An APE that proofreads.
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Anti-HIV Agents↗
Biomedical subjects
Josef Jiricny
Publications and source records attributed to Josef Jiricny.
Identification and functional characterization of the promoter region of the human MSH6 gene.
Postreplicative mismatch repair (MMR) corrects polymerase errors arising during DNA replication. Consistent with this role, the Saccharomyces cerevisiae MMR genes MSH2, MSH6, and PMS1 were reported to be transcriptionally upregulated during late G(1) phase of the cell cycle. Surprisingly, despite the high degree of conservation of the MMR system in evolution, the human MMR genes studied to date, MSH2, MLH1, and PMS2, appear to be transcribed from classical housekeeping promoters, and the amounts of the polypeptides encoded by them fluctuate little during the cell cycle. Only the amounts of the 160-kDa MSH6 protein were reported to vary, both during development and following stimulation of cell growth. Moreover, transcription of this gene was found to be downregulated by CpG methylation of the promoter region in a subset of clones treated with alkylating agents. In an attempt to understand the molecular basis underlying these phenomena, we isolated the 5' region of the MSH6 gene and subjected it to functional analysis. We now show that the MSH6 gene is also transcribed from a classical housekeeping gene promoter. Despite housing putative binding sites for the transcription factors AP1, NF-kappaB, and MTF-1, the MSH6 promoter failed to respond to ionizing radiation or heavy metals. Interestingly, MSH6 transcription was upregulated during late G(1) phase, even though the levels of the protein remained essentially constant during the cell cycle.
5' Flanking Region↗
Endometrial and colorectal tumors from patients with hereditary nonpolyposis colon cancer display different patterns of microsatellite instability.
The colorectum and uterine endometrium are the two most commonly affected organs in hereditary nonpolyposis colon cancer (HNPCC), but the genetic basis of organ selection is poorly understood. As tumorigenesis in HNPCC is driven by deficient DNA mismatch repair (MMR), we compared its typical consequence, instability at microsatellite sequences, in colorectal and endometrial cancers from patients with identical predisposing mutations in the MMR genes MLH1 or MSH2. Analysis of non-coding (BAT25, BAT26, and BAT40) and coding mononucleotide repeats (MSH6, MSH3, MLH3, BAX, IGF2R, TGF beta RII, and PTEN), as well as MLH1- and MSH2-linked dinucleotide repeats (D3S1611 and CA7) revealed significant differences, both quantitative and qualitative, between the two tumor types. Whereas colorectal cancers displayed a predominant pattern consisting of instability at the BAT loci (in 89% of tumors), TGF beta RII (73%), dinucleotide repeats (70%), MSH3 (43%), and BAX (30%), no such single pattern was discernible in endometrial cancers. Instead, the pattern was more heterogeneous and involved a lower proportion of unstable markers per tumor (mean 0.27 for endometrial cancers versus 0.45 for colorectal cancers, P < 0.001) and shorter allelic shifts for BAT markers (average 5.1 bp for unstable endometrial cancers versus 9.3 bp for colorectal cancers, P < 0.001). Among the individual putative "target" loci, PTEN instability was associated with endometrial cancers and TGF beta RII instability with colon cancers. The different instability profiles in endometrial and colorectal cancers despite identical genetic predisposition underlines organ-specific differences that may be important determinants of the HNPCC tumor spectrum.
Adaptor Proteins, Signal Transducing↗
High-frequency microsatellite instability is associated with defective DNA mismatch repair in human melanoma.
Hereditary nonpolyposis colorectal cancers and a steadily increasing number of sporadic tumors display microsatellite instability. In colorectal tumors, high-frequency microsatellite instability is strictly associated with inactivation of the DNA mismatch repair genes hMSH2, hMLH1, or hPMS2, whereas mutations in the mismatch repair gene hMSH6 have been identified in a subset of tumors with low-frequency microsatellite instability. In addition to epithelial tumors of the colon, endometrium, and ovary, microsatellite instability has been reported to occur also in sporadic melanoma. The relationship between microsatellite instability and mismatch repair in melanoma cells, however, has not been investigated so far. In this study, we analyzed microsatellite instability, mismatch repair activity, and expression of the hMSH2, hMSH6, hMLH1, and hPMS2 proteins in five melanoma cell lines and in tumor specimens from which the cells were derived. Four cell lines displayed normal levels of mismatch repair activity and expressed all the mismatch repair proteins. The extracts of the fifth cell line lacked the hMLH1 and hPMS2 proteins, and were correspondingly deficient in the repair of DNA mismatches. This line displayed high-frequency microsatellite instability, whereas the four mismatch-repair-proficient cell lines displayed either no or low-frequency microsatellite instability. These findings could be confirmed in the tumor specimens, in that only the tumor that did not express hMLH1 and hPMS2 displayed high-frequency microsatellite instability. Our data are consistent with the hypothesis that in melanoma, similarly to epithelial tumors, only the high-frequency microsatellite instability phenotype is strictly dependent on a defective mismatch repair system. Further studies on a large series of tumor specimens are required to establish the frequency of mismatch repair loss in human melanoma.
Base Pair Mismatch↗
Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system.
BACKGROUND & AIMS: Germline mutations in the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 are associated with susceptibility to hereditary nonpolyposis colorectal cancer (HNPCC). Because a significant proportion of hMLH1 mutations are missense, the assessment of their pathogenic role may be difficult. To date, functional analysis of missense mutations has been performed primarily in Saccharomyces cerevisiae. The aim of this study was to examine the biochemical properties of hMLH1 protein variants in a human expression system. METHODS: The HNPCC-related hMLH1 mutations T117M, V185G, R217C, G244D, R265C, V326A, and K618T, the polymorphisms I219V and R265H, and a hMLH1 splicing variant lacking exon 9 and 10 (hMLH1 Delta 9/10) were cloned. On transfection of these constructs into human 293T cells, which do not express hMLH1 because of promoter hypermethylation, the hMLH1 protein variants were analyzed by Western blotting and in a MMR assay. RESULTS: Transfection was successful for all hMLH1 constructs. As anticipated, the mutations K618T and T117M, which affect the highly conserved domains of hMLH1 that are necessary for interaction with hPMS2 or for adenosine triphosphate (ATP) binding, respectively, affected protein stability or its ability to complement MMR-deficient 293T-cell extracts. The V185G, G244D, and Delta 9/10 variants were also unable to complement MMR in 293T cells, whereas hMLH1 proteins carrying the I219V, R265H, R265C, R217C, and V326A mutations were MMR competent. CONCLUSIONS: These data show that the pathogenic role of hMLH1 missense mutations and splicing variants can be assessed by analyzing the biochemical properties of their protein products in a homologous expression system.
Adaptor Proteins, Signal Transducing↗
Functional analysis of MLH1 mutations linked to hereditary nonpolyposis colon cancer.
Hereditary nonpolyposis colon cancer (HNPCC) is associated with malfunction of postreplicative mismatch repair (MMR). While a majority of HNPCC-associated mutations in the MMR genes MLH1, MSH2, or MSH6 genes cause truncations-and thus loss of function--of the respective polypeptides, little is currently known about the biochemical defects associated with nontruncating mutations. We studied the interactions of six MLH1 variants, carrying either missense mutations or in-frame deletions, with normal PMS2 and tested the functionality of these heterodimers of MLH1 and PMS2 (MutL(alpha)) in an in vitro MMR assay. Three MLH1 carboxy-terminal mutations, consisting of internal deletions of exon 16 (amino acids 578-632) or exon 17 (amino acids 633-663), or a missense R659P mutation in exon 17, affected the formation of a functional MutL(alpha). Interestingly, mutations C77R and I107R in the amino-terminal part of MLH1 did not affect its heterodimerization with PMS2. The complexes MLH1(C77R)/PMS2 and MLH1(I107R)/PMS2, however, failed to complement a MMR-deficient extract lacking a functional MutL(alpha). As all these five mutations were identified in typical HNPCC families and produce nonfunctional proteins, they can be considered disease-causing. In contrast, the third amino-terminal mutation S93G did not affect the heterodimerization, and the MLH1(S93G)/PMS2 variant was functional in the in vitro MMR assay, given thus the nature of the HNPCC family in question. Although the missense mutation segregates with the disease, the mean age of onset in the family is unusually high (approximately 65 years).
Adaptor Proteins, Signal Transducing↗
Mismatch repair and DNA damage signalling.
Postreplicative mismatch repair (MMR) increases the fidelity of DNA replication by up to three orders of magnitude, through correcting DNA polymerase errors that escaped proofreading. MMR also controls homologous recombination (HR) by aborting strand exchange between divergent DNA sequences. In recent years, MMR has also been implicated in the response of mammalian cells to DNA damaging agents. Thus, MMR-deficient cells were shown to be around 100-fold more resistant to killing by methylating agents of the S(N)1type than cells with functional MMR. In the case of cisplatin, the sensitivity difference was lower, typically two- to three-fold, but was observed in all matched MMR-proficient and -deficient cell pairs. More controversial is the role of MMR in cellular response to other DNA damaging agents, such as ionizing radiation (IR), topoisomerase poisons, antimetabolites, UV radiation and DNA intercalators. The MMR-dependent DNA damage signalling pathways activated by the above agents are also ill-defined. To date, signalling cascades involving the Ataxia telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), as well as the stress-activated kinases JNK/SAPK and p38alpha have been linked with methylating agent and 6-thioguanine (TG) treatments, while cisplatin damage was reported to activate the c-Abl and JNK/SAPK kinases in MMR-dependent manner. MMR defects are found in several different cancer types, both familiar and sporadic, and it is possible that the involvement of the MMR system in DNA damage signalling play an important role in transformation. The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.
Animals↗