Li G, Modrich P.. Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs. Proc Natl Acad Sci USA 92: 1950-1954

Duke University, Durham, North Carolina, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/1995; 92(6):1950-4. DOI: 10.1073/pnas.92.6.1950
Source: PubMed


Hypermutable H6 colorectal tumor cells are defective in strand-specific mismatch repair and bear defects in both alleles of the hMLH1 gene. We have purified to near homogeneity an activity from HeLa cells that complements H6 nuclear extracts to restore repair proficiency on a set of heteroduplex DNAs representing the eight base-base mismatches as well as a number of slipped-strand, insertion/deletion mispairs. This activity behaves as a single species during fractionation and copurifies with proteins of 85 and 110 kDa. Microsequence analysis demonstrated both of these proteins to be homologs of bacterial MutL, with the former corresponding to the hMLH1 product and the latter to the product of hPMS2 or a closely related gene. The 1:1 molar stoichiometry of the two polypeptides and their hydrodynamic behavior indicate formation of a heterodimer, which we have designated hMutL alpha. These observations indicate that interactions between members of the family of human MutL homologs may be restricted.

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    • "This strongly supports the pathogenic potential of the p.Cys680Arg variant. The MLH1 protein exists predominately in a complex with PMS2 also known as the MutLa heterodimer (Li and Modrich 1995). The formation of a MutLa complex is essential for MMR activity (Baker et al. 1995, 1996; Edelmann et al. 1996) and, therefore, the failure of LS- MLH1 proteins to associate with PMS2 could result in "
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    ABSTRACT: In clinical genetic diagnostics, it is difficult to predict whether genetic mutations that do not greatly alter the primary sequence of the encoded protein causing unknown functional effects on cognate proteins lead to development of disease. Here, we report the clinical identification of c.2038 T>C missense mutation in exon 18 of the human MLH1 gene and biochemically characterization of the p.Cys680Arg mutant MLH1 protein to implicate it in the pathogenicity of the Lynch syndrome (LS). We show that the mutation is deficient in DNA mismatch repair and, therefore, contributing to LS in the carriers.
    07/2014; 2(4). DOI:10.1002/mgg3.80
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    • "In another approach, extracts from cell lines lacking a defined MMR protein are complemented with the missing MMR protein [Holmes et al., 1990; Li and Modrich, 1995]. The complemented extracts are added to an artificial substrate that carries a mismatch within a restriction site, interfering with its cleavage. "
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    ABSTRACT: Lynch syndrome (LS) is caused by germline mutations in DNA mismatch repair (MMR) genes and is the most prevalent hereditary colorectal cancer syndrome. A significant proportion of variants identified in MMR and other common cancer susceptibility genes are missense or noncoding changes whose consequences for pathogenicity cannot be easily interpreted. Such variants are designated as "variants of uncertain significance" (VUS). Management of LS can be significantly improved by identifying individuals who carry a pathogenic variant and thus benefit from screening, preventive, and therapeutic measures. Also, identifying family members that do not carry the variant is important so they can be released from the intensive surveillance. Determining which genetic variants are pathogenic and which are neutral is a major challenge in clinical genetics. The profound mechanistic knowledge on the genetics and biochemistry of MMR enables the development and use of targeted assays to evaluate the pathogenicity of variants found in suspected patients with LS. We describe different approaches for the functional analysis of MMR gene VUS and propose development of a validated diagnostic framework. Furthermore, we call attention to common misconceptions about functional assays and endorse development of an integrated approach comprising validated assays for diagnosis of VUS in patients suspected of LS.
    Human Mutation 12/2012; 33(12). DOI:10.1002/humu.22168 · 5.14 Impact Factor
    • "During DNA synthesis in S-phase, O 6 MeG mispairs with thymine [28], this mismatch is a substrate for MMR and is bound by the heterodimer MutSa [29], comprised of MSH2 and MSH6. After recruitment of the heterodimer MutLa [30], comprised of PMS2 and MLH1, and exonuclease I [31] thymine is excised, but because of the mispairing properties of O 6 MeG, still present in the DNA, thymine is simply reinserted during the re-synthesis of the double-stranded DNA. As this remains a substrate for MMR, the futile removal and re-insertion of thymine will occur causing the formation of long persistent stretches of single-stranded DNA [32] until the cell again enters S-phase. "
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    ABSTRACT: DNA damaging agents are potent inducers of cell death triggered by apoptosis. Since these agents induce a plethora of different DNA lesions, it is firstly important to identify the specific lesions responsible for initiating apoptosis before the apoptotic executing pathways can be elucidated. Here, we describe specific DNA lesions that have been identified as apoptosis triggers, their repair and the signaling provoked by them. We discuss methylating agents such as temozolomide, ionizing radiation and cisplatin, all of them are important in cancer therapy. We show that the potentially lethal events for the cell are O(6)-methylguanine adducts that are converted by mismatch repair into DNA double-strand breaks (DSBs), non-repaired N-methylpurines and abasic sites as well as bulky adducts that block DNA replication leading to DSBs that are also directly induced following ionizing radiation. Transcriptional inhibition may also contribute to apoptosis. Cells are equipped with sensors that detect DNA damage and relay the signal via kinases to executors, who on their turn evoke a process that inhibits cell cycle progression and provokes DNA repair or, if this fails, activate the receptor and/or mitochondrial apoptotic cascade. The main DNA damage recognition factors MRN and the PI3 kinases ATM, ATR and DNA-PK, which phosphorylate a multitude of proteins and thus induce the DNA damage response (DDR), will be discussed as well as the downstream players p53, NF-κB, Akt and survivin. We review data and models describing the signaling from DNA damage to the apoptosis executing machinery and discuss the complex interplay between cell survival and death.
    Cancer letters 01/2012; 332(2). DOI:10.1016/j.canlet.2012.01.007 · 5.62 Impact Factor
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