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Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

Smith CE, Mendillo ML, Bowen N, Hombauer H, Campbell CS, Desai A, Putnam CD, Kolodner RD - PLoS Genet. (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

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.

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Metal coordination mutations eliminate the ability of Mlh1-Pms1 to nick closed circular DNA.Endonuclease reactions to nick closed circular DNA were performed with Mlh1-Pms1 alone or with Mlh1-Pms1, PCNA and RFC-Δ1N. A. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Pms1 amino acid changes. B. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Mlh1 amino acid changes. 100% cleavage of the 100 ng of pRS425 in the assay is 2.2 fmoles of cleavage events.
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pgen-1003869-g002: Metal coordination mutations eliminate the ability of Mlh1-Pms1 to nick closed circular DNA.Endonuclease reactions to nick closed circular DNA were performed with Mlh1-Pms1 alone or with Mlh1-Pms1, PCNA and RFC-Δ1N. A. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Pms1 amino acid changes. B. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Mlh1 amino acid changes. 100% cleavage of the 100 ng of pRS425 in the assay is 2.2 fmoles of cleavage events.

Mentions: Previous studies have shown that S. cerevisiae Mlh1-Pms1 has a metal-dependent endonuclease activity that can be stimulated by RFC and PCNA [26]. To determine if the dominant pms1 mutations affecting metal ligating amino acids disrupt the endonuclease function of Mlh1-Pms1, we expressed and purified the S. cerevisiae wild-type Mlh1-Pms1 complex and the mutant Mlh1-Pms1-G683E, Mlh1-Pms1-C817R, Mlh1-Pms1-C848S, and Mlh1-Pms1-H850R complexes and assayed the ability of these complexes to nick supercoiled pRS425 plasmid DNA with or without accessory factors RFC-Δ1N and PCNA (Figure 2A). Wild-type Mlh1-Pms1 alone showed little endonuclease activity. However, addition of PCNA and RFC-Δ1N to reactions containing wild-type Mlh1-Pms1 resulted in a 20-fold increase in endonuclease activity resulting in cleavage of nearly half of the original substrate DNA. The newly identified Mlh1-Pms1 mutant proteins and the previously studied Mlh1-Pms1-E707K mutant protein did not exhibit any PCNA and RFC-Δ1N stimulated endonuclease activity, with the exception of the Mlh1-Pms1-H850R mutant protein (Figure 2A). An explanation of the ability of the Mlh1-Pms1-H850R mutant protein to nick supercoiled DNA is provided in the “Discussion”. These results support the idea that loss of metal coordination by Pms1 inhibits the endonuclease activity of Mlh1-Pms1.


Dominant mutations in S. cerevisiae PMS1 identify the Mlh1-Pms1 endonuclease active site and an exonuclease 1-independent mismatch repair pathway.

Smith CE, Mendillo ML, Bowen N, Hombauer H, Campbell CS, Desai A, Putnam CD, Kolodner RD - PLoS Genet. (2013)

Metal coordination mutations eliminate the ability of Mlh1-Pms1 to nick closed circular DNA.Endonuclease reactions to nick closed circular DNA were performed with Mlh1-Pms1 alone or with Mlh1-Pms1, PCNA and RFC-Δ1N. A. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Pms1 amino acid changes. B. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Mlh1 amino acid changes. 100% cleavage of the 100 ng of pRS425 in the assay is 2.2 fmoles of cleavage events.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814310&req=5

pgen-1003869-g002: Metal coordination mutations eliminate the ability of Mlh1-Pms1 to nick closed circular DNA.Endonuclease reactions to nick closed circular DNA were performed with Mlh1-Pms1 alone or with Mlh1-Pms1, PCNA and RFC-Δ1N. A. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Pms1 amino acid changes. B. Percentage of wild-type cleaved product formed by wild-type Mlh1-Pms1 and mutant Mlh1-Pms1 complexes containing the indicated Mlh1 amino acid changes. 100% cleavage of the 100 ng of pRS425 in the assay is 2.2 fmoles of cleavage events.
Mentions: Previous studies have shown that S. cerevisiae Mlh1-Pms1 has a metal-dependent endonuclease activity that can be stimulated by RFC and PCNA [26]. To determine if the dominant pms1 mutations affecting metal ligating amino acids disrupt the endonuclease function of Mlh1-Pms1, we expressed and purified the S. cerevisiae wild-type Mlh1-Pms1 complex and the mutant Mlh1-Pms1-G683E, Mlh1-Pms1-C817R, Mlh1-Pms1-C848S, and Mlh1-Pms1-H850R complexes and assayed the ability of these complexes to nick supercoiled pRS425 plasmid DNA with or without accessory factors RFC-Δ1N and PCNA (Figure 2A). Wild-type Mlh1-Pms1 alone showed little endonuclease activity. However, addition of PCNA and RFC-Δ1N to reactions containing wild-type Mlh1-Pms1 resulted in a 20-fold increase in endonuclease activity resulting in cleavage of nearly half of the original substrate DNA. The newly identified Mlh1-Pms1 mutant proteins and the previously studied Mlh1-Pms1-E707K mutant protein did not exhibit any PCNA and RFC-Δ1N stimulated endonuclease activity, with the exception of the Mlh1-Pms1-H850R mutant protein (Figure 2A). An explanation of the ability of the Mlh1-Pms1-H850R mutant protein to nick supercoiled DNA is provided in the “Discussion”. These results support the idea that loss of metal coordination by Pms1 inhibits the endonuclease activity of Mlh1-Pms1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, La Jolla, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus