Reconstitution of 5′-Directed Human Mismatch Repair in a Purified System

Abstract

This paper reports reconstitution of 5'-nick-directed mismatch repair using purified human proteins. The reconstituted system includes MutSalpha or MutSbeta, MutLalpha, RPA, EXO1, HMGB1, PCNA, RFC, polymerase delta, and ligase I. In this system, MutSbeta plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSalpha, which efficiently corrects both types of heteroduplexes. MutLalpha reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLalpha, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSalpha-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLalpha-mediated excision termination past mismatch. Evidence is provided that efficient repair of a single mismatch requires multiple molecules of MutSalpha-MutLalpha complex. These data suggest a model for human mismatch repair involving coordinated initiation and termination of mismatch-provoked excision.

DNA mismatch repair: Molecular mechanism, cancer, and ageing

“…1 and discussion below), and reconstitutions in the Li and Modrich laboratories of MMR reactions from purified proteins possess many of the key features associated with MMR in vivo (Constantin et al, 2005;Dzantiev et al, 2004;Zhang et al, 2005). These studies were predicated on a large body of earlier work that identified individual components from active fractions of cell extracts and characterized partial reactions (reviewed in Jiricny, 2006).…”

“…These studies were predicated on a large body of earlier work that identified individual components from active fractions of cell extracts and characterized partial reactions (reviewed in Jiricny, 2006). Zhang et al (2005) have demonstrated MMR of a G-T mismatch in 5′-directed repair reactions containing MutSα, MutLα, RPA, EXO1, PCNA, RFC, HMGB1, DNA polymerase δ, and DNA ligase I (Yuan et al, 2004). Substitution of MutSβ for MutSα allowed repair of a 3 nt IDL.…”

DNA Replication to Aid Somatic Hypermutation

“…In mammals, MMR is mediated by proteins homologous to prokaryotic MMR factors, such as Msh2, Msh3 and Msh6 and MutL homologues (Mlh) 1, Mlh3, postmeiotic segregation increased (Pms)1 and Pms2. Consistent with the role of Msh2-Msh6 as the sensor for single-nucleotide mismatches (Kunkel and Erie, 2005;Zhang et al, 2005), mice deficient in Msh2 and/or Msh6 exhibit an overall decreased frequency of mutations. In these mice, residual mutations show a significantly altered spectrum, with decreased mutations at dA/dT and concurrent increased mutations at dC/dG (Table 2) (Rada et al, 1998;Martin and Scharff, 2002;Martomo et al, 2004;Rada et al, 2004).…”

Mammalian mismatch repair: error-free or error-prone?

“…The minimal human MMR system was reconstituted from its individual purified components MutSα or MutSβ, MutLα, EXO1, RFC, PCNA, RPA, polymerase-δ and DNA ligase I several years ago [4,5] and has been extensively reviewed [6][7][8][9][10][11]. We shall therefore discuss only the key features of the system and focus on recent work that yielded novel mechanistic insights into this important pathway of DNA metabolism.…”