ABSTRACT
In eukaryotic post-replicative mismatch repair, MutS homologs (MSH) detect mismatches and recruit MLH complexes to nick the newly replicated DNA strand upon activation by the replication processivity clamp, PCNA. This incision enables mismatch removal and DNA repair. Biasing MLH endonuclease activity to the newly replicated DNA strand is crucial for repair. In reconstituted in vitro assays, PCNA is loaded at pre-existing discontinuities and orients the major MLH endonuclease Mlh1-Pms1/MLH1-PMS2 (yeast/human) to nick the discontinuous strand. In vivo, newly replicated DNA transiently contains discontinuities which are critical for efficient mismatch repair. How these discontinuities are preserved as strand discrimination signals during the window of time where mismatch repair occurs is unknown. Here, we demonstrate that yeast Mlh1-Pms1 uses ATP binding to recognize DNA discontinuities. This complex does not efficiently interact with PCNA, which partially suppresses ATPase activity, and prevents dissociation from the discontinuity. These data suggest that in addition to initiating mismatch repair by nicking newly replicated DNA, Mlh1-Pms1 protects strand discrimination signals, aiding in maintaining its own strand discrimination signposts. Our findings also highlight the significance of Mlh1-Pms1's ATPase activity for inducing DNA dissociation, as mutant proteins deficient in this function become immobilized on DNA post-incision, explaining in vivo phenotypes.
ABSTRACT
In eukaryotic mismatch repair, MutS homologs recognize mismatches and recruit the MutLα endonuclease which introduces a nick in the newly replicated, error-containing DNA strand. The nick occurs in response to the mismatch, but at a site up to several hundred base pairs away. The MutLα nick promotes mismatch excision by an exonuclease (Exo1) or removal by the strand displacement activity of a DNA polymerase which may work in conjunction with a flap endonuclease. Models have suggested that MutL homolog endonucleases form oligomeric complexes which facilitate and are activated by strand capture mechanisms, although such models have never been explicitly tested. We present evidence that the mismatch repair MutLα endonuclease is activated by DNA-DNA associations and that it can use this property to overcome DNA torsional barriers. Using DNA ligation and pull-down experiments, we determined that the MutLα endonuclease associates two DNA duplexes. Using nuclease assays, we determined that this activity stimulates MutLα's endonuclease function. We also observe that MutLα enhances a topoisomerase without nicking the DNA itself. Our data provide a mechanistic explanation for how MutL proteins interact with DNA during mismatch repair, and how MutL homologs participate in other processes, such as recombination and trinucleotide repeat expansions.
