A new DNA binding site in the mismatch repair MutS protein involved in the interaction with DNA replication structures

Congreso; LV Reunión Anual de SAIB y Conferencia de la PABMB; 2019

MutS maintains genomic stability by recognizing mispaired nucleotides (MMs) and triggering the Mismatch Repair (MMR) pathway. Findings from our laboratory have demonstrated that the repair factor also contributes to DNA replication fidelity by regulating the access of the mutagenic DNA polymerase IV to replication sites. In this work, we showed for the first time that MutS from Pseudomonas aeruginosa can interact with DNA structures present at replication sites, i.e. primed DNA (pDNA). Moreover, MutS suffered a conformational change upon binding to a pDNA containing a GT mismatch (GT-pDNA), resulting in a more compact and stable protein structure, as determined by native gel electrophoresis, circular dichroism and trypsin digestion assays. This structural rearrangement was not observed when MutS was associated with pDNA and the MMR double-stranded DNA substrates, GT-dsDNA and dsDNA. We hypothesized that the MutS conformational change induced by its association with the mismatched pDNA could result from binding to new protein residues. In fact, using the nucleic acid binding prediction BindUP software and DNA-protein docking analysis, we identified a new DNA binding surface in MutS. Within this novel site, Arg275 appeared to directly contact the 3?-OH end of the pDNA. Mutation of this residue to Glu abolished DNA binding in vitro and produced a mutator phenotype in vivo, indicating a key role of this novel binding site in the activity of MutS. We are testing if the structural change induced by the mismatched replication substrate and the new DNA binding site are important for MutS gaining access to replication sites. In conclusion, our results reveal a novel DNA interaction site in MutS, which may play an important role in the control of Pol IV access to replication sites by MutS.