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.
Highlights
Slipped-DNAs are mutagenic intermediates in disease-causing trinucleotide repeat instability; their processing is not well understood
Most mammalian studies of mismatch repair in trinucleotide repeats (TNRs) instability have focused on the roles of MSH2 and MSH3, with very few having investigated a role for MLH1 and its binding partners: PMS2, PMS1, and MLH3
In human cell line assays focused upon TNR contractions, transcription-induced complementary (CTG)50 and (CAG) contractions were decreased by MSH2 and MSH3 knockdown, and yet contractions were increased with a knockdown of MLH1 or PMS2 [8, 18]
Summary
Slipped-DNAs are mutagenic intermediates in disease-causing trinucleotide repeat instability; their processing is not well understood. Because MMR proteins repair short insertion-deletion loops, it is possible that their mutagenic role in TNR instability is the result of attempts to process CTG/CAG repeat structures. Following the recognition of unpaired or damaged DNA by either the MutS␣ (MSH2-MSH6) or MutS (MSH2-MSH3) complex, other proteins are recruited to initiate and complete repair Such factors include the MutL homologs hMutL␣ (hMLH1-hPMS2), hMutL (hMLH1-hPMS1), and hMutL␥ (hMLH1-hMLH3), where the latter two complexes play a minor role in mismatch repair relative to hMutL␣ [10]. We found that hMutL␣, but not hMutL or hMutL␥, is required for the repair of short slipouts and plays a partial role in the limited repair of clustered short slip-outs, similar to hMutS
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