Abstract
MutS homologs identify base-pairing errors made in DNA during replication and initiate their repair. In the presence of adenosine triphosphate, MutS induces DNA bending upon mismatch recognition and subsequently undergoes conformational transitions that promote its interaction with MutL to signal repair. In the absence of MutL, these transitions lead to formation of a MutS mobile clamp that can move along the DNA. Previous single-molecule FRET (smFRET) studies characterized the dynamics of MutS DNA-binding domains during these transitions. Here, we use protein–DNA and DNA–DNA smFRET to monitor DNA conformational changes, and we use kinetic analyses to correlate DNA and protein conformational changes to one another and to the steps on the pathway to mobile clamp formation. The results reveal multiple sequential structural changes in both MutS and DNA, and they suggest that DNA dynamics play a critical role in the formation of the MutS mobile clamp. Taking these findings together with data from our previous studies, we propose a unified model of coordinated MutS and DNA conformational changes wherein initiation of mismatch repair is governed by a balance of DNA bending/unbending energetics and MutS conformational changes coupled to its nucleotide binding properties.
Highlights
E) DNA dissociation kinetics were measured by mixing AF555-MutS-E315C and Cy5-T-bulge with excess unlabeled T-bulge DNA in the absence of nucleotides or with ATP or ADP in the stopped flow, and monitoring decrease in Cy5 acceptor fluorescence over time
F) ATP hydrolysis and phosphate (Pi) release rates were measured for wild type and unlabeled or labeled MutS-E315C in the absence or presence of a 37 bp Tbulge DNA using a Pi-binding MDCC-PBP reporter assay, as described previously [6,8]
Mixing MutS with ATP in the stopped-flow in the absence of DNA results in a burst of ATP hydrolysis and Pi release followed by a linear steady state phase
Summary
ATP and ADP accelerate DNA unbending to 1.9 and 1.5 s-1, respectively, as reported previously for wild type MutS [6,7]. E) DNA dissociation kinetics were measured by mixing AF555-MutS-E315C and Cy5-T-bulge with excess unlabeled T-bulge DNA in the absence of nucleotides or with ATP or ADP in the stopped flow, and monitoring decrease in Cy5 acceptor fluorescence over time (final concentrations: 0.1 μM MutS dimer, 40 nM Cy5-T-bulge, 2 μM unlabeled T-bulge, 0.5 mM ATP or ADP, 50 mM Tris-HCl, pH 7.5, 100 mM sodium acetate, 5 mM MgCl2, 40 oC).
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