MutS-DNA Complex Research Articles

MutS recognition of mismatches within primed DNA replication intermediates

MutS initiates mismatch repair by recognizing mismatches in newly replicated DNA. Specific interactions between MutS and mismatches within double-stranded DNA promote ADP-ATP exchange and a conformational change into a sliding clamp. Here, we demonstrated that MutS from Pseudomonas aeruginosa associates with primed DNA replication intermediates. The predicted structure of this MutS-DNA complex revealed a new DNA binding site, in which Asn 279 and Arg 272 appeared to directly interact with the 3′-OH terminus of primed DNA. Mutation of these residues resulted in a noticeable defect in the interaction of MutS with primed DNA substrates. Remarkably, MutS interaction with a mismatch within primed DNA induced a compaction of the protein structure and impaired the formation of an ATP-bound sliding clamp. Our findings reveal a novel DNA binding mode, conformational change and intramolecular signaling for MutS recognition of mismatches within primed DNA structures.

Mechanism of formation of a toroid around DNA by the mismatch sensor protein.

The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS–DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS–DNA complex.

Kinetic Analysis of Interactions between MutS, MutL and DNA During Initiation of DNA Mismatch Repair

Mismatch repair (MMR) is essential for correcting base-pairing errors in DNA. MutS protein recognizes mis-paired bases in DNA and recruits MutL to signal excision and re-synthesis of the mismatched strand. The mechanism of MMR is under active investigation_especially the transient events involved in mismatch recognition and initiation of repair. We are utilizing transient kinetic methods coupled with fluorescence spectroscopy to determine the mechanisms of action of MutS and MutL from Thermus aquaticus. We have built a kinetic model of the ATPase-linked actions of MutS on DNA whereby initial rapid encounter between the two establishes a weak binding equilibrium (KD1 = 5 μM), followed by slow formation of a mismatch-specific MutS-DNA complex with the DNA in bent conformation (kconf ∼ 30 s-1 and KD2 = 5 nM). ATP binding to MutS-DNA complex (kON = 0.5 μM-1s-1) is followed by two slow steps involving conformational changes in MutS that correspond to unbending of DNA at the mismatch site (∼ 3 s-1), and subsequent MutS release from the mismatch (∼ 0.5 s-1). To understand the mechanism of MMR during the transition between mismatch recognition and initiation of excision, we plan kinetic analysis of the concerted actions of MutS and MutL on DNA. We are developing fluorescence-based assays to monitor MutL interactions with DNA and with the MutS-DNA complex. Currently, we are testing the fluorescent nucleoside analog 6-methyl-isoxanthopterin (6-MI), paired with cytosine and located adjacent to a mismatch. This reporter can be used to analyze MutS dynamics at the mismatch site and its communication with MutL, thus enabling novel mechanistic insights into the mechanism of initiation of MMR.

MutS and DNA dynamics during mismatch recognition and repair.

Mismatch repair (MMR) is essential for correcting base‐pairing errors in DNA. MutS protein recognizes mispaired bases in DNA and signals excision and resynthesis of the mismatched strand. The DNA binding and ATPase activities of MutS are under active investigation‐especially the transient events involved in mismatch recognition and initiation of repair. We utilize transient kinetic methods coupled with fluorescence spectroscopy to determine the mechanisms of action of Thermus aquaticus MutS. Our results indicate a two‐step DNA binding mechanism for MutS, wherein an initial rapid encounter establishes a weak binding equilibrium (KD1 = 3 μM), followed by slow formation of a mismatch‐specific MutS‐DNA complex with the DNA in bent conformation (kconf ~ 20 s−1and KD2 = 5 nM). ATP binding to MutS‐DNA complex (kON = 0.3 μM−1s−1) is followed by two slow steps involving conformational changes in MutS and the mismatch site in DNA (~ 2 s−1) followed by MutS release from the mismatch (~ 0.5 s−1). With detailed kinetic analysis, we are developing a comprehensive view of MutS actions on DNA, and gaining novel mechanistic insights into the mechanism of initiation of MMR.

Understanding the Kinetic Mechanism of MutS during Mismatch Recognition and Initiation of Repair

DNA mismatch repair (MMR) is essential for correcting errors generated in DNA during replication. Abnormal expression or function(s) of MMR proteins give rise to a mutator phenotype, increasing cancer susceptibility. MMR initiates when MutS recognizes a mismatch and binds it with high affinity. MutS then binds ATP and forms a sliding clamp to signal initiation of downstream repair steps. The DNA-binding and ATPase activities of MutS are under active investigation, in particular the events involved in transition between recognition of errors and initiation of repair. We are utilizing stopped-flow analysis of fluorescently labeled protein as well as DNA to determine the kinetic mechanism of Thermus aquaticus MutS. Our results indicate that MutS rapidly interacts with mismatches to form an initial weak complex (KD1 = 2 µM), followed by conformational changes in both DNA and MutS to form a high affinity MutS-DNA complex with the DNA kinked at the mismatch site (kconf ∼ 20 s-1 and KD2 = 15 nM). In absence of mismatched DNA, MutS rapidly switches between two conformations in response to ATP binding and hydrolysis. ATP hydrolysis is suppressed in mismatch-bound MutS, and our data suggest that after ATP binding (kON = 0.2 µM-1s-1), the MutS-DNA complex undergoes at least two conformational changes (kobs1 ∼ 3 s-1 and kobs2 ∼ 0.3s-1) possibly related to DNA unbending and formation of the MutS sliding clamp, respectively. By monitoring the reaction via fluorescent reporters on both DNA and protein, we are developing a comprehensive view of MutS actions on DNA and gaining novel mechanistic insights into the initiation of mismatch repair.

Sculpting of DNA at Abasic Sites by DNA Glycosylase Homolog Mag2

Modifications and loss of bases are frequent types of DNA lesions, often handled by the base excision repair (BER) pathway. BER is initiated by DNA glycosylases, generating abasic (AP) sites that are subsequently cleaved by AP endonucleases, which further pass on nicked DNA to downstream DNA polymerases and ligases. The coordinated handover of cytotoxic intermediates between different BER enzymes is most likely facilitated by the DNA conformation. Here, we present the atomic structure of Schizosaccharomyces pombe Mag2 in complex with DNA to reveal an unexpected structural basis for nonenzymatic AP site recognition with an unflipped AP site. Two surface-exposed loops intercalate and widen the DNA minor groove to generate a DNA conformation previously only found in the mismatch repair MutS-DNA complex. Consequently, the molecular role of Mag2 appears to be AP site recognition and protection, while possibly facilitating damage signaling by structurally sculpting the DNA substrate.

Covalently trapping MutS on DNA to study DNA mismatch recognition and signaling

The DNA repair protein MutS forms clamp-like structures on DNA that search for and recognize base mismatches leading to ATP-transformed signaling clamps. In this study, the mobile MutS clamps were trapped on DNA in a functional state using single-cysteine variants of MutS and thiol-modified homoduplex or heteroduplex DNA. This approach allows stabilization of various transient MutS-DNA complexes and will enable their structural and functional analysis.

Base-Flipping Mechanism in Postmismatch Recognition by MutS

DNA mismatch recognition and repair is vital for preserving the fidelity of the genome. Conserved across prokaryotes and eukaryotes, MutS is the primary protein that is responsible for recognizing a variety of DNA mismatches. From molecular dynamics simulations of the Escherichia coli MutS-DNA complex, we describe significant conformational dynamics in the DNA surrounding a G·T mismatch that involves weakening of the basepair hydrogen bonding in the basepair adjacent to the mismatch and, in one simulation, complete base opening via the major groove. The energetics of base flipping was further examined with Hamiltonian replica exchange free energy calculations revealing a stable flipped-out state with an initial barrier of ∼2 kcal/mol. Furthermore, we observe changes in the local DNA structure as well as in the MutS structure that appear to be correlated with base flipping. Our results suggest a role of base flipping as part of the repair initiation mechanism most likely leading to sliding-clamp formation.

Finding the Right ‘Mis’Match: Millisecond Conformational Dynamics of MutS-DNA Complex During DNA Damage Recognition

Errors in replication and recombination cause base-pair mismatches and insertion-deletion loops (IDLs) in DNA, which, if unchecked, lead to mutations implicated in cancer and heart disease. Thus, repair pathways that detect such errors and initiate their repair are vital to preserving DNA integrity. This study focuses on prokaryotic T. aquaticus (Taq) MutS and its eukaryotic homolog S. cerevisiae MutSα, both proteins that recognize mispairs and IDLs in DNA and recruit downstream proteins to rectify the damage. Structural studies have shown that MutS-bound DNA is bent at the mispair/IDL site. However, the underlying mechanism of recognition of these target sites is not well understood. In particular, the role of DNA flexibility at the target site in the recognition mechanism remains unclear. We employ nanosecond laser temperature jump (T-jump) to perturb the MutS-DNA complex, and monitor the conformational relaxation dynamics on microsecond-to-milliseconds time-scales relevant to the formation of the initial recognition complex that triggers DNA repair. We have carried out equilibrium and kinetic measurements on DNA labeled with 2-aminopurine (2AP) adjoining a T-bulge, in complex with Taq MutS andMutSα. Equilibrium fluorescence measurements as a function of increasing temperature reveal a sharp increase in 2AP fluorescence at ∼40°C for MutSα, and at ∼70°C for Taq MutS, indicating a significant conformational change in the MutS-DNA complex near their optimal physiological temperatures. Relaxation kinetics monitored with 2AP fluorescence in response to a rapid T-jump show relaxation kinetics in Taq MutS near 70°C, occurring with a time constant of a ∼10 milliseconds. This rapid phase has not been observed in previous dynamics studies, and we propose that it may correspond to the transition from a non-specific MutS-DNA complex to a specific complex that signals initiation of repair.

Recognition and Signaling in DNA Mismatch Repair: MD Studies of MutS Complexes with DNA and ATP

The MutS family of DNA binding proteins has been reported to play a critical role in mismatch repair (MMR). Crystal structures of MutS (Escherichia coli and Thermus aquaticus) as well MSH homologs including human MutSα reveal intricate and complex multi-domain protein structures comprised of greater than 1,500 residues. The DNA binding domain of these proteins recognizes mispaired or unpaired bases. It has been proposed that this recognition event results in the release of a signal that travels from the DNA binding domain to the ATPase site. While much has been learned from previous binding studies of MutS, the contribution of the protein dynamics on MutS complex formation and intraprotein communication events are not fully resolved at the atomic level. In this study, state-of-the-art molecular dynamics (MD) simulations are used to investigate the dynamical processes that occur during the interactions with DNA and ATP substrates. In particular, we are interested in how the DNA mismatch recognition/binding event is signaled, triggering the initiation of DNA repair. The computational challenge represented by the size and complexity of MutS-DNA complexes provides an opportunity to develop MD approaches for large multi-component biological systems.

Mechanism of MutS Searching for DNA Mismatches and Signaling Repair

DNA mismatch repair is initiated by the recognition of mismatches by MutS proteins. The mechanism by which MutS searches for and recognizes mismatches and subsequently signals repair remains poorly understood. We used single-molecule analyses of atomic force microscopy images of MutS-DNA complexes, coupled with biochemical assays, to determine the distributions of conformational states, the DNA binding affinities, and the ATPase activities of wild type and two mutants of MutS, with alanine substitutions in the conserved Phe-Xaa-Glu mismatch recognition motif. We find that on homoduplex DNA, the conserved Glu, but not the Phe, facilitates MutS-induced DNA bending, whereas at mismatches, both Phe and Glu promote the formation of an unbent conformation. The data reveal an unusual role for the Phe residue in that it promotes the unbending, not bending, of DNA at mismatch sites. In addition, formation of the specific unbent MutS-DNA conformation at mismatches appears to be required for the inhibition of ATP hydrolysis by MutS that signals initiation of repair. These results provide a structural explanation for the mechanism by which MutS searches for and recognizes mismatches and for the observed phenotypes of mutants with substitutions in the Phe-Xaa-Glu motif.

Detection of point mutation and insertion mutations in DNA using a quartz crystal microbalance and MutS, a mismatch binding protein.

MutS protein is a mismatch binding protein that recognizes mispaired and unpaired base(s) in DNA. In this study, we incorporate the MutS protein-based mutation recognition into quartz crystal microbalance (QCM) measurements for DNA single-base substitution mutation and 1-4 base(s) insertion (or deletion) mutation detection. The method involves the immobilization of single-stranded probe DNA on a QCM surface, the hybridization of target DNA to form homoduplex or heteroduplex DNA, and finally the application of MutS protein for the mutation recognition. By measuring the MutS binding signal, DNA containing a T:G mismatch or unpaired base(s) is(are) discriminated against perfectly matched DNA at target concentrations ranging from 1nM to 5 microM. Furthermore, the QCM damping behavior upon MutS-DNA complex formation is studied using a Network Analyzer. The measured motional resistance changes per coupled MutS unit mass (deltaR/deltaf) are found to be indicative of the viscoelastic or structural properties of the bound protein, corresponding to different binding mechanisms. In addition, the deltaR/deltaf values vary remarkably when the MutS protein binds at different distances away from the QCM surface. Thus, these values can be used as a "fingerprint" for MutS mismatch recognition and also used to quantitatively locate the mutation site.

MutS-DNA interactions and DNase protection analysis with surface plasmon resonance.

Elucidation of the mechanisms of recognition of mispaired DNA by MutS and subsequent repair by the multicomponent mismatch repair machinery presents a fascinating challenge (). ATP binding or hydrolysis are implicated in several steps of the process, including enabling MutS to dissociate from the mismatch, probably through a conformational change () and for MutSL-DNA interactions (,). In addition to mismatch recognition, the protein has significant affinity for normal DNA. This may be important for the protein to scan DNA in search of a mismatch and for its proposed translocation activity. It can be expected, and especially once a MutS-DNA complex structure is solved, that numerous mutants will be generated to study the recognition mechanism. To examine stable and transient DNA interactions of the native and mutant proteins in various conformational states, assays that enable detailed kinetics are desirable. Instrumentation providing such capability is a worthwhile complement to classic assays, such as filter binding and electrophoretic mobility shift assays. Here, we describe techniques for studying the interaction of MutS and MutL with immobilized DNA using surface plasmon resonance and the BIAcore instrument. Such techniques should be readily adaptable to studies of other mismatch binding proteins (MMBP) or other DNA damage recognition proteins.

ATP hydrolysis-dependent formation of a dynamic ternary nucleoprotein complex with MutS and MutL.

Functional interactions of Escherichia coli MutS and MutL in mismatch repair are dependent on ATP. In this study, we show that MutS and MutL associate with immobilised DNA in a manner dependent on ATP hydrolysis and with an ATP concentration near the solution K m of the ATPase of MutS. After removal of MutS, MutL and ATP, much of the protein in this ternary complex is not stably associated, with MutL leaving the complex more rapidly than MutS. The rapid dissociation reveals a dynamic interaction with concurrent rapid association and dissociation of proteins from the DNA. Analysis by surface plasmon resonance showed that the DNA interacting with dynamically bound protein was more resistant to nuclease digestion than the DNA in MutS-DNA complexes. Non-hydrolysable analogs of ATP inhibit the formation of this dynamic complex, but permit formation of a second type of ternary complex with MutS and MutL stably bound to the immobilised DNA.