Abstract
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 δ. Both reactions required Mg(2+) and Mn(2+) 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.
Highlights
Biochemical analysis of S. cerevisiae mismatch repair (MMR) mutants has been limited by a lack of reconstituted MMR reactions
Mlh1-Pms1-dependent MMR Catalyzed by Purified S. cerevisiae Proteins—In a previous study, we demonstrated that a combination of six purified S. cerevisiae proteins including Msh2-Msh6, exonuclease 1 (Exo1), replication protein A (RPA), DNA polymerase ␦, proliferating cell nuclear antigen (PCNA), and replication factor C (RFC)-⌬1N catalyzed mismatch-dependent repair of a mispaired substrate containing a nick at either an NaeI site 343 bp 5Ј or at an AflIII site 442 bp 3Ј (i.e. 2,479 bp 5Ј) from the mispair [57]
Three reaction parameters were investigated including: 1) reducing the amounts of Exo1 to prevent mispair-dependent 5Ј 3 3Ј excision initiating at the AflIII site from reaching the mispair, which would mask any repair initiating from nicks introduced by Mlh1-Pms1; 2) separating the reaction into two reaction stages to allow for the interaction of Mlh1-Pms1 with substrate DNA; and 3) inclusion of Mn2ϩ in the reactions at the concentrations previously used to support the Mn2ϩ-dependent nicking activity of Mlh1-Pms1 [41, 42, 44] in addition to Mg2ϩ, to achieve optimal Mlh1-Pms1 endonuclease activation
Summary
Biochemical analysis of S. cerevisiae MMR mutants has been limited by a lack of reconstituted MMR reactions. We describe the reconstitution of a mispair-dependent Mlh1-Pms endonuclease activation reaction requiring Msh2-Msh (or Msh2Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3 nickdirected MMR reaction requiring Msh2-Msh (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase ␦. Both reactions required Mg2؉ and Mn2؉ for optimal activity. Mutant Mlh1-Pms and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1Pms endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should make it possible to better study both Exo1independent and Exo1-dependent MMR
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