Mismatch repair deficiency associated with overexpression of the MSH3 gene.
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.
Marra
G
G
Institute for Medical Radiobiology, August Forel-Strasse 7, CH-8029 Zürich, Switzerland.
Iaccarino
I
I
Lettieri
T
T
Roscilli
G
G
Delmastro
P
P
Jiricny
J
J
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D005816:Genetic Complementation Test; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D009692:Nucleic Acid Heteroduplexes; D011994:Recombinant Proteins / Q000235:genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D000964:Antimetabolites, Antineoplastic; D004268:DNA-Binding Proteins; D008727:Methotrexate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D000074081:MutS Homolog 3 Protein; D009692:Nucleic Acid Heteroduplexes; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; C078444:MSH3 protein, human; C421109:multidrug resistance-associated protein 1