Single-molecule views of MutS on mismatched DNA

摘要: Abstract Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability conversely increase diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction initiated by MutS homolog (MSH) binding to the mismatched nucleotides. MSH mismatch-binding signal then transmitted immediate downstream MutL (MLH/PMS) components ultimately distant strand scission site where excision begins. transmission controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) Polarization Total Internal Reflection (smP-TIRF) examine interactions dynamic behaviors Thermus aquaticus (TaqMutS) particles on DNA. determined TaqMutS forms an incipient clamp search in ∼1 s intervals 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while continuous contact with helical duplex When encounters it lingers ∼3 s exchange bound ADP ATP (ADP → ATP exchange). induces extremely stable conformation (∼10 min) slides off moves along adjacent driven simply 1D diffusion. ATP-bound sliding clamps rotate freely discontinuous visualization train proteins suggests dissociation from permits multiple mismatch-dependent loading events. These direct observations provided critical clues into understanding molecular MMR.