Mismatch repair deficiency associated with overexpression of the MSH3 gene - PubMed (original) (raw)

Mismatch repair deficiency associated with overexpression of the MSH3 gene

G Marra et al. Proc Natl Acad Sci U S A. 1998.

Abstract

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

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Figures

Figure 1

Figure 1

Mismatch- and IDL-binding activities in extracts of CHO, CHO R, HEC59, HL60, and HL60R cells. The extracts were incubated with radioactively labeled oligonucleotide duplexes (see Materials and Methods) either perfectly complementary (G/C) or containing a single mispair (G/T) or an IDL containing two extrahelical thymines (-TT-) (10). (Left and Center) The bandshift experiments were carried out with cytoplasmic extracts of CHO, CHO R, HEC59, HL60, and HL60R cells. The electrophoretic mobility of the protein/DNA complexes was compared with those formed upon incubation of the oligonucleotide substrates with the purified recombinant hMutSα (lane α) or hMutSβ (lane β). Note that, unlike the purified recombinant hMSH3/hMSH2 heterodimer, the hMutSβ present in human cell extracts gave rise to two distinct protein/DNA complexes (lane HL60R/TT). Neither of these complexes comigrates with hMutSα-bound substrates (e.g., lane HL60R/G/T) (see also text). (Right) Supershift experiments using the anti-hMSH6 mAb 66H6. Addition of the mAb to the binding mixtures containing extracts of HL60 or HL60R cells retarded the mobility of the oligonucleotide probes bound by hMutSα. The figure is an autoradiograph of a nondenaturing 6% polyacrylamide gel run in TAE buffer.

Figure 2

Figure 2

Immunoblot of MSH2, MSH3, and MSH6 polypeptides present in extracts of HeLa, CHO, CHO R, HEC59, HL60, and HL60R cell lines. The band migrating just below MSH3 in the hamster cell extracts is caused by a protein cross-reacting with the polyclonal anti-hMSH3 antiserum. The figure is a Western blot of cytoplasmic extracts stained with rabbit polyclonal antisera raised against the three proteins. The extracts were prepared as described (21), and 20 μg per lane were loaded on a 7.5% denaturing SDS-polyacrylamide gel. The proteins were transferred onto nitrocellulose membranes, which were then stained with the indicated antisera. hMutSα and hMutSβ, purified recombinant heterodimers included for reference.

Figure 3

Figure 3

Efficiency of mismatch correction in extracts of HEC59, CHO, CHO R, HL60, and HL60R cell lines. The extracts were supplemented with purified recombinant hMutSα or hMutSβ as indicated below the columns (see also Materials and Methods). (a) Repair efficiency of the G/G mispair and of the two nucleotide IDL in HEC59 extracts supplemented or not with hMutSα or hMutSβ. (b) As in a, except that CHO and CHO R extracts were used. (c) As in b, except that HL60 and HL60R extracts were used.

Figure 4

Figure 4

Partial proteolysis of hMSH2 and hMSH6 within the context of hMutSα. Purified recombinant hMutSα was incubated (from left to right) with 0, 5, 10, 50, 100, and 600 ng trypsin as described in Materials and Methods. The products were separated on SDS/PAGE, transferred onto nitrocellulose membranes, and visualized with polyclonal anti-hMSH2 (Lower) and anti-hMSH6 (Upper) antisera.

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