Affiliation:
1. Department of Molecular and Human Genetics 1 and
2. Howard Hughes Medical Institute, 2 Baylor College of Medicine, Houston, Texas 77030
Abstract
ABSTRACT
We have previously described the use of homologous recombination and CRE-
loxP
-mediated marker recycling to generate mouse embryonic stem (ES) cell lines homozygous for mutations at the
Msh3
,
Msh2
, and both
Msh3
and
Msh2
loci (2). In this study, we describe the analysis of these ES cells with respect to processes known to be affected by DNA mismatch repair. ES cells homozygous for the
Msh2
mutation displayed increased resistance to killing by the cytotoxic drug 6-thioguanine (6TG), indicating that the 6TG cytotoxic mechanism is mediated by Msh2. The mutation rate of the herpes simplex virus thymidine kinase 1 (HSV-
tk1
) gene was unchanged in Msh3-deficient ES cell lines but markedly elevated in Msh2-deficient and
Msh3 Msh2
double-mutant cells. Notably, the HSV-
tk1
mutation rate was 11-fold higher, on average, than that of the hypoxanthine-guanine phosphoribosyl transferase (
Hprt
) locus in Msh2-deficient cells. Sequence analysis of HSV-
tk1
mutants from these cells indicated the presence of a frameshift hotspot within the HSV-
tk1
coding region. Msh3-deficient cells displayed a modest (16-fold) elevation in the instability of a dinucleotide repeat, whereas Msh2-deficient and
Msh2 Msh3
double-mutant cells displayed markedly increased levels of repeat instability. Targeting frequencies of nonisogenic vectors were elevated in Msh2-deficient ES cell lines, confirming the role of Msh2 in blocking recombination between diverged sequences (homeologous recombination) in mammalian cells. These results are consistent with accumulating data from other laboratories and support the current model of DNA mismatch repair in mammalian cells.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Cited by
46 articles.
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