| Post-replication DNA mismatch repair (MMR) targets the DNA replication errors that skip the proofreading by DNA polymerase, and can decrease the replication error rate from 10−7 to 10−10 . Two poorly understood processes in MMR are related to repair specificity and the large spatial separation between the mismatch site and the strand discrimination site. How does recognition of a mismatch by MutS trigger excision repair and lead to high repair specificity? How do the protein-DNA, protein-protein complexes communicate between the mismatch site and the strand-discrimination site, which can be thousands of base pair apart?; To fully understand the dynamic process of initiation events in DNA MMR, we have done AFM imaging in air on E. coli MutS-DNA, MutS-MutL-DNA, and yeast MLH1-PMS1-DNA complexes.; Based on the comparison of the MutS-induced DNA bending observed at specific and nonspecific sites (without any nucleotide or in the presence of ADP), we propose that MutS bends the DNA during the search of a mismatch. Upon specific recognition, MutS undergoes a conformational change to a bent initial recognition complex (IRC). Finally, the bent IRC undergoes a further conformational change to an unbent ultimate recognition complex (URC). We propose that the unbent URC is the precursor that leads to downstream events.; We observed mismatch and ATP dependent formation of multiple MutS proteins and large MutS-MutL protein assemblies on a single piece of DNA which span ∼200 by around the mismatch. We suggest that multiple loading of MutS is a signal amplification step in initiation of DNA MMR. We propose that formation of large MutS-MutL complexes facilitates the communication between the mismatch site and the strand discrimination site. These observations provide new insights into how DNA MMR overcomes the low mismatch binding affinity by MutS and achieves higher repair efficiency.; We have shown that yeast MLH1-PMS1 has at least one independent DNA binding site on each of its subunits, and can bind to either one or two strands of duplex DNA cooperatively. In addition, using computer simulation of cooperative protein assembly on DNA, we estimated the DNA binding site size for MLH1-PMS1 on dsDNA to be ∼17–23 bp. |
