Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

Abstract

A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispaircontaining DNA substrates. A mixture of MutS homolog 2 (Msh2)-MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5′ or 3′ strand interruption with different efficiencies. The Msh2-MutS homolog 3 mispair recognition protein could substitute for the Msh2-Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1-postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5′ or 3′ strand interruption occurred by mispair binding-dependent 5′ excision and subsequent resynthesis with excision tracts of up to ∼2.9 kb occurring during the repair of the substrate with a 3′ strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.DNA replication fidelity | genome instability | mutator phenotype | cancer | mutagenesis

Reconstitution of Saccharomyces cerevisiae DNA polymerase ε-dependent mismatch repair with purified proteins

“…A role for Pol δ is primarily supported by fractionation studies in which MMR proficiency was restored to a depleted extract by the addition of a single protein, either fractions containing Pol δ or purified Pol δ (50). Importantly, Pol δ is sufficient to support in reconstituted MMR reactions (25)(26)(27)31). Purified DNA polymerase α (Pol α) could not substitute for Pol δ in these studies, suggesting that Pol α cannot not act by itself in MMR (50).…”

“…In contrast, the repair tracts of the S. cerevisiae protein reactions can be up to ∼3 kb long and there may be up to 10-12 repair events per molecule of Exo1 (25). In a second type of reaction, a combination of Msh2-Msh6 or Msh2-Msh3, MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) (called Mlh1-Pms2 in humans), Exo1, Pol δ, RPA, PCNA, and RFC promotes the repair of a circular mispaired substrate containing a nick on the 3′ side of the mispair (25,26). In this reaction, the Mlh1-Pms1…”

“…Two types of mispair-dependent excision/repair reactions have been reconstituted with human and S. cerevisiae proteins. In the first type of reaction, a combination of one of the mispair recognition factors MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or Msh2-MutS homolog 3 (Msh3), exonuclease 1 (Exo1), DNA polymerase δ (Pol δ), the single-stranded DNA binding protein replication protein A (RPA), proliferating cell nuclear antigen (PCNA), and the PCNA loading factor replication factor C (RFC) catalyze the repair of a circular mispaired substrate containing a single-strand break (referred to as a nick) on the 5′ side of the mispair (25)(26)(27). In this reaction, the mispair recognition factors stimulate excision by Exo1 from the nick past the mispair to produce a gap that is filled in by Pol δ, PCNA, and RFC repairing the mispair (25,28).…”

“…In the first type of reaction, a combination of one of the mispair recognition factors MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or Msh2-MutS homolog 3 (Msh3), exonuclease 1 (Exo1), DNA polymerase δ (Pol δ), the single-stranded DNA binding protein replication protein A (RPA), proliferating cell nuclear antigen (PCNA), and the PCNA loading factor replication factor C (RFC) catalyze the repair of a circular mispaired substrate containing a single-strand break (referred to as a nick) on the 5′ side of the mispair (25)(26)(27). In this reaction, the mispair recognition factors stimulate excision by Exo1 from the nick past the mispair to produce a gap that is filled in by Pol δ, PCNA, and RFC repairing the mispair (25,28). In the human protein reactions, the mismatched DNA substrates contain a 5′ nick that is 128 bp from the mispair, resulting in excision tracts that are relatively short, and typically there is only one repair event per molecule of Exo1 (26)(27)(28).…”

“…In the human protein reactions, the mismatched DNA substrates contain a 5′ nick that is 128 bp from the mispair, resulting in excision tracts that are relatively short, and typically there is only one repair event per molecule of Exo1 (26)(27)(28). In contrast, the repair tracts of the S. cerevisiae protein reactions can be up to ∼3 kb long and there may be up to 10-12 repair events per molecule of Exo1 (25). In a second type of reaction, a combination of Msh2-Msh6 or Msh2-Msh3, MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) (called Mlh1-Pms2 in humans), Exo1, Pol δ, RPA, PCNA, and RFC promotes the repair of a circular mispaired substrate containing a nick on the 3′ side of the mispair (25,26).…”