PCNA, the trimeric replication sliding clamp is believed to coordinate various DNA metabolic processes via
its interactions with components of replication, repair, and recombination systems. In human mismatch repair, PCNA has been implicated at two stages in the reaction: it is required for activation of the MutLα endonuclease (32
) but is also necessary for the repair synthesis step of the reaction (3
), functions that presumably depend on its known ability to interact with multiple mismatch repair factors, including MutSα, MutLα, Exo1 and DNA polymerase δ. However, the conformational nature of these complexes and the contributions of individual interactions to various steps of the reaction remain largely uncertain. We have addressed these issues for the human MutSα-PCNA complex. Previous studies with yeast proteins led to the suggestion that MutSα and PCNA associate to form an active mispair recognition complex, which has a higher affinity for heteroduplex DNA, and that PCNA is released upon mismatch binding (7
). By contrast, we observe that human MutSα binds heteroduplex DNA with comparable affinity and specificity in the absence or presence of PCNA. Consistent with these observations, mismatch-bound MutSα has an affinity for PCNA that is very similar to that of free MutSα.
A key function of PCNA in mismatch repair is its role in activation of the latent endonuclease of MutLα (32
). This endonuclease, which also depends on MutSα and RFC for its activation, introduces additional breaks into the discontinuous strand of a nicked heteroduplex that serve as entry sites for Exo1. Despite the severe nature of the PCNA interaction defect conferred by MutSαΔ12 and MutSαΔ341 mutations, MutSαΔ12 supports MutLα activation and both mutants are essentially fully active in their abilities to support mismatch-provoked excision in a reconstituted system. Furthermore, MutSαΔ12 supports normal levels of mismatch-provoked excision upon supplementation of MutSα-deficient nuclear extracts. Thus, the interaction between MutSα and PCNA is not critical for early steps of mismatch repair, suggesting that PCNA interaction with other activities such as MutLα and/or Exo1 account for involvement of the replication clamp in early steps of the reaction. Indeed, a recent study has suggested an important effector role for PCNA in function of the MutLα endonuclease (41
While removal of the PCNA interaction motif from MutSα has little if any effect on MutLα endonuclease activation or mismatch-provoked excision steps of the reaction, MutSαΔ12 and MutSαΔ341 both display a limited but reproducible defect in 5′-, but not 3′-directed mismatch repair. Because we have been unable to detect a corresponding defect in 5′-directed mismatch-provoked excision (scored under conditions of DNA synthesis block), we infer that this partial 5′-repair defect is manifested at the repair synthesis step. This defect, which reduces the rate of the reaction ~50%, may indicate MutSα participation in the DNA synthesis step of 5′-directed repair. It is also noteworthy that the nature of this defect is consistent with the significant but limited mutability increase that has been observed upon alanine substitution of conserved residues within the PCNA interaction motif of MSH6 (6
). In contrast to our results with Δ12 and Δ341 MutSα variants, removal of the 77 N-terminal residues of human MSH6 has been shown to substantially reduce the mismatch repair activity MutSα in human cell extracts (8
). It is possible that the MSH6Δ77 mutation interferes with the interaction of MutSα with mismatch repair factors other than PCNA or compromises function of the protein in a manner unrelated to its inability to interact with PCNA.
The nature of PCNA lends itself to models wherein a single homotrimeric ring can interact with up to three molecules of a binding partner. In fact, human PCNA is capable of binding 3 molecules of p21 or Fen1 (35
). However, the several different experimental approaches used here demonstrate that the interaction stoichiometry of human MutSα with the PCNA trimer is restricted to 1:1, even under conditions of large MutSα excess. Given the trivalent nature of PCNA, the 1:1 stoichiometry limitation may be due to steric factors, although it is important to note that while the MutSα·PCNA complex cannot bind a second molecule of MutSα, the PCNA component of the complex might be capable of simultaneous interaction with another repair or replication activity.
Our low-resolution SAXS models reveal that MutSα and PCNA associate in an end-to-end fashion in solution to form an elongated complex in which the DNA binding channels of the two proteins are not aligned, but rather are orthogonal to the long axis of the complex. While contrary to expectation, a similar extended conformation has been demonstrated for the Sulfolobus solfataricus
ligase·PCNA complex in solution (37
). Because MSH6 residues 1-341 are not present in MutSαΔ341, for which the crystal structure has been determined (14
), the structure of the N-terminal MSH6 domain has been uncertain. However, recent NMR studies have demonstrated that residues 89-194 of human MSH6 adopt a globular conformation (Sophie Zinn-Justin, personal communication; PDB accession 2GFU). Our SAXS reconstructions with native MutSα and the MutSα·PCNA complex are also consistent with the idea that the N-terminal domain of MSH6 has globular features and interacts with PCNA to form a complex with significantly defined conformational character. By contrast, SAXS studies of an N-terminal fragment of yeast MSH6 (residues 1-304) have indicated that this polypeptide exists in an extended unstructured conformation, leading Shell et al. (43
) to conclude that yeast MutSα interacts with PCNA by virtue of a highly extended unstructured tether. While the Shell et al. study included SAXS data collection on full-length yeast MutSα in the absence or presence of PCNA, surface reconstructions for these polypeptide complexes were not described. It is also noteworthy that while the yeast MSH6 N-terminal segment supports PCNA interaction with 3:1 stoichiometry (43
), native yeast MutSα, probably interacts with the yeast PCNA trimer with 1:1 stoichiometry (7
). The significance of these apparent differences in the nature of the human and yeast MutSα·PCNA complexes thus remain to be resolved.