Journal Article

Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme

Jing-Fan-Xu, Qiu-Ping Yang, James Y.J. Chen, Marrit R. van Baalen and Ih-Chang Hsu

in Carcinogenesis

Volume 17, issue 2, pages 321-326
Published in print February 1996 | ISSN: 0143-3334
Published online February 1996 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/17.2.321
Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme

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The Escherichia coli MutY gene was cloned into a modified pET-11 plasmid which was then transfected into an E.coli HMS174 host for overproduction of the MutY mismatch repair (MR) enzyme. Approximately 30–50% of the total cellular protein in the transformed HMS174 cells was isopropyl-β-D-thiogalactoside-induced MutY protein, as estimated from the staining intensity on an SDS-PAGE gel following electrophoresis. The MutY protein was purified to near homogeneity by cellulose phosphate ion-exchange chrome purified MutY protein had enzyme activities which cleaved the A of a G/A mismatch at the 3' end of the first phosphodiester bond and then the 5' end of the second phosphodiester bond of the A. It also cut the A of a C/A mismatch, but to a much lesser extent, and the activity was DNA sequence-dependent. The reliability of the assay in determining the site and nature of a DNA mutation was examined in human tumor DNA samples with known or unknown p53 mutations. In the assay, polymerase chain reaction-amplified DNA fragments from normal and mutated p53 genes were mixed, denatured and annealed to generate mismatches of G/A or C/A for cleavage by the MutY MR enzyme. The assay results revealed the site and nature of known G: C ↔ T: A mutations. In addition, a previously unknown G: C to T: A mutation, which was misread in the sequencing analysis of a tumor DNA preparation, was identified by this assay.

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Subjects: Clinical Cytogenetics and Molecular Genetics

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