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

Abstract

Mammalian and Saccharomyces cerevisiae mismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5' to 3' excision by exonuclease 1 (Exo1) from a single-strand break 5' to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3' to the mispair, to make nicks 5' to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of the S. cerevisiae mispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR using S. cerevisiae proteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5' nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3' to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.

Keywords: DNA excision; DNA repair; DNA replication fidelity; genome instability; mutator phenotype.

Fig. 1.

pBluescript-based substrates for detecting MMR in vitro. (A) Sequence of the polylinker region between the ApaI and BamHI sites of different substrates indicating the mispair, the restriction sites in each strand, and the plasmid from which each strand was derived. N, nicked strand; C, continuous strand. (B) Map of the 5′ NaeI-nicked +T substrate and (C) map of the 3′ AflIII-nicked +T substrate showing the positions of the various features used in the assays and the relevant distances between key sites. The mispair is indicated by the arrowhead.

Fig. 2.

Time course of DNA polymerase ε-dependent MMR of the NaeI-nicked +1 (+T) substrate. Assays of 5′ nick-directed repair of the +1 (+T) substrate containing a nick at the NaeI site were performed for the indicated times as described in Materials and Methods. The presence/absence of Msh2–Msh6 is as indicated. (A) Repair was detected by digestion with PstI and ScaI, and the repair products were visualized after agarose gel electrophoresis, and (B) the repair products seen on the gels were quantified as described in Materials and Methods. MW, molecular weight markers; arrows, markers for repair products. One hundred percent repair is repair of 100 ng or 52.75 fmol of substrate.

Fig. 3.

Protein requirements for 5′ nick-directed MMR of the NaeI-nicked +T substrate. (A) Assays of 5′ nick-directed repair of the +1 (+T) substrate containing a nick at the NaeI site containing the indicated amounts of DNA Pol ε. (B) Assays of 5′ nick-directed repair of the +1 (+T) substrate containing the indicated amounts of Pol ε as in A, with or without PCNA and RFC-Δ1N as indicated. Relative repair of 1.0 was the amount of repair observed at 800 fmol of Pol ε. (C) Assays of 5′ nick-directed repair of the +1 (+T) substrate containing a nick at the NaeI site in which different proteins were omitted or substituted as indicated. MW, molecular weight markers; arrows, markers for repair products.

Fig. 4.

Time course and protein requirements for 5′ nick-directed repair of the AflIII-nicked +T substrate. (A) The 5′ nick-directed repair reactions with the +1 (+T) substrate containing a nick at the AflIII site were performed for the indicated times with the presence/absence of Msh2–Msh6 as indicated. (B) The 5′ nick-directed repair reactions with the +1 (+T) substrate containing a nick at the AflIII site in which different proteins were omitted or substituted as indicated. MW, molecular weight markers; arrows, markers for repair products.

Fig. 5.

Time course of DNA polymerase ε- and Mlh1–Pms1-dependent MMR of the AflIII-nicked +T substrate. Two-stage repair reactions with the +1 (+T) substrate containing a 3′ nick at the AflIII site were performed for the indicated times as described in Materials and Methods. (A) Repair was detected by digestion with PstI and ScaI, and the repair products were visualized after agarose gel electrophoresis, and (B) the repair products seen on the gels were quantified as described in Materials and Methods. MW, molecular weight markers; arrows, markers for repair products.

Fig. 6.

Protein requirements for DNA polymerase ε- and Mlh1–Pms1-dependent repair of the AflIII-nicked +1 (+T) substrate. Two-stage repair reactions with the +1 (+T) substrate containing a 3′ nick at the AflIII site were performed in which different proteins were omitted or substituted as indicated. The amount of repair was quantified as described in Materials and Methods and normalized to the amount of repair seen in complete reactions. The average of at least three independent experiments is presented; the error bars indicate the SE.