Dominant Mutations in S. cerevisiae PMS1 Identify the Mlh1-Pms1 Endonuclease Active Site and an Exonuclease 1-Independent Mismatch Repair Pathway

Abstract

Lynch syndrome (hereditary nonpolypsis colorectal cancer or HNPCC) is a common cancer predisposition syndrome. Predisposition to cancer in this syndrome results from increased accumulation of mutations due to defective mismatch repair (MMR) caused by a mutation in one of the mismatch repair genes MLH1, MSH2, MSH6 or PMS2/scPMS1. To better understand the function of Mlh1-Pms1 in MMR, we used Saccharomyces cerevisiae to identify six pms1 mutations (pms1-G683E, pms1-C817R, pms1-C848S, pms1-H850R, pms1-H703A and pms1-E707A) that were weakly dominant in wild-type cells, which surprisingly caused a strong MMR defect when present on low copy plasmids in an exo1Δ mutant. Molecular modeling showed these mutations caused amino acid substitutions in the metal coordination pocket of the Pms1 endonuclease active site and biochemical studies showed that they inactivated the endonuclease activity. This model of Mlh1-Pms1 suggested that the Mlh1-FERC motif contributes to the endonuclease active site. Consistent with this, the mlh1-E767stp mutation caused both MMR and endonuclease defects similar to those caused by the dominant pms1 mutations whereas mutations affecting the predicted metal coordinating residue Mlh1-C769 had no effect. These studies establish that the Mlh1-Pms1 endonuclease is required for MMR in a previously uncharacterized Exo1-independent MMR pathway.

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

“…Mlh1-Pms1 was not required for repair of either substrate (Fig. 3A), even though our Mlh1-Pms1 preparations have RFC-PCNA-stimulated endonuclease activity (45). The lack of an Mlh1-Pms1 requirement is consistent with the fact that both substrates are repaired by a 5′ excision reaction (see The NaeI and AflIII Substrates Are Repaired by Short and Long Patch 5′ to 3′ Excision Repair, Respectively).…”

“…The lack of an Mlh1-Pms1 requirement in the S. cerevisiae system, even though our Mlh1-Pms1 preparations are fully active for RFC-PCNA-dependent endonuclease activity (45), conflicted with the results of some human MMR studies where an Mlh1-Pms2 endonuclease-dependent MMR reaction can occur (17). The most likely explanations for this lack of an Mlh1-Pms1 requirement are that the Mlh1-Pms1 endonuclease is not active under the reaction conditions used here or that the potent 5′ excision-based MMR reaction seen with the AflIII substrate containing a 3′ nick outcompetes any Mlh1-Pms1 endonucleasedependent reaction.…”

Mispair-specific Recruitment of the Mlh1-Pms1 Complex Identifies Repair Substrates of the Saccharomyces cerevisiae Msh2-Msh3 Complex

“…The plasmids used for overexpression of the Msh2-Msh3 mutant complexes were pRDK354 with pRDK1769 and pRDK1768 for MSH3-K158E-FLAG and MSH3-R195D-FLAG, respectively, which were constructed from the plasmid pRDK1596 using the GeneArt sitedirected mutagenesis system (Invitrogen) to introduce the nucleotide changes A472G and AGG583_585GAT, respectively. The plasmids used for overexpression of the Mlh1-Pms1 complex were pRDK573 (2 P GAL1-10 -MLH1 TRP1) and pRDK1099 (2 P GAL1-10 -PMS1-FLAG LEU2) (64,65). All plasmids were periodically verified by DNA sequencing to ensure the absence of unwanted mutations.…”

“…Purification of Mlh1-Pms1-S. cerevisiae Mlh1-Pms1 was overproduced in the S. cerevisiae strain RDKY1293 using the plasmids pRDK573 and pRDK1099 and purified to Ͼ98% purity as previously described (64,65).…”

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

“…endonuclease is activated in a mispair-dependent fashion by a combination of Msh2-Msh6 or Msh2-Msh3, PCNA, and RFC to generate DNA nicks 5′ to the mispair (29)(30)(31)(32)(33). Once the 5′ nicks are formed, repair appears to occur as observed in the 5′ nickdirected MMR reactions.…”