Nucleic Acids Research, Vol 27, Issue 3 736-742, Copyright © 1999 by Oxford University Press
AB Clark, ME Cook, HT Tran, DA Gordenin, MA Resnick and TA Kunkel
Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or
hMSH2*hMSH3 binds to mismatches. Here we perform functional analyses of
these human protein complexes in yeast. We use a sensitive genetic system
wherein the rate of single-base deletions in a homopolymeric run in the
LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild- type
strain. Expression of the human proteins alone or in combination does not
reduce the mutation rate of the msh2 strain, and expression of the
individual human proteins does not increase the low mutation rate of a
wild-type strain. However, co-expression of hMSH2 and hMSH6 in wild-type
yeast increases the mutation rate 4000-fold, while co- expression of hMSH2
and hMSH3 elevates the rate 5-fold. Analysis of cell extracts indicates
that the proteins are expressed and bind to mismatched DNA. The results
suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent
correction of replication slippage errors in yeast. Expression of hMSH6
with hMSH2 containing a proline substituted for a conserved Arg524
eliminates the mutator effect and reduces mismatch binding. The analogous
mutation in humans is associated with microsatellite instability, defective
MMR and cancer, illustrating the utility of the yeast system for studying
human disease alleles.
ARTICLES
Functional analysis of human MutSalpha and MutSbeta complexes in yeast
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233,Research Triangle Park, NC 27709, USA.
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