Human mismatch repair protein hMutLα is required to repair short slipped-DNAs of trinucleotide repeats

Abstract

Mismatch repair (MMR) is required for proper maintenance of the genome by protecting against mutations. The mismatch repair system has also been implicated as a driver of certain mutations, including disease-associated trinucleotide repeat instability. We recently revealed a requirement of hMutSβ in the repair of short slip-outs containing a single CTG repeat unit (1). The involvement of other MMR proteins in short trinucleotide repeat slip-out repair is unknown. Here we show that hMutLα is required for the highly efficient in vitro repair of single CTG repeat slip-outs, to the same degree as hMutSβ. HEK293T cell extracts, deficient in hMLH1, are unable to process single-repeat slip-outs, but are functional when complemented with hMutLα. The MMR-deficient hMLH1 mutant, T117M, which has a point mutation proximal to the ATP-binding domain, is defective in slip-out repair, further supporting a requirement for hMLH1 in the processing of short slip-outs and possibly the involvement of hMHL1 ATPase activity. Extracts of hPMS2-deficient HEC-1-A cells, which express hMLH1, hMLH3, and hPMS1, are only functional when complemented with hMutLα, indicating that neither hMutLβ nor hMutLγ is sufficient to repair short slip-outs. The resolution of clustered short slip-outs, which are poorly repaired, was partially dependent upon a functional hMutLα. The joint involvement of hMutSβ and hMutLα suggests that repeat instability may be the result of aberrant outcomes of repair attempts.

FIGURE 1.

Western blot analysis of the complementation of HEK293T (MLH1-deficient) cell extracts with hMLH1 and its variants hPMS2, and actin.

FIGURE 2.

Southern blot analysis of the repair of single CTG slip-out substrates by hMLH1-proficient/-deficient cell extracts. Repair efficiencies are the percentage of the “repaired/linear” fragment compared with all repeat-containing fragments in the lane (n = 3). Graph shows starting background (white bars) and repair (checkered bars). Substrates contain 5′- (A) and 3′-nicks (B) on the CTG strand. The repair fragment has (CTG)47·(CAG)47, whereas the slipped heteroduplex SI-DNA fragment has (CTG)48·(CAG)47.

FIGURE 3.

hPMS2 is required to repair short slipped-DNAs. A, Western blot analysis of the complementation of HEC-1-A (hPMS2-deficient, proficient in both hMutLβ and hMutLγ (16, 43, 44)) cell extracts. B and C, Southern blot analysis of the repair of single CTG slip-out substrates by hPMS2-proficient/-deficient HEC-1-A cell extracts. Substrates contain 5′- (B) or 3′-nicks (C). The repair fragment has (CTG)47(CAG)47, whereas the S-DNA fragment has (CTG)48(CAG)47.

FIGURE 4.

Southern blot analysis of the repair of multiple clustered slip-outs (S-DNAs) by hMLH1-proficient HeLa extracts and hMLH1-deficient and complemented 293T cell extracts. Arrowhead indicates S-DNA which has hMLH1-dependent repair. The repair fragment has perfectly paired (CTG)50·(CAG)50, whereas the homoduplex slipped S-DNA fragments have multiple clustered short slip-outs on either strand of the (CTG)50·(CAG)50 tract.