Determining the site and nature of DNA mutations with the cloned MutY mismatch repair enzyme.
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-beta-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 chromatography followed by gel filtration chromatography. The 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.
Xu
J F
JF
Department of Pathology, School of Medicine, University of Maryland at Baltimore 21201, USA.
Yang
Q P
QP
Chen
J Y
JY
van Baalen
M R
MR
Hsu
I C
IC
D001483:Base Sequence; D045647:DNA Glycosylases; D011088:DNA Ligases / Q000096:biosynthesis / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D017354:Point Mutation / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016159:Tumor Suppressor Protein p53; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D011088:DNA Ligases