Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System

作者：Smith Catherine E; Bowen Nikki; Graham William J; Goellner Eva M; Srivatsan Anjana; Kolodner Richard D^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2015, 290(35): 21580-21590.

DOI：10.1074/jbc.M115.662189

• DNA recombination
• DNA repair
• DNA replication
• mutagenesis
• proliferating cell nuclear antigen (PCNA)
• replication factor C (RFC)
• yeast genetics
• exonuclease 1
• genome instability
• Msh2-Msh6

Background: Biochemical analysis of S. cerevisiae MMR mutants has been limited by a lack of reconstituted MMR reactions. Results: 3 nick-directed Mlh1-Pms1-dependent endonuclease and reconstituted MMR reactions were developed. Conclusion: 3 nick-directed MMR required the Mlh1-Pms1 endonuclease and was eliminated by mutations inactivating Exo1-independent MMR. Significance: The reconstituted MMR reactions facilitated analysis of uncharacterized MMR mutants and the mechanism of Exo1-independent MMR.
Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5 nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3 nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase . Both reactions required Mg2+ and Mn2+ for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.

• 出版日期2015-8-28