Coordinating Multi-Protein Mismatch Repair by Managing Diffusion Mechanics on the DNA

Kim, Daehyung,Fishel, Richard,Lee, Jong-Bong Elsevier 2018 Journal of molecular biology Vol.430 No.22

<P><B>Abstract</B></P> <P>DNA mismatch repair (MMR) corrects DNA base-pairing errors that occur during DNA replication. MMR catalyzes strand-specific DNA degradation and resynthesis by dynamic molecular coordination of sequential downstream pathways. The temporal and mechanistic order of molecular events is essential to insure interactions in MMR that occur over long distances on the DNA. Biophysical real-time studies of highly conserved components on mismatched DNA have shed light on the mechanics of MMR. Single-molecule imaging has visualized stochastically coordinated MMR interactions that are based on thermal fluctuation-driven motions. In this review, we describe the role of diffusivity and stochasticity in MMR beginning with mismatch recognition through strand-specific excision. We conclude with a perspective of the possible research directions that should solve the remaining questions in MMR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> MSH (MutS homologs) bound to a mispaired nucleotide switches its conformation to a sliding clamp by ATP binding. </LI> <LI> Freely diffusing ATP-bound MSH sliding clamp recruits MLH/PMS (MutL homologs). </LI> <LI> The diffusing complex of ATP-bound MSH and MLH/PMS activates strand excision at a distant strand break. </LI> <LI> ATP-bound MSH sliding clamp is a key player for the initiation and termination of DNA mismatch repair. </LI> <LI> The stochastic coordination by thermal diffusion of mismatch repair components eventually results in robust repair events. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Stochastic Processes and Component Plasticity Governing DNA Mismatch Repair

Liu, Jiaquan,Lee, Jong-Bong,Fishel, Richard Elsevier 2018 Journal of molecular biology Vol.430 No.22

<P><B>Abstract</B></P> <P>DNA mismatch repair (MMR) is a DNA excision–resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We review the history of mismatch repair, the development of models describing mismatch repair mechanisms and recent single-molecule studies that have contributed to our understanding of these models/mechanisms. </LI> </UL> </P> <P><B>Graphical Abstract</B></P> <P>[DISPLAY OMISSION]</P>

Widespread nuclease contamination in commonly used oxygen-scavenging systems

Senavirathne, Gayan,Liu, Jiaquan,Lopez Jr, Miguel A,Hanne, Jeungphill,Martin-Lopez, Juana,Lee, Jong-Bong,Yoder, Kristine E,Fishel, Richard Nature Publishing Group 2015 NATURE METHODS Vol. No.

ATP Alters the Diffusion Mechanics of MutS on Mismatched DNA

Cho, Won-Ki,Jeong, Cherlhyun,Kim, Daehyung,Chang, Minhyeok,Song, Kyung-Mi,Hanne, Jeungphill,Ban, Changill,Fishel, Richard,Lee, Jong-Bong Elsevier 2012 Structure Vol.20 No.7

<P><B>Summary</B></P><P>The mismatch repair (MMR) initiation protein MutS forms at least two types of sliding clamps on DNA: a transient mismatch searching clamp (∼1 s) and an unusually stable (∼600 s) ATP-bound clamp that recruits downstream MMR components. Remarkably, direct visualization of single MutS particles on mismatched DNA has not been reported. We have combined real-time particle tracking with fluorescence resonance energy transfer (FRET) to image MutS diffusion dynamics on DNA containing a single mismatch. We show searching MutS rotates during diffusion independent of ionic strength or flow rate, suggesting continuous contact with the DNA backbone. In contrast, ATP-bound MutS clamps that are visually and successively released from the mismatch spin freely around the DNA, and their diffusion is affected by ionic strength and flow rate. These observations show that ATP binding alters the MutS diffusion mechanics on DNA, which has a number of implications for the mechanism of MMR.</P> <P><B>Graphical Abstract</B></P><P><ce:figure id='dfig1'></ce:figure></P><P><B>Highlights</B></P><P>► The development and use of smFlow-FRET and smPolarization-TIRF microscopy ► Lesion-searching MutS rotationally diffuses in continuous contact with the DNA ► ATP-bound MutS spins freely and in discontinuous contact with the DNA backbone ► Direct visualization of multiple ATP-bound MutS clamps that diffuse along the DNA</P>

Single-molecule views of MutS on mismatched DNA

Lee, Jong-Bong,Cho, Won-Ki,Park, Jonghyun,Jeon, Yongmoon,Kim, Daehyung,Lee, Seung Hwan,Fishel, Richard Elsevier 2014 DNA repair Vol.20 No.-

<P><B>Abstract</B></P> <P>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 <I>Thermus aquaticus</I> MutS (TaqMutS) particles on mismatched DNA. We determined that TaqMutS forms an incipient clamp to search for a mismatch in ∼1s 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 ∼3s to exchange bound ADP for ATP (ADP→ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10min) 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.</P>

Single-Molecule Studies on EXOI Excision during DNA Mismatch Repair

Jeon, Y.,Kim, D.,Martin-Lopez, J.,Lee, R.,Oh, J.,Hanne, J.,Fishel, R.,Lee, J.B. Biophysical Society ; Published for the Biophysica 2016 Biophysical journal Vol.110 No.3

Cascading MutS and MutL sliding clamps control DNA diffusion to activate mismatch repair

Liu, Jiaquan,Hanne, Jeungphill,Britton, Brooke M.,Bennett, Jared,Kim, Daehyung,Lee, Jong-Bong,Fishel, Richard Nature Publishing Group, a division of Macmillan P 2016 Nature Vol.539 No.7630

<P>Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage(1). Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis(2). Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10-40% of related sporadic tumours(3). However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS-EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS-EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS-EcMutL-EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.</P>

Muts Homolog Sliding Clamps Shield the DNA from Binding Proteins

Hanne, Jeungphill,Britton, Brooke M.,Park, Jonghyun,Liu, Jiaquan,Martin-Lopez, Juana,Jones, Nathan,Schoffner, Matthew,Klajner, Piotr,Bundschuh, Ralf,Lee, Jong-Bong,Fishel, Richard Published for the Biophysical Society by the Rocke 2019 Biophysical journal Vol.116 No.3

Dynamic control of strand excision during human DNA mismatch repair

Jeon, Yongmoon,Kim, Daehyung,Martí,n-Lí,ó,pez, Juana V.,Lee, Ryanggeun,Oh, Jungsic,Hanne, Jeungphill,Fishel, Richard,Lee, Jong-Bong National Academy of Sciences 2016 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.113 No.12

<P>Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' -> 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.</P>

MutS homolog sliding clamps shield the DNA from binding proteins

Hanne, Jeungphill,Britton, Brooke M.,Park, Jonghyun,Liu, Jiaquan,Martí,n-Lí,ó,pez, Juana,Jones, Nathan,Schoffner, Matthew,Klajner, Piotr,Bundschuh, Ralf,Lee, Jong-Bong,Fishel, Richar American Society for Biochemistry and Molecular Bi 2018 The Journal of biological chemistry Vol.293 No.37

<P>Sliding clamps on DNA consist of evolutionarily conserved enzymes that coordinate DNA replication, repair, and the cellular DNA damage response. MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recognizing mispaired nucleotides and in the presence of ATP form stable sliding clamps that randomly diffuse along the DNA. The MSH sliding clamps subsequently load MutL homolog (MLH/PMS) proteins that form a second extremely stable sliding clamp, which together coordinate downstream MMR components with the excision-initiation site that may be hundreds to thousands of nucleotides distant from the mismatch. Specific or nonspecific binding of other proteins to the DNA between the mismatch and the distant excision-initiation site could conceivably obstruct the free diffusion of these MMR sliding clamps, inhibiting their ability to initiate repair. Here, we employed bulk biochemical analysis, single-molecule fluorescence imaging, and mathematical modeling to determine how sliding clamps might overcome such hindrances along the DNA. Using both bacterial and human MSH proteins, we found that increasing the number of MSH sliding clamps on a DNA decreased the association of the Escherichia coli transcriptional repressor LacI to its cognate promoter LacO. Our results suggest a simple mechanism whereby thermal diffusion of MSH sliding clamps along the DNA alters the association kinetics of other DNA-binding proteins over extended distances. These observations appear generally applicable to any stable sliding clamp that forms on DNA.</P>