance in vivo. Here, we show that mutations impacting this endonuclease motif also significantly affect MMR-dependent suppression of homeologous recombination in yeast and responses to S(n)1-type methylating agents in both yeast and mammalian cells. Thus, our in vivo results suggest that the endonuclease activity of MutLalpha is important not only in MMR-dependent mutation avoidance but also for recombination and damage response functions.

[1]  P. Karran,et al.  Genomic instability and tolerance to alkylating agents. , 1996, Cancer surveys.

[2]  P. Tran,et al.  Functional Studies on the Candidate ATPase Domains of Saccharomyces cerevisiae MutLα , 2000, Molecular and Cellular Biology.

[3]  R. Fishel.  Signaling mismatch repair in cancer , 1999, Nature Medicine.

[4]  A. Bellacosa.  Functional interactions and signaling properties of mammalian DNA mismatch repair proteins , 2001, Cell Death and Differentiation.

[5]  M. Turker,et al.  A mouse kidney cell line with a G:C --> C:G transversion mutator phenotype. , 2002, Mutation research.

[6]  A. Bellacosa.  Role of MED1 (MBD4) Gene in DNA repair and human cancer , 2001, Journal of cellular physiology.

[7]  S. Dudley,et al.  Novel PMS1 Alleles Preferentially Affect the Repair of Primer Strand Loops during DNA Replication , 2005, Molecular and Cellular Biology.

[8]  R. Rothstein,et al.  An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisiae. , 1999, Genetics.

[9]  R. Liskay,et al.  The human MLH1 cDNA complements DNA mismatch repair defects in Mlh1-deficient mouse embryonic fibroblasts. , 1999, Cancer research.

[10]  P. Modrich,et al.  Mechanism of 5'-directed excision in human mismatch repair. , 2003, Molecular cell.

[11]  T. Prolla,et al.  Dual requirement in yeast DNA mismatch repair for MLH1 and PMS1, two homologs of the bacterial mutL gene , 1994, Molecular and cellular biology.

[12]  Q. Zhan,et al.  The mismatch repair system is required for S-phase checkpoint activation , 2003, Nature Genetics.

[13]  P. Hsieh,et al.  ATR Kinase Activation Mediated by MutSα and MutLα in Response to Cytotoxic O6-Methylguanine Adducts , 2006 .

[14]  P. Modrich,et al.  Endonucleolytic Function of MutLα in Human Mismatch Repair , 2006, Cell.

[15]  R. Rothstein,et al.  Holliday Junctions Accumulate in Replication Mutants via a RecA Homolog-Independent Mechanism , 1997, Cell.

[16]  Ludovic C. Gillet,et al.  Homologous Recombination Rescues Mismatch-Repair-Dependent Cytotoxicity of SN1-Type Methylating Agents in S. cerevisiae , 2005, Current Biology.

[17]  S. Jinks-Robertson,et al.  Role of mismatch repair in the fidelity of RAD51- and RAD59-dependent recombination in Saccharomyces cerevisiae. , 2003, Genetics.

[18]  M. Hawn,et al.  Evidence for a connection between the mismatch repair system and the G2 cell cycle checkpoint. , 1995, Cancer research.

[19]  Josef Jiricny,et al.  Identification of hMutLβ, a Heterodimer of hMLH1 and hPMS1* , 1999, The Journal of Biological Chemistry.

[20]  P. Modrich,et al.  Mismatch repair in replication fidelity, genetic recombination, and cancer biology. , 1996, Annual review of biochemistry.

[21]  T. Kunkel,et al.  Resistance to 6-thioguanine in mismatch repair-deficient human cancer cell lines correlates with an increase in induced mutations at the HPRT locus. , 1998, Carcinogenesis.

[22]  C. Ban,et al.  DNA mismatch repair system , 2006, The FEBS journal.

[23]  S. Jinks-Robertson,et al.  Regulation of mitotic homeologous recombination in yeast. Functions of mismatch repair and nucleotide excision repair genes. , 2000, Genetics.

[24]  T. Prolla,et al.  Functional domains of the Saccharomyces cerevisiae Mlh1p and Pms1p DNA mismatch repair proteins and their relevance to human hereditary nonpolyposis colorectal cancer-associated mutations , 1997, Molecular and cellular biology.

[25]  J. Jiricny,et al.  Mismatch repair and DNA damage signalling. , 2004, DNA repair.

[26]  P. Modrich.  Mechanisms in Eukaryotic Mismatch Repair* , 2006, Journal of Biological Chemistry.

[27]  W. Edelmann,et al.  Mismatch repair proteins as sensors of alkylation DNA damage. , 2006, Cancer cell.

[28]  P. Modrich,et al.  Human Mismatch Repair , 2005, Journal of Biological Chemistry.

[29]  A. Tomkinson,et al.  Reconstitution of 5′-Directed Human Mismatch Repair in a Purified System , 2005, Cell.

[30]  T. Kunkel,et al.  Purification of eukaryotic MutL homologs from Saccharomyces cerevisiae using self-affinity technology. , 2001, Protein Expression and Purification.

[31]  C. A. Coulson,et al.  The distribution of the numbers of mutants in bacterial populations , 1949, Journal of Genetics.

[32]  R. Liskay,et al.  Contribution of Human Mlh1 and Pms2 ATPase Activities to DNA Mismatch Repair* , 2002, The Journal of Biological Chemistry.

[33]  Mimi Y. Kim,et al.  Dominant effects of an Msh6 missense mutation on DNA repair and cancer susceptibility. , 2004, Cancer cell.

[34]  Robin J. Leach,et al.  Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer , 1993, Cell.

[35]  T. Petes,et al.  Alleles of the yeast Pms1 mismatch-repair gene that differentially affect recombination- and replication-related processes. , 2002, Genetics.

[36]  R. Liskay,et al.  The E705K mutation in hPMS2 exerts recessive, not dominant, effects on mismatch repair. , 2007, Cancer letters.

[37]  R. Fleischmann,et al.  Mutation of a mutL homolog in hereditary colon cancer. , 1994, Science.

[38]  T. Kunkel,et al.  Purification of Eukaryotic MutL Homologs from Saccharomyces cerevisiae Using Self-Cleaving Affinity Technology , 2001 .

[39]  O. Ozier-Kalogeropoulos,et al.  A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. , 1993, Nucleic acids research.

[40]  F. Sherman.  Getting started with yeast. , 1991, Methods in enzymology.

[41]  P. Modrich,et al.  DNA Mismatch Repair: Functions and Mechanisms , 2006 .

[42]  D. Ward,et al.  Mutation in the DNA mismatch repair gene homologue hMLH 1 is associated with hereditary non-polyposis colon cancer , 1994, Nature.

[43]  N. Copeland,et al.  The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. , 1993, Cell.

[44]  J. Jiricny.  The multifaceted mismatch-repair system , 2006, Nature Reviews Molecular Cell Biology.

[45]  S. Jinks-Robertson,et al.  Mismatch Repair Proteins Regulate Heteroduplex Formation during Mitotic Recombination in Yeast , 1998, Molecular and Cellular Biology.

[46]  T. Waters,et al.  Cytotoxic mechanism of 6-thioguanine: hMutSalpha, the human mismatch binding heterodimer, binds to DNA containing S6-methylthioguanine. , 1997, Biochemistry.

[47]  L. Edelmann,et al.  Loss of DNA mismatch repair function and cancer predisposition in the mouse: Animal models for human hereditary nonpolyposis colorectal cancer , 2004, American journal of medical genetics. Part C, Seminars in medical genetics.

[48]  P. Modrich,et al.  Mismatch Repair-dependent Iterative Excision at Irreparable O6-Methylguanine Lesions in Human Nuclear Extracts* , 2006, Journal of Biological Chemistry.

[49]  B. Harfe,et al.  Mismatch repair proteins and mitotic genome stability. , 2000, Mutation research.

[50]  Rodney Rothstein,et al.  Elevated recombination rates in transcriptionally active DNA , 1989, Cell.

[51]  R. Liskay,et al.  Mammalian DNA mismatch repair. , 1999, Annual review of genetics.

[52]  B. Harfe,et al.  DNA mismatch repair and genetic instability. , 2000, Annual review of genetics.