title: Single-molecule views of MutS on mismatched DNA.
abstract: Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic 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 that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ~1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ~3 s to exchange bound ADP for ATP (ADP→ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (~10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR.
authors.affiliation: Department of Physics, POSTECH, Pohang 790-784, Republic of Korea; School of Interdisciplinary Bioscience & Bioengineering, POSTECH, Pohang 790-784, Republic of Korea. Electronic address: jblee@postech.ac.kr.
authors.forename: Jong-Bong
authors.initials: JB
authors.lastname: Lee
authors.affiliation: Department of Physics, POSTECH, Pohang 790-784, Republic of Korea.
authors.forename: Won-Ki
authors.initials: WK
authors.lastname: Cho
authors.affiliation: Department of Physics, POSTECH, Pohang 790-784, Republic of Korea.
authors.forename: Jonghyun
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Physics, POSTECH, Pohang 790-784, Republic of Korea.
authors.forename: Yongmoon
authors.initials: Y
authors.lastname: Jeon
authors.affiliation: Department of Physics, POSTECH, Pohang 790-784, Republic of Korea.
authors.forename: Daehyung
authors.initials: D
authors.lastname: Kim
authors.affiliation: School of Interdisciplinary Bioscience & Bioengineering, POSTECH, Pohang 790-784, Republic of Korea.
authors.forename: Seung Hwan
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, United States; Physics Department, The Ohio State University, Columbus, OH 43210, United States. Electronic address: rfishel@osu.edu.
authors.forename: Richard
authors.initials: R
authors.lastname: Fishel
mesh_terms: D000818:Animals; D053843:DNA Mismatch Repair; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008856:Microscopy, Fluorescence; D051719:MutS DNA Mismatch-Binding Protein / Q000737:chemistry*
chemical_list: D051719:MutS DNA Mismatch-Binding Protein
keywords: DNA mismatch repair; Diffusion mechanism; MutS; Single particle tracking; Single-molecule FRET; Single-molecule polarization