The mismatch repair protein MutS safeguards genomic integrity by finding and initiating repair of basepairing errors in DNA. Single-molecule studies show MutS diffusing on DNA, presumably scanning for mispaired/unpaired bases, and crystal structures show a characteristic "mismatch-recognition" complex with DNA enclosed within MutS and kinked at the site of error. But how MutS goes from scanning thousands of Watson-Crick basepairs to recognizing rare mismatches remains unanswered, largely because atomic-resolution data on the search process are lacking. Here, 10 μs all-atom molecular dynamics simulations of Thermus aquaticus MutS bound to homoduplex DNA and T-bulge DNA illuminate the structural dynamics underlying the search mechanism. MutS-DNA interactions constitute a multistep mechanism to check DNA over two helical turns for its 1) shape, through contacts with the sugar-phosphate backbone, 2) conformational flexibility, through bending/unbending engineered by large-scale motions of the clamp domain, and 3) local deformability, through basepair destabilizing contacts. Thus, MutS can localize a potential target by indirect readout due to lower energetic costs of bending mismatched DNA and identify a site that distorts easily due to weaker base stacking and pairing as a mismatch. The MutS signature Phe-X-Glu motif can then lock in the mismatch-recognition complex to initiate repair.

Msh4-Msh5 or MutSy belongs to the MutS family of repair proteins that are mainly involved in post-replicative mismatch repair for the purpose of maintaining genome integrity. However, MutSy plays a different role than the other members of its family by facilitating crossover formation during meiotic recombination in many eukaryotic organisms. Failure to form crossovers leads to improper segregation of chromosomes during meiosis, which can lead to infertility and birth defects. Understanding the structural and functional intricacies of this protein can help to elucidate how it functions in crossover formation and meiotic recombination.Previously, our lab has shown that MutSy binds to Holliday junctions (HJ) and HJ-like structures with high affinity, in contrast to DNA duplexes to which the protein binds with 100- fold weaker affinity.

Msh4-Msh5 or MutSgamma is a protein belonging to the MutS family of DNA repair proteins that are mainly involved in post-replicative mismatch repair for the purpose of maintaining genome integrity. Msh4-Msh5 has a different role in which the protein facilitates crossover formation during meiotic recombination in many eukaryotic organisms. Failure to form crossovers results in improper segregation of chromosomes during meiosis, which can lead to infertility and birth defects. Understanding the structural and functional intricacies of this protein will help to elucidate how it functions in crossover formation and meiotic recombination. Previously, our lab has shown that Msh4-Msh5 binds to Holliday junctions (HJ) and HJ-like structures such as 3’ overhangs, single-stranded forks, and D-loops with high affinity, as opposed to DNA duplexes to which the protein binds with much weaker affinity. Generally, our FRET measurements show Msh4-Msh5 displaces the single strand in single-strand containing substrates and induces stacked junction-like structures for substrates more closely resembling Holliday junctions. We are investigating how the presence of nucleotides affects the DNA binding properties of S. cerevisiae Msh4-Msh5. Our approach uses a combination of fluorescently-labeled substrates and nucleotides to determine dissociation constants in the presence of different recombination intermediates while also examining protein-induced conformational changes. We are pairing these results with kinetic studies of ATP hydrolysis in the presence of the same DNA recombination intermediates to gain a clearer understanding of how Msh4-Msh5 ATPase activity is coupled with DNA and protein conformational changes. Through this work, we are investigating the potential molecular switch mechanism of Msh4-Msh5 by which other members of the MutS family have been show to function, while comparing our results from studying S. cerevisiae Msh4-Msh5 to previous results reporting on human Msh4-Msh5.

DNA mismatch repair systems (MMR) are important in reducing post replicative errors and preventing recombination between homologous sequences. Mismatch repair is highly conserved in most organisms and is initiated by the MutS homodimer in prokaryotes. In eukaryotes, small insertion/deletion loops (IDL), and single base pair mismatches are recognized by the MutS homolog Msh2‐Msh6. Prior research in our lab and by others (Marsischky G. T., Lee S., Griffith J., and Kolodner R. D. (1999) J.Biol.Chem. 274: 7200–7206 doi:10.1074/jbc.274.11.7200) determined the binding affinity of the junction with Msh2‐Msh6 with a Kd = 12.9 ± 1.3 nM, which is similar to the binding affinity for a G:T duplex, suggesting that the Msh2‐Msh6 Holliday Junction interaction is specific. Our investigation aims to understand the Msh2‐Msh6‐junction binding interaction by studying nucleotide utilization upon binding and determining any conformational changes associated with complex formation and ATP hydrolysis. We use the well‐characterized J3 Holliday junction with 17 base pair arms to characterize binding. Malachite green assays measuring inorganic phosphate were performed to obtain the rate of ATP hydrolysis in the presence of Msh2‐Msh6, J3 junction, and other DNA substrates. Msh2‐Msh6 alone has a Kcat = 0.27s−1 in the presence of 100mM NaCl, with the Kcat weakly increasing with a G:T mismatch 34‐mer duplex and the J3 junction to 0.47s−1and 0.6s−1 respectively. We will also report on junction conformational changes associated with protein binding and how protein affinity for the junction is modulated by nucleotide binding.Support or Funding InformationASBMB Undergraduate Research Grant.

DNA mismatch repair (MMR), the guardian of the genome, com-mences when MutS identifies a mismatch and recruits MutL to nick the error-containing strand, allowing excision and DNA resynthe-sis. Dominant MMR models posit that after mismatch recognition, ATP converts MutS to a hydrolysis-independent, diffusive mobile clamp that no longer recognizes the mismatch. Little is known about the postrecognition MutS mobile clamp and its interactions with MutL. Two disparate frameworks have been proposed: One in which MutS-MutL complexes remain mobile on the DNA, and one in which MutL stops MutS movement. Here we use single-molecule FRET to follow the postrecognition states of MutS and the impact of MutL on its properties. In contrast to current think-ing, we find that after the initial mobile clamp formation event, MutS undergoes frequent cycles of mismatch rebinding and mobile clamp reformation without releasing DNA. Notably, ATP hy-drolysis is required to alter the conformation of MutS such that it can recognize the mismatch again instead of bypassing it; thus, ATP hydrolysis licenses the MutS mobile clamp to rebind the mismatch. Moreover, interaction with MutL can both trap MutS at the mismatch en route to mobile clamp formation and stop movement of the mobile clamp on DNA. MutS's frequent rebinding of the mismatch, which increases its residence time in the vicinity of the mismatch, coupled with MutL's ability to trap MutS, should increase the prob-ability that MutS-MutL MMR initiation complexes localize near the mismatch.

The UvrA(2) dimer finds lesions in DNA and initiates nucleotide excision repair. Each UvrA monomer contains two essential ATPase sites: proximal (P) and distal (D). The manner whereby their activities enable UvrA(2) damage sensing and response remains to be clarified. We report three key findings from the first pre-steady state kinetic analysis of each site. Absent DNA, a P-2ATP-D-2ADP species accumulates when the low-affinity proximal sites bind ATP and enable rapid ATP hydrolysis and phosphate release by the high-affinity distal sites, and ADP release limits catalytic turnover. Native DNA stimulates ATP hydrolysis by all four sites, causing UvrA(2) to transition through a different species, P-2ADP-D-2ADP. Lesion-containing DNA changes the mechanism again, suppressing ATP hydrolysis by the proximal sites while distal sites cycle through hydrolysis and ADP release, to populate proximal ATP-bound species, P-2ATP-D-empty and P-2ATP-D-2ATP. Thus, damaged and native DNA trigger distinct ATPase site activities, which could explain why UvrA(2) forms stable complexes with UvrB on damaged DNA compared with weaker, more dynamic complexes on native DNA. Such specific coupling between the DNA substrate and the ATPase mechanism of each site provides new insights into how UvrA(2) utilizes ATP for lesion search, recognition and repair.

The mismatch repair (MMR) pathway maintains genome integrity by correcting errors such as mismatched base pairs formed during DNA replication. In MMR, Msh2-Msh6, a heterodimeric protein, targets single base mismatches and small insertion/deletion loops for repair. By incorporating the fluorescent nucleoside base analog 6-methylisoxanthopterin (6-MI) at or adjacent to a mismatch site to probe the structural and dynamic elements of the mismatch, we address how Msh2-Msh6 recognizes these mismatches for repair within the context of matched DNA. Fluorescence quantum yield and rotational correlation time measurements indicate that local base dynamics linearly correlate with Saccharomyces cerevisiae Msh2-Msh6 binding affinity where the protein exhibits a higher affinity (KD ≤ 25 nM) for mismatches that have a significant amount of dynamic motion. Energy transfer measurements measuring global DNA bending find that mismatches that are both well and poorly recognized by Msh2-Msh6 experience the same amount of protein-induced bending. Finally, base-specific dynamics coupled with protein-induced blue shifts in peak emission strongly support the crystallographic model of directional binding, in which Phe 432 of Msh6 intercalates 3' of the mismatch. These results imply an important role for local base dynamics in the initial recognition step of MMR.

The authors noted an error in the 550-bp DNA substrate used in our experiments shown in Figs 3–5, 6A–C, and 7. The synthetic oligos annealed into the gapped pUC-19 segment included an additional six non-homologous bases at the 30. This resulted in a six base flap of single-strand DNA overhanging the duplex region. Note, the erroneous oligos that we used are also published elsewhere (Robertson AB, Matson SW, 2012, J Biol Chem, 287: 32953–32966 and Robertson AB, 2007, Ph.D. Thesis). Based upon the identical results we reported using either this flap-containing 550-bp DNA or the 50-bp DNA, which did not contain any single-strand flaps, the conclusion that MutS undergoes conformational changes when interacting with mismatched bases is not affected. We further conclude that a six base single-strand flap located 12 bases 30 from a mismatch does not alter MutS behavior at a mismatch. Finally, we report that we have confirmed these interpretations by duplicating the results in our paper using contiguous, 550-bp duplexes created with correct oligos to fill the gapped DNA (Qiu R, Sakato M, Sacho EJ, Wilkins H, Zhang X, Modrich P, Hingorani MM, Erie DA, Weninger KR, 2015, Proc Natl Acad Sci USA, 112: 10914– 10919). We apologize for any inconvenience caused by this error.

RNA-DNA hybrid primers synthesized by low fidelity DNA polymerase α to initiate eukaryotic lagging strand synthesis must be removed efficiently during Okazaki fragment (OF) maturation to complete DNA replication. In this process, each OF primer is displaced and the resulting 5'-single-stranded flap is cleaved by structure-specific 5'-nucleases, mainly Flap Endonuclease 1 (FEN1), to generate a ligatable nick. At least two models have been proposed to describe primer removal, namely short- and long-flap pathways that involve FEN1 or FEN1 along with Replication Protein A (RPA) and Dna2 helicase/nuclease, respectively. We addressed the question of pathway choice by studying the kinetic mechanism of FEN1 action on short- and long-flap DNA substrates. Using single molecule FRET and rapid quench-flow bulk cleavage assays, we showed that unlike short-flap substrates, which are bound, bent and cleaved within the first encounter between FEN1 and DNA, long-flap substrates can escape cleavage even after DNA binding and bending. Notably, FEN1 can access both substrates in the presence of RPA, but bending and cleavage of long-flap DNA is specifically inhibited. We propose that FEN1 attempts to process both short and long flaps, but occasional missed cleavage of the latter allows RPA binding and triggers the long-flap OF maturation pathway.

Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair, and recombination and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap: template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps, from substrate binding to product release, during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that whereas hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap: template junction, indicating that local DNA distortion (e.g. disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34 and Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93 and Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.

In many organisms, MutSγ plays a role in meiotic recombination, facilitating crossover formation between homologous chromosomes. Failure to form crossovers leads to improper segregation of chromosomes and aneuploidy, which in humans result in infertility and birth defects. To improve current understanding of MutSγ function, this study investigates the binding affinities and structures of MutSγ in complex with DNA substrates that model homologous recombination intermediates. For these studies, we overexpressed and isolated from Escherichia coli the yeast MutSγ protein Saccharomyces cerevisiae (Sc) Msh4-Msh5. Sc Msh4-Msh5 binds Holliday junction (HJ)-like substrates, 3' overhangs, single-stranded (ss) forks, and the displacement loop with nanomolar affinity. The weakest binding affinities are detected for an intact duplex and open-junction construct. Similar to the human protein, Sc Msh4-Msh5 exhibits the highest affinity for the HJ with a Kd < 0.4 nM in solution. Energy-transfer experiments further demonstrate that DNA structure is modulated by the binding interaction with the largest changes associated with substrates containing an ss end. Upon binding, Sc Msh4-Msh5 displaces the ss away from the duplex in most of the ss-containing intermediates, potentially enabling the binding of RPA and other proteins. In the case of the junction-like intermediates, Msh4-Msh5 binding either stabilizes the existing stacked structure or induces formation of the stacked X conformation. Significantly, we find that upon binding, Msh4-Msh5 stacks an open-junction construct to the same extent as the standard junction. Stabilization of the junction in the stacked conformation is generally refractory to branch migration, which is consistent with a potential role for MutSγ to stabilize HJs and prevent branch migration until resolution by MutLγ. The different binding modalities observed suggest that Msh4-Msh5 not only binds to and stabilizes stacked junctions but also participates in meiotic recombination before junction formation through the stabilization of single-end invasion intermediates.

The Mismatch Repair (MMR) pathway is responsible for correcting errors in DNA and is thus essential for maintaining genome stability in all living organisms. Several studies have shown that mutations causing defects in MMR protein structure and function lead to hypermutation. The increased level of mutations results in serious consequences for human health, highlighted by Lynch Syndrome (LS), a hereditary predisposition to colorectal and other cancers (1). The first protein in the MMR pathway, MutS, recognizes mispaired bases or short insertion‐deletion loops (IDLs) and utilizes its ATPase activity to initiate DNA repair (2). A long‐term study in the Hingorani laboratory examines cancer‐linked single amino acid mutants of the human MutS protein (using Thermus aquaticus MutS as a model system) in order to understand how the changes affect protein structure and function, and alter MMR. This work involves kinetic analysis of MutS activities, and related dynamic conformational changes in the protein as it utilizes ATP to work on DNA. The results will enable construction of a complete mechanism of action for each mutant protein, and help us determine whether the mutants are compromised in their ability to target mismatches, and/or bind and hydrolyze ATP, and/or undergo necessary conformational changes, and therefore understand why they disrupt DNA mismatch repair and give rise to Lynch Syndrome. My project focuses on fluorescence‐based stopped flow kinetic experiments to monitor the conformational changes of wild type MutS and several LS‐linked mutants as they bind +T insertion‐containing DNA and catalyze ATP hydrolysis. In addition to generating new information on each mutant, the findings will advance the proof‐of‐principle concept that detailed analysis of the structure, dynamics, and catalytic activities of individual MutS mutants is both feasible and can reveal critical information for understanding the molecular basis of Lynch Syndrome.Support or Funding InformationNational Institutes of Health R15 GM114743; The Beckman Scholars Program, Arnold and Mabel Beckman FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Nucleotide excision repair (NER) protects genomic DNA from a wide variety of lesions, including nucleotide adducts and pyrimidine dimers. In bacteria, NER is initiated by the UvrA2(B2) scanning complex when UvrA2 locates a lesion and UvrB binding confirms lesion recognition. Each monomer in the UvrA2 dimer has two ATPase sites (proximal and distal), and previous studies indicate that each site plays a distinct role in lesion detection. These studies, however, have lacked the resolution needed to determine the temporal order and stoichiometry of ATP binding, hydrolysis and product release at all sites. Hence, there is uncertainty about how the four ATPase sites work together to drive UvrA2 actions during lesion detection and initiation of NER. Our goal is to address this question by first determining the UvrA2 ATPase mechanism on and off a lesion. To this end, we have initiated kinetic analysis of wild type B. stearothermophilius UvrA2 and Walker A and Walker B ATPase mutants. Thus far, our results show that when UvrA2 is not bound to DNA, ATP binding to the proximal sites permits ATP hydrolysis at the distal sites; however, ADP is released slowly leaving the latter predominantly ADP-bound. Contact with undamaged DNA accelerates the ATPase cycle at both the proximal and distal sites, but the latter still appear predominantly ADP-bound. Contact with damaged DNA also accelerates the ATPase cycle, but ADP release from the distal sites is stimulated such that ATP hydrolysis becomes the slowest step in the reaction. Thus, when UvrA2 binds a lesion it switches from a predominantly distal ADP-bound to ATP-bound state. These distinct responses allow us to begin building a model of how UvrA2 ATPase activity is coupled to lesion search, recognition and response.

Flap Endonuclease 1 (FEN1) recognizes and cleaves 5′ single‐stranded DNA flaps to create nicked duplex products during DNA metabolic processes such as Okazaki fragment maturation, long‐patch base excision repair and telomere maintenance, and therefore has an essential role in genome duplication and stability (1). Defects in human FEN1 are associated with cancer and other genetic diseases (2). Hence, there is considerable interest in hFEN1 as a therapeutic target and diagnostic or prognostic biomarker (3–5), which can be aided by a detailed mechanistic understanding of the enzyme.Previous studies have shown that hFEN1 bends its DNA substrate, and suggest that unpairing of two bases in the duplex beyond the 5′ flap helps position the scissile phosphate in the active site for cleavage (6,7). These studies have largely been performed under equilibrium conditions and in the absence of Mg2+, which is indispensible for catalysis. To study the hFEN1 mechanism under catalytically competent conditions, we utilized transient kinetic approaches and monitored individual events in the millisecond time scale, including DNA binding and bending, base unpairing and cleavage, which elucidated the order of events and rate‐determining step(s) in the reaction. Furthermore, we examined the role of several conserved active‐site residues in the reaction mechanism. A FRET assay revealed that hFEN1 binds and bends the DNA substrate rapidly at a diffusion‐limited rate (109 M−1s−1), indicating that these events occur simultaneously. Lack of Mg2+ and mutations in the active site do not affect this binding/bending rate, thus this initial event does not depend on specific contacts within the catalytic pocket. A 2‐aminopurine fluorescence‐based assay revealed unpairing of base(s) at the flap junction at a much slower rate of 20 s−1. Importantly, quench‐flow measurements revealed that base unpairing limits the cleavage rate to 20 s−1. A subsequent event, likely related to product release, limits the overall steady state turnover rate to 1 s−1. Mutations in active site residues D34, Y40, K93, R100, E160 and D181 exhibit differential effects on base unpairing and cleavage. The results show that base unpairing is a pre‐requisite for flap cleavage, and clarify the contributions of these conserved residues toward an electrostatic environment that stimulates precise cleavage of the 5′ flap. The findings also apply to other 5′‐nucleases that share similar active site residues and a two‐metal ion dependent reaction mechanism (8,9).

Mismatch repair (MMR) is an essential, evolutionarily conserved pathway that maintains genome stability by correcting base-pairing errors in DNA. Here we examine the sequence and structure of MutS MMR protein to decipher the amino acid framework underlying its two key activities-recognizing mismatches in DNA and using ATP to initiate repair. Statistical coupling analysis (SCA) identified a network (sector) of coevolved amino acids in the MutS protein family. The potential functional significance of this SCA sector was assessed by performing molecular dynamics (MD) simulations for alanine mutants of the top 5% of 160 residues in the distribution, and control nonsector residues. The effects on three independent metrics were monitored: (i) MutS domain conformational dynamics, (ii) hydrogen bonding between MutS and DNA/ATP, and (iii) relative ATP binding free energy. Each measure revealed that sector residues contribute more substantively to MutS structure-function than nonsector residues. Notably, sector mutations disrupted MutS contacts with DNA and/or ATP from a distance via contiguous pathways and correlated motions, supporting the idea that SCA can identify amino acid networks underlying allosteric communication. The combined SCA/MD approach yielded novel, experimentally testable hypotheses for unknown roles of many residues distributed across MutS, including some implicated in Lynch cancer syndrome.

DNA polymerases depend on circular sliding clamps for processive replication. Clamps must be loaded onto primer-template DNA (ptDNA) by clamp loaders that open and close clamps around ptDNA in an ATP-fueled reaction. All clamp loaders share a core structure in which five subunits form a spiral chamber that binds the clamp at its base in a twisted open form and encloses ptDNA within, while binding and hydrolyzing ATP to topologically link the clamp and ptDNA. To understand how clamp loaders perform this complex task, here we focused on conserved arginines that might play a central coordinating role in the mechanism because they can alternately contact ptDNA or Walker B glutamate in the ATPase site and lie close to the clamp loader-clamp-binding interface. We mutated Arg-84, Arg-88, and Arg-101 in the ATPase-active B, C, and D subunits of Saccharomyces cerevisiae replication factor C (RFC) clamp loader, respectively, and assessed the impact on multiple transient events in the reaction: proliferating cell nuclear antigen (PCNA) clamp binding/opening/closure/release, ptDNA binding/release, and ATP hydrolysis/product release. The results show that these arginines relay critical information between the PCNA-binding, DNA-binding, and ATPase sites at all steps of the reaction, particularly at a checkpoint before RFC commits to ATP hydrolysis. Moreover, their actions are subunit-specific with RFC-C Arg-88 serving as an accelerator that enables rapid ATP hydrolysis upon contact with ptDNA and RFC-D Arg-101 serving as a brake that confers specificity for ptDNA as the correct substrate for loading PCNA.