Phosphorylated hMSH6: DNA mismatch versus DNA damage recognition

Department of Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis (Impact Factor: 3.68). 10/2010; 706(1-2):36-45. DOI: 10.1016/j.mrfmmm.2010.10.008
Source: PubMed


DNA mismatch repair (MMR) maintains genomic integrity by correction of mispaired bases and insertion-deletion loops. The MMR pathway can also trigger a DNA damage response upon binding of MutSα to specific DNA lesions such as O(6)methylguanine (O(6)meG). Limited information is available regarding cellular regulation of these two different pathways. Within this report, we demonstrate that phosphorylated hMSH6 increases in concentration in the presence of a G:T mismatch, as compared to an O(6)meG:T lesion. TPA, a kinase activator, enhances the phosphorylation of hMSH6 and binding of hMutSα to a G:T mismatch, though not to O(6)meG:T. UCN-01, a kinase inhibitor, decreases both phosphorylation of hMSH6 and binding of hMutSα to G:T and O(6)meG:T. HeLa MR cells, pretreated with UCN-01 and exposed to MNNG, undergo activation of Cdk1 and mitosis despite phosphorylation of Chk1 and inactivating phosphorylation of Cdc25c. These results indicate that UCN-01 may inhibit an alternative cell cycle arrest pathway associated with the MMR pathway that does not involve Cdc25c. In addition, recombinant hMutSα containing hMSH6 mutated at an N-terminal cluster of four phosphoserines exhibits decreased phosphorylation and decreased binding of hMutSα to G:T and O(6)meG:T. Taken together, these results suggest a model in which the amount of phosphorylated hMSH6 bound to DNA is dependent on the presence of either a DNA mismatch or DNA alkylation damage. We hypothesize that both phosphorylation of hMSH6 and total concentration of bound hMutSα are involved in cellular signaling of either DNA mismatch repair or MMR-dependent damage recognition activities.

Download full-text


Available from: Kandace J Williams,
  • Source
    • "Briefly, 100 fmol of the DNA substrates were incubated in the presence of binding buffer [25 mM HEPES (pH 7.8), 5 mM MgCl2, 80 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1 mg/ml bovine serum albumin (BSA)] with 100 nM purified MSH2-MSH6 complex at room temperature for 30 min along with poly dI-dC competitor DNA (20-fold excess). The detailed protocol for the purification of the complex is described (36). Reactions were resolved on a 4% polyacrylamide gel at 4°C, dried and exposed with X-ray film for autoradiography. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Base excision repair (BER) and mismatch repair (MMR) pathways play an important role in modulating cis-Diamminedichloroplatinum (II) (cisplatin) cytotoxicity. In this article, we identified a novel mechanistic role of both BER and MMR pathways in mediating cellular responses to cisplatin treatment. Cells defective in BER or MMR display a cisplatin-resistant phenotype. Targeting both BER and MMR pathways resulted in no additional resistance to cisplatin, suggesting that BER and MMR play epistatic roles in mediating cisplatin cytotoxicity. Using a DNA Polymerase β (Polβ) variant deficient in polymerase activity (D256A), we demonstrate that MMR acts downstream of BER and is dependent on the polymerase activity of Polβ in mediating cisplatin cytotoxicity. MSH2 preferentially binds a cisplatin interstrand cross-link (ICL) DNA substrate containing a mismatch compared with a cisplatin ICL substrate without a mismatch, suggesting a novel mutagenic role of Polβ in activating MMR in response to cisplatin. Collectively, these results provide the first mechanistic model for BER and MMR functioning within the same pathway to mediate cisplatin sensitivity via non-productive ICL processing. In this model, MMR participation in non-productive cisplatin ICL processing is downstream of BER processing and dependent on Polβ misincorporation at cisplatin ICL sites, which results in persistent cisplatin ICLs and sensitivity to cisplatin.
    Nucleic Acids Research 06/2013; 41(15). DOI:10.1093/nar/gkt479 · 9.11 Impact Factor
  • Source

    DNA Repair, 11/2011; , ISBN: 978-953-307-697-3
  • [Show abstract] [Hide abstract]
    ABSTRACT: The field of DNA mismatch repair (MMR) has rapidly expanded after the discovery of the MutHLS repair system in bacteria. By the mid 1990s yeast and human homologues to bacterial MutL and MutS had been identified and their contribution to hereditary non-polyposis colorectal cancer (HNPCC; Lynch Syndrome) was under intense investigation. The human MutS homologue 6 protein (hMSH6), was first reported in 1995 as a G:T binding partner (GTBP) of hMSH2, forming the hMutSα mismatch-binding complex. Signal transduction from each DNA-bound hMutSα complex is accomplished by the hMutLα heterodimer (hMLH1 and hPMS2). Molecular mechanisms and cellular regulation of individual MMR proteins are now areas of intensive research. This review will focus on molecular mechanisms associated with mismatch binding, as well as emerging evidence that MutSα, and in particular, MSH6, is a key protein in MMR-dependent DNA damage response and communication with other DNA repair pathways within the cell. MSH6 is unstable in the absence of MSH2, however it is the DNA lesion-binding partner of this heterodimer. MSH6, but not MSH2, has a conserved Phe-X-Glu motif that recognizes and binds several different DNA structural distortions, initiating different cellular responses. hMSH6 also contains the nuclear localization sequences required to shuttle hMutSα into the nucleus. For example, upon binding to O(6)meG:T, MSH6 triggers a DNA damage response that involves altered phosphorylation within the N-terminal disordered domain of this unique protein. While many investigations have focused on MMR as a post-replication DNA repair mechanism, MMR proteins are expressed and active in all phases of the cell cycle. There is much more to be discovered about regulatory cellular roles that require the presence of MutSα and, in particular, MSH6.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/2013; 743-744. DOI:10.1016/j.mrfmmm.2012.12.008 · 3.68 Impact Factor
Show more