Extrahelical (CAG)/(CTG) triplet repeat elements support proliferating cell nuclear antigen loading and MutLa endonuclease activation

Abstract

MutLa endonuclease can be activated on covalently continuous DNA that contains a MutSa- or MutSβ-recognizable lesion and a helix perturbation that supports proliferating cell nuclear antigen (PCNA) loading by replication factor C, providing a potential mechanism for triggering mismatch repair on nonreplicating DNA. Because mouse models for somatic expansion of disease-associated (CAG)n/(CTG)n triplet repeat sequences have implicated both MutSβ and MutLa and have suggested that expansions can occur in the absence of replication, we have asked whether an extrahelical (CAG)n or (CTG)n element is sufficient to trigger MutLa activation. (CAG)n and (CTG)n extrusions in relaxed closed circular DNA do in fact support MutSβ-, replication factor C-, and PCNA-dependent activation of MutLa endonuclease, which can incise either DNA strand. Extrahelical elements of two or three repeat units are the preferred substrates for MutLa activation, and extrusions of this size also serve as moderately effective sites for loading the PCNA clamp. Relaxed heteroduplex DNA containing a two or three-repeat unit extrusion also triggers MutSβ- and MutLa-endonuclease- dependent mismatch repair in nuclear extracts of human cells. This reaction occurs without obvious strand bias at about 10% the rate of that observed with otherwise identical nicked heteroduplex DNA. These findings provide a mechanism for initiation of triplet repeat processing in nonreplicating DNA that is consistent with several features of the model of Gomes-Pereira et al. [Gomes-PereiraM, FortuneMT, IngramL,McAbney JP,Monckton DG (2004) HumMol Genet 13(16):1815-1825]. They may also have implications for triplet repeat processing at a replication fork.