Mismatch repair (MMR) ensures the fidelity of DNA replication, initiates the cellular response to certain classes of DNA damage, and has been implicated in the generation of immune diversity. Each of these functions depends on MutSalpha (MSH2*MSH6 heterodimer). Inactivation of this protein complex is responsible for tumor development in about half of known hereditary nonpolyposis colorectal cancer kindreds and also occurs in sporadic tumors in a variety of tissues. Here, we describe a series of crystal structures of human MutSalpha bound to different DNA substrates, each known to elicit one of the diverse biological responses of the MMR pathway. All lesions are recognized in a similar manner, indicating that diversity of MutSalpha-dependent responses to DNA lesions is generated in events downstream of this lesion recognition step. This study also allows rigorous mapping of cancer-causing mutations and furthermore suggests structural pathways for allosteric communication between different regions within the heterodimer.

Mismatch repair rectifies DNA biosynthetic errors, mediates several recombination-associated phenomena, and in mammalian cells participates in an early step of the cellular response to certain classes of DNA damage (reviewed in Refs. 1 and 2). Defects in the human pathway are the cause of hereditary nonpolyposis colorectal cancer and have been implicated in the development of a subset of sporadic tumors.A strand-specific mismatch repair reaction that is presumably responsible for replication error correction has been studied in mammalian cell extracts and in several purified systems. A number of activities have been implicated in this pathway, including MutS␣ (MSH2⅐MSH6 heterodimer), MutS␤ (MSH2⅐MSH3), MutL␣ (MLH1⅐PMS2), Exo1, replication protein A, HMGB1, PCNA, 2 RFC, and DNA polymerase ␦. This reaction can be conceptually divided into three steps: mismatch recognition, excision, and repair DNA synthesis/ligation. The replication clamp PCNA functions at several stages of this multistep reaction. It participates in the DNA synthesis phase of repair (3), consistent with its function as a cofactor for DNA polymerase ␦ (4). PCNA is also necessary during an early step of the reaction (5) and has been shown to interact with activities implicated in early steps of repair (6 -11). However, the significance of these various PCNA interactions is not clear. Analysis of PCNA involvement in mismatch repair has emphasized the MutS␣⅐PCNA complex, which has been suggested to be a key intermediate in mismatch repair. A conserved QXX(L/I)XXFF motif near the N terminus of MSH6 has been implicated in this interaction (6 -8). A human MutS␣ variant lacking the MSH6 N-terminal 77 amino acids including the QXX(L/I)XXFF motif is severely compromised in its ability to support mismatch repair in vitro (8), but alanine substitution of conserved residues within the PCNA interaction motif of yeast Msh6 results in only a modest increase in mutability (6, 7). The implications of the MutS␣-PCNA interaction for the mechanism of mismatch repair are also uncertain. Biochemical analyses have indicated that yeast PCNA enhances the specific affinity of yeast MutS␣ for a mismatch (7), but a subsequent study concluded that although PCNA, MutS␣, and homoduplex DNA form a stable ternary complex, binding to a mismatch leads to disruption of the MutS␣-PCNA interaction (12). These two conclusions appear to be at odds with thermodynamic expectations: if PCNA increases the affinity of MutS␣ for a mispair, then a mismatch should increase the affinity of PCNA for MutS␣.This study further clarifies the nature and function of the human MutS␣-PCNA interaction. We show that MutS␣ binds PCNA with a stoichiometry of 1:1, adopting an elongated conformation in solution, and that the affinity of this interaction is not significantly affected by mismatch binding. Abrogation of the MutS␣-PCNA interaction has little effect on 5Ј-or 3Ј-directed mismatch-provoked excision. However, 5Ј-directed repair is partially attenuated under these conditions. EXPERIMENTAL PROCEDURESMut...

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