Much of our knowledge about MMR comes from genetic studies and in vitro reconstitution experiments. Although a few attempts to visualize MMR proteins under conditions of normal DNA replication in vivo have been reported, these studies have been complicated by the use of overexpressed levels of MMR proteins (Elez et al., 2010
) or the use of partially- or non-functional tagged MMR proteins (Smith et al., 2001
), and only report a limited analysis (Elez et al., 2010
; Kleczkowska et al., 2001
; Smith et al., 2001
). Here, we used fully functional GFP or mCherry tagged versions of the MMR proteins Msh2, Msh6 and Pms1, expressed at native levels, to visualize Msh2-Msh6 and Mlh1-Pms1 complexes in living S. cerevisiae
cells. We found that Msh2-Msh6 formed nuclear foci in S-phase cells that colocalized with replication factories. This colocalization was completely dependent on the interaction between the Msh6 N-terminal PIP box and PCNA, and the N-terminus of Msh6 was sufficient for this interaction. Mutations in POL2
that increased the frequency of mispairs, a rad52Δ
mutation that eliminates recombination and replication-dependent recombination intermediates and an msh3Δ
mutation that eliminates targeting of Msh2 to recombination intermediates did not affect the frequency of Msh2-Msh6 foci. In addition, Rad52 foci, which form at recombination and repair intermediates, occur in S-phase cells at less than 10% of the frequency of Msh2-Msh6 foci (Lisby et al., 2001
). Therefore, Msh2-Msh6 foci are not assembled in response to mispaired bases or recombination and repair intermediates, but represent a mispair recognition system that is constitutively present as an integral component of a significant proportion of replication factories.
The formation of Msh2-Msh6 foci and their localization to replication factories depended on the interaction between the PIP box at the Msh6 N-terminus and PCNA. However, mutations that eliminate the interaction between Msh6 and PCNA only cause a 10–15% reduction in the efficiency of MMR. These results suggest there are additional redundant pathway(s) for mispair recognition that do not involve the formation of Msh2-Msh6 foci coupled to replication factories. These alternative pathways require Exo1 and an internal region of the Msh6 N-terminal leader sequence, as MMR in exo1Δ
mutants was completely dependent on the interaction between Msh6 and PCNA. The observation that the MMR pathway coupled to DNA replication becomes essential for preventing the accumulation of mutations in the absence of Exo1 is consistent with the previous identification of mutations that inactivate MMR only in the absence of Exo1 (Amin et al., 2001
), one of which was a deletion of POL32
that encodes a non-essential subunit of DNA Polymerase δ. Furthermore, lagging strand MMR was significantly more dependent on EXO1
than leading strand MMR, providing additional evidence for a specific MMR pathway(s) that depends on Exo1. An alternative explanation is that EXO1
deletion and Msh6-PCNA interaction-defective mutants exhibit a synergistic MMR defect by extending the time that is required to complete MMR past the time it takes to reinitiate S-phase in the next cell cycle.
We also used live-cell imaging to visualize MMR proteins that function downstream from mismatch recognition. We found that Mlh1-Pms1 also formed distinct S-phase nuclear foci, but these foci rarely colocalized with Msh2-Msh6 foci or replication factories, and did not colocalize with telomeres, centromeres, the nucleolus or the nuclear periphery. Four main lines of evidence indicate that these Mlh1-Pms1 foci represent sites where active MMR is taking place. First, Mlh1-Pms1 foci were completely abolished by mutations that eliminate the Msh2-Msh6 and Msh2-Msh3 mispair recognition complexes or mispair recognition. In addition, msh6 mutations that do not affect mispair recognition but prevent sliding clamp formation and the interaction with Mlh1-Pms1 eliminated the Mlh1-Pms1 foci. Second, the frequency of Mlh1-Pms1 foci was increased by mutations in POL2 or POL3 that increase the frequency of mispaired bases in cells. Third, mutations that inactivate MMR downstream of the interaction of Mlh1-Pms1 with Msh2-Msh6, including a Pms1 endonuclease mutation and an exo1Δ mutation, resulted in increased levels of Mlh1-Pms1 foci. Fourth, a rad52Δ mutation that eliminates recombination and replication-dependent recombination intermediates had no affect on Pms1 foci. These results are consistent with Mlh1-Pms1 foci being an active intermediate during MMR.
The observation that Mlh1-Pms1 foci do not colocalize with Msh2-Msh6 foci or contained sub-stoichiometric amounts of Msh2-Msh6 that was below the limits of detection was surprising. One possible explanation for this is that the action, or a step in the action, of Mlh1-Pms1 in MMR is temporally separable from the mispair-dependent interaction of Msh2-Msh6 with Mlh1-Pms1 during MMR. Alternatively, once a mispaired base is recognized by Msh2-Msh6, one or a few molecules of Msh2-Msh6 catalytically load multiple Mlh1-Pms1 complexes onto the DNA resulting in the formation Mlh1-Pms1 foci. These mechanisms are inconsistent with previous ideas proposing that multiple Msh2-Msh6 complexes are recruited at the mispair site, with each one of them able to interact with one Mlh1-Pms1 heterodimer. A number of studies have reported that MutL and Mlh1-Pms1 can interact with DNA, although the biological significance of this has been questioned (Park et al., 2010
); however, the correlation of Mlh1-Pms1 foci with mispair levels suggests that the interaction of Mlh1-Pms1 with DNA may reflect a mechanistic step in MMR.
We found that the msh6-G1142D mutation, which results in a mutant Msh2-Msh6 complex that forms mispair dependent ternary complexes with Mlh1-Pms1 but does not form sliding clamps, eliminated the formation of Mlh1-Pms1 foci. This result indicates that the ability of Msh2-Msh6 to interact with Mlh1-Pms1 is not sufficient for Mlh1-Pms1 foci formation. Rather, it is likely that conformational changes of Msh2-Msh6 that occur after mismatch recognition and Mlh1-Pms1 recruitment are essential for loading multiple Mlh1-Pms1 complexes. We speculate that loading of multiple Mlh1-Pms1 complexes at the site of the mispair is a crucial step necessary to guarantee the subsequent degradation and repair of the mispair-containing strand. This mechanism shares similarities with the one proposed for double strand break repair, where phosphorylation of several H2A histone molecules (γ-H2AX) adjacent to the site of the break, marks and amplifies the signal to ensure subsequent repair.
A model summarizing our results is presented in . In this model, Msh2-Msh6 can locate mismatches in two ways. The first, is as a component of replication factories, where it acts as a sensor coupled to DNA replication by PCNA. Since DNA replication mediates the disassembly of chromatin, this coupling of MMR to replication provides a mechanism by which MMR is able to overcome the barriers to repair presented by chromatin structure. Alternatively, Msh2-Msh6 can scan the genome for mispairs independently of its association with replication factories. The nature of this second pathway is unclear. When a mispair is encountered, Msh2-Msh6 loads multiple molecules of Mlh1-Pms1 onto DNA, Mlh1-Pms1 is activated and Exo1, or other excision functions, is recruited for removal of the mispair-containing DNA strand followed by DNA resynthesis and ligation. The ability to visualize and quantify MMR intermediates provides an assay that can be used for identification and analysis of MMR components in the alternative MMR pathways.
Figure 6 Model of MMR pathways. Replication- and repair-associated foci are intermediates of MMR. At least two independent pathways act in preventing the accumulation of mispairs; one is coupled to the replication machinery through the interaction between Msh6 (more ...)