Mapping the Minimal Domain of hMSH-2 Sufficient for Binding Mismatched Oligonucleotides

doi:10.1006/bbrc.1997.6211

Biochemical and Biophysical Research Communications

Volume 232, Issue 1, 6 March 1997, Pages 10-13

https://doi.org/10.1006/bbrc.1997.6211 Get rights and content

Abstract

The human MSH-2 gene product is a member of a highly conserved family of proteins involved in post-replication mismatch repair. Germline mutations in this gene have been implicated in hereditary non-poly- posis colorectal cancer (HNPCC). Alterations in the coding region of the hMSH-2 gene result in a mutator phenotype with marked instability of microsatellite sequences, indicative of a deficiency in DNA repair. We have previously shown that a region of high homology between MutS proteins of different species containing a nucleotide binding domain, is sufficient to bind DNA containing specific mismatched residues. In order to determine the minimal domain of hMSH-2 necessary for binding mismatch-containing oligonucleotides, deletion analysis of the $C$ -terminal region was performed. We have constructed a 5′ and 3′ deletion series, expressed each deletion as a bacterial fusion protein and assessed it for ATPase activity and its ability to identify mismatch containing DNA. Here we demonstrate that a 585 bp fragment encoding 195 amino acids within the $C$ -terminal domain of hMSH-2 is sufficient to bind to DNA containing mismatches.

References (22)

A. Whitehouse et al.
Biochem. Biophys. Res. Commun.
(1996)
H.T. Lynch et al.
Gastroentrology
(1993)
F.S. Leach et al.
Cell
(1993)
R.D. Kolodner et al.
Genomics
(1994)
M.G. Dunlop
Br. J. Surgery
(1992)
S. Parker et al.
CA Cancer J. Clin.
(1996)
L.A. Loeb
Cancer Res.
(1994)
R. Parsons et al.
Cell
(1993)
C.E. Bronner et al.
Nature
(1994)
R.D. Kolodner et al.
Cancer Res.
(1994)

There are more references available in the full text version of this article.

Cited by (10)

Presymptomatic diagnosis using a deletion of a single codon in families with hereditary non-polyposis colorectal cancer
2005, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
The diagnosis of hereditary non-polyposis colorectal cancer (HNPCC) is often confirmed by a mutation in one of several mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Presymptomatic diagnosis requires the identification of a mutation causing the disease. Three different deletions of a single amino acid codon have previously been published as assumed pathogenic. The objective of this study was to determine if an MSH2 3 base pair in-frame deletion (N596del) could be used in presymptomatic screening of at-risk individuals. We report on five HNPCC families with the N596del mutation, identified after mutation screening of MSH2 and MLH1. All patients in the families were haplotyped using markers flanking the MSH2 gene. The haplotypes revealed that the five families with high probability descended from only two founders. The N596del segregated with the HNPCC phenotype with lod scores of 3.2 and 2.0 at the recombination fraction of 0.0 in the two founder families. Sequencing of MSH2 and MLH1 did not reveal other pathogenic mutations, and N596del was not identified in 50 healthy controls. The mutation has previously been found expressed in mRNA, and is located in a conserved domain. The results support the hypothesis that N596del is the disease causing mutation and not a clinically silent variation. On this basis, the application of the MSH2 N596del mutation, in presymptomatic screening of HNPCC families, is recommended.
DNA mismatch repair enzyme activity and gene expression in prostate cancer
2001, Biochemical and Biophysical Research Communications
Microsatellite instability (MSI) of short repetitive sequences in human chromosomal DNA can result from defective DNA mismatch repair function in tumor cells. We hypothesize that DNA mismatch repair (MMR) activity is down-regulated during prostatic carcinogenesis. To test this hypothesis, MMR activities and mismatch repair-related genes were analyzed in five different prostate cancer cell lines. Our results demonstrate that MMR activities were decreased as compared to MMR proficient HeLa cells. Interestingly, LNCaP, PC-3 and DU145 had much lower MMR activities as compared to DUPro and TSUPr1. The MMR-related genes (hMLH1, hPMS1, hPMS2, hMSH2, hMSH3, hMSH6) showed mRNA transcripts in all prostate cancer cell lines. However, Western blotting showed decreased or absent hMLH1 protein expression in PC-3, DU145, DUPro and TSUPr1 cells. Similarly, the hMSH2 protein expression was low or absent in DU145 and LNCaP cells. This is the first report that demonstrates decreased MMR activities is associated with low expression of hMLH1, hMSH2 and other MMR-related proteins in prostate cancer.
DNA binding and protein-protein interaction sites in MutS, a mismatched DNA recognition protein from Thermus thermophilus HB8
2000, Journal of Biological Chemistry
Citation Excerpt :
T. thermophilus MutS, as well as the other MutS homologues, possesses weak ATPase activity (14, 19, 35-37). It has been reported that the activity requires the Walker's nucleotide-binding motif which is located on the C-terminal region of the protein (14, 15, 18). A C-terminal mutant of S. typhimurium MutS containing a modified Walker's A-type motif bound to heteroduplex DNA with reduced affinity (14).
The mismatch repair system repairs mismatched base pairs, which are caused by either DNA replication errors, DNA damage, or genetic recombination. Mismatch repair begins with the recognition of mismatched base pairs in DNA by MutS. Protein denaturation and limited proteolysis experiments suggest thatThermus thermophilus MutS can be divided into three structural domains as follows: A (N-terminal domain), B (central domain), and C (C-terminal domain) (Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K., and Kuramitsu, S. (1998)Nucleic Acids Res. 26, 4153–4159). To investigate the functions of each domain in detail, truncated genes corresponding to the domains were designed. The gene products were overproduced inEscherichia coli, purified, and assayed for various activities. The MutS-MutS protein interaction site was determined by size-exclusion chromatography to be located in the B domain. The B domain was also found to possess nonspecific double-stranded DNA-binding ability. The C domain, which contains a Walker's A-type nucleotide-binding motif, demonstrated ATPase activity and specific DNA recognition of mismatched base pairs. These ATPase and specific DNA binding activities were found to be dependent upon C domain dimerization.
DNA mismatch binding in human lung tumor cell lines
1999, Lung Cancer
Defects in mismatch repair (MMR) genes have been involved in several types of sporadic and hereditary cancers. In order to elucidate the role of MMR in human lung carcinogenesis we examined DNA mismatch binding in cell-free extracts of seven lung tumor cell lines and five corresponding lymphoblastoid cell lines from lung cancer patients. Using the technique of bandshift assay we have demonstrated that 2/7 of the tumor cell lines are aberrant in binding to specific DNA mismatches while all lymphoblastoid cell lines were proficient in binding to all tested mismatches. Both extracts were aberrant in binding to G/T mismatch whereas one of the cell lines showed deficiency in binding to the C:A mismatches as well. Immunoblotting analysis showed that all known DNA mismatch repair (MMR) proteins were present in these extracts. The cell line deficient in binding to both G:T and C:A mismatches showed microsatellite instability (MSI) in tumor DNA and higher resistance to the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). This report indicates that DNA mismatch binding deficiencies may be implicated in at least a subgroup of human lung cancer.
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2001, Genome
Loss of mismatch repair activity in simian virus 40 large T antigen-immortalized BPH-1 human prostatic epithelial cell line
2001, Molecular Carcinogenesis

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¹: Corresponding author. Fax: 044 113 2444475; Email: msjaw@ stjames.leeds.ac.uk.

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Regular ArticleMapping the Minimal Domain of hMSH-2 Sufficient for Binding Mismatched Oligonucleotides

Abstract

Biochem. Biophys. Res. Commun.

Gastroentrology

Cell

Genomics

Br. J. Surgery

CA Cancer J. Clin.

Cancer Res.

Cell

Nature

Cancer Res.

Regular Article
Mapping the Minimal Domain of hMSH-2 Sufficient for Binding Mismatched Oligonucleotides