The mismatch repair system is an evolutionarily conserved pathway that is vital in maintaining genomic integrity and cellular stability. It is involved in post-replication DNA repair and recombination processes and its inactivation is strongly linked to increased tumor development. The Saccharomyces cerevisiae MutSα or Msh2-Msh6 heterodimer is a post-replicative mismatch repair protein that recognizes and corrects single base mismatches and small insertion-deletion loops. Interestingly, Msh2-Msh6 binds DNA Holliday Junctions (HJ) with the same affinity as DNA mismatches; however, the specifics of this binding interaction and the key residues involved are relatively unknown. To investigate the orientation in which Msh2-Msh6 binds to HJs, the protein was photocrosslinked to DNA junction constructs with 5- bromouridine (BrU), which forms a stable crosslink to proteins upon exposure to 254 nm UV light. Incorporation of BrU on different strands of a homologous junction did not reveal any binding asymmetry; although probe locations further from the junction center reduce crosslinking efficiency. The Msh6 subunit has been shown through crystallography to use a Phe-X-Glu motif for mismatch recognition. Crosslinking studies and fluorescence binding assays performed with mutant proteins suggest that this motif is not needed for junction recognition. To identify the residues important for the junction interaction, we are employing mass spectrometry to determine the protein residues crosslinked to BrU. In parallel, we have been incorporating a non-natural amino acid pBpA to photocrosslink the protein to the DNA and identify protein residues important for junction binding. Our preliminary results suggest Msh2-Msh6 may bind Holliday junctions in a different manner from DNA single base mismatches.

The mismatch repair (MMR) protein MutSα (Msh2-Msh6), known for improving the fidelity of DNA replication, also improves the fidelity of homologous recombination (HR). Msh2-Msh6 and its fellow MMR proteins are responsible for suppressing homologous recombination when excessive base mispairing is present between the participating chromosomes. In MMR, Msh2-Msh6 recognizes small insertion/deletion loops and single base pair mismatches while scanning DNA post-replication. Interestingly, Msh2-Msh6 binds to Holliday Junctions (HJs) with an affinity comparable to that of mismatched duplex DNA (Kd =15 nM).

DNA Holliday Junction (HJ) formation and resolution is requisite for maintaining genomic stability in processes such as replication fork reversal and double-strand break repair. If HJs are not resolved, chromosome disjunction and aneuploidy result, hallmarks of tumor cells. To understand the structural features that lead to processing of these four-stranded joint molecule structures, we seek to identify structural and dynamic features unique to the central junction core. We incorporate the fluorescent guanine analog 6-methylisoxanthopterin (6-MI) at ten different locations throughout a model HJ structure to obtain site-specific information regarding the structure and dynamics of bases relative to those in a comparable sequence context in duplex DNA. These comparisons were accomplished through measuring fluorescence lifetime, relative brightness, fluorescence anisotropy, and thermodynamic stability, along with fluorescence quenching assays. These time-resolved and steady-state fluorescence measurements demonstrate that the structural distortions imposed by strand crossing result in increased solvent exposure, less stacking of bases and greater extrahelical nature of bases within the junction core. The 6-MI base analogs in the junction reflect these structural changes through an increase in intensity relative to those in the duplex. Molecular dynamics simulations performed using a model HJ indicate the primary sources of deformation are in the shift and twist parameters of the bases at the central junction step. These results suggest that junction-binding proteins may use the unique structure and dynamics of the bases at the core for recognition.

The mismatch repair system is an evolutionarily conserved pathway that is vital in maintaining genomic integrity and cellular stability. It is involved in post-replication DNA repair and recombination processes and its inactivation is strongly linked to increased tumor development. The Saccharomyces cerevisiae MutSa or Msh2-Msh6 heterodimer is a post-replicative mismatch repair protein that recognizes and corrects single base mismatches and small insertion-deletion loops. Interestingly, Msh2-Msh6 binds DNA Holliday Junctions (HJ) with the same affinity as DNA mismatches; however, the specifics of this binding interaction and the key residues involved are relatively unknown. To investigate the orientation in which Msh2-Msh6 binds to HJs, the protein was photocrosslinked to DNA junction constructs with 5- bromouridine (BrU), which forms a stable crosslink to proteins upon exposure to 254nm UV light. Incorporation of BrU on different strands of a homologous junction did not reveal any binding asymmetry; although probe locations further from the junction center reduce crosslinking efficiency. The Msh6 subunit has been shown through crystallography to use a Phe-X-Glu motif for mismatch recognition. Crosslinking studies and fluorescence binding assays performed with mutant proteins suggest that this motif is not needed for junction recognition. To identify the residues important for the junction interaction, we are employing mass spectrometry to determine the protein residues crosslinked to BrU. In parallel, we have been incorporating a non-natural amino acid pBpA to photocrosslink the protein to the DNA and identify protein residues important for junction binding. Our preliminary results suggest Msh2-Msh6 may bind Holliday junctions in a different manner from DNA single base mismatches.

Integration host factor (IHF) is a nucleoid-associated protein involved in DNA packaging, integration of viral DNA and recombination. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence, inducing a bend of ~160° upon binding. We determined that IHF binds to DNA Four-way or Holliday junctions (HJ) with high affinity regardless of the presence of the consensus sequence, signifying a structure-based mechanism of recognition. Junctions, important intermediates in DNA repair and homologous recombination, are dynamic and can adopt either an open or stacked conformation, where the open conformation facilitates branch migration and strand exchange. Using ensemble and single molecule Förster resonance energy transfer (FRET) methods, we investigated IHF-induced changes in the population distribution of junction conformations and determined that IHF binding shifts the population to the open conformation. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Protein binding alters junction conformational dynamics, as cross correlation analyses reveal the protein slows the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+. Stopped flow kinetic experiments provide evidence for two binding steps, a rapid, initial binding step followed by a slower step potentially associated with a conformational change. These measurements also confirm that the protein remains bound to the junction during the conformer transitions and further suggest that the protein forms a partially dissociated state that allows junction arms to be dynamic. These findings, which demonstrate that IHF binds HJs with high affinity and stabilizes junctions in the open conformation, suggest that IHF may play multiple roles in the processes of integration and recombination in addition to stabilizing bacterial biofilms.

Msh2‐Msh6 is a eukaryotic ATP‐dependent mismatch repair (MMR) protein that plays a key role in maintaining the fidelity of the genetic code. The protein recognizes and initiates repair for single base pair mismatches and short insertion deletion loops (IDL). Msh2‐Msh6 functions via a molecular switch mechanism, whereby ATP hydrolysis and ADP/ATP exchange facilitates the binding and release of the protein from the mismatch. Previous research has revealed that the protein binds G:T mismatch DNA with the highest affinity. Interestingly, in‐solution binding assays have revealed that Msh2‐Msh6 binds Holliday Junction DNA, a key recombination intermediate, with 1:1 stoichiometry and a similarly high affinity. However, the functional importance and structural details of this interaction are unclear. Thus far, X‐ray crystallography and Molecular Dynamics (MD) simulations are limited to the protein’s interactions with mismatch DNA only. To address the lack of structural information, we modeled the Msh2‐Msh6‐HJ interaction using the human Msh2‐Msh6 crystal structure in which we have docked in junction DNA instead of the mismatched DNA. The junction DNA used in all experiments and simulations is J3, a non‐migrating junction with 17 base pair long arms. These MD simulations will help refine the structural model and assess significant interactions between the protein and DNA. Furthermore, ATPase assays and equilibrium binding assays are used to elucidate the protein’s ATP hydrolysis activity and binding affinity in the presence of G:T mismatch DNA, J3 junction DNA, and J3 junction DNA containing a G:T mismatch. Preliminary experiments show that relative to the apoprotein’s basal ATPase activity, ATP hydrolysis is weakly stimulated when Msh2‐Msh6 is bound to G:T mismatch and J3. Gel mobility shift assays (GMSA) reveal binding affinities in the range of 9.1±2.6 nM to 23.5±11.8 nM for G:T mismatch, J3 junction, and mismatched J3 junction. These data illustrate that Msh2‐Msh6 not only binds G:T mismatch and J3 with similarly high affinity, but also that introducing a mismatch in J3 does not modulate the protein’s affinity for the junction. In addition, the GMSA data suggest that Msh2‐Msh6‐HJ binding may take on multiple conformations, consistent with previous Förster resonance energy transfer (FRET) calculations that show Msh2‐Msh6 opening the junction after binding.

Förster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

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.

Crossing over is essential for chromosome segregation during meiosis. Protein modification by SUMO is implicated in crossover control, but pertinent targets have remained elusive. Here we identify Msh4 as a target of SUMO-mediated crossover regulation. Msh4 and Msh5 constitute the MutSγ complex, which stabilizes joint-molecule (JM) recombination intermediates and facilitates their resolution into crossovers. Msh4 SUMOylation enhances these processes to ensure that each chromosome pair acquires at least one crossover. Msh4 is directly targeted by E2 conjugase Ubc9, initially becoming mono-SUMOylated in response to DNA double-strand breaks, then multi/poly-SUMOylated forms arise as homologs fully engage. Mechanistically, SUMOylation fosters interaction between Msh4 and Msh5. We infer that initial SUMOylation of Msh4 enhances assembly of MutSγ in anticipation of JM formation, while secondary SUMOylation may promote downstream functions. Regulation of Msh4 by SUMO is distinct and independent of its previously described stabilization by phosphorylation, defining MutSγ as a hub for crossover control.

Integration host factor (IHF) is an E. coli nucleoid-associated protein that was initially discovered in the integration of bacterial phage DNA. IHF binds with nanomolar affinity to duplex DNA containing a 13 bp consensus sequence and induces a bend of ~160° upon binding. Our previous results have shown that IHF binds to DNA 4WJ that do not contain the consensus sequence with high affinity. DNA Four-Way Junctions (4WJ) are important intermediates in double-strand break repair and homologous recombination. These junctions can adopt either an open or stacked conformation, in which the open conformation facilitates the process of branch migration and strand exchange. In this study, we investigated the IHF-induced changes in the population distribution of junction conformations, using ensemble and single molecule Förster resonance energy transfer (FRET) methods. Our steady state and time-resolved FRET data indicated that under conditions stabilizing the stacked junction conformation, upon binding, IHF induces the open conformation of junction. Single-molecule FRET (smFRET) experiments, performed with total internal reflection fluorescence microscopy, measured population distributions of the junction and showed clear opening of the junctions upon IHF binding. Further analysis of smFRET dynamics revealed that even in the presence of protein, the junctions remain dynamic as fast transitions are observed for the protein-bound open state. Cross correlation analyses reveal that the protein slows down the transition rate at 1 mM Mg2+ but accelerates the transition rate at 10 mM Mg2+, consistent with stabilization of the open conformation. These findings which show that IHF binds to 4WJ with high affinity, induces the open conformation and alters conformational dynamics, suggest that IHF may play multiple roles in the processes of integration and recombination.

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.

The Holliday junction is a four‐way DNA structure formed during the process of double strand break repair and DNA recombination in meiosis. Branch migration of the junction is a vital molecular process for the transfer of genetic information from parents to offspring. Understanding this process relative to base pair composition can provide a better picture of the overall structure of the junction and its connection to short inverted repeats that have been linked to chromosome instability. We used a fluorescent nucleoside analog, 4‐amino‐6methyl‐8‐(2‐deoxy‐beta‐d‐ribofuranosyl)‐7(8H)‐pteridone (6‐MAP), to obtain site‐specific information about the melting process, through incorporation of the probe at distinct sites throughout a 34 bp immobile junction. Under our conditions, the junction primarily adopts a conformation where the arms stack co‐axially on each other. We have examined the stability of the junction arms by incorporating the probe at equivalent positions on each arm. From these measurements we have found that the junction follows a 'pseudo‐duplex' model of denaturation, in which one of the coaxially stacked pair of arms melts at a lower temperature relative to the other. Our results also point to a sequential melting pattern in which positions at the end of the arms and the center destabilize before the interior of the arms. The relative proximity to the junction center appears to be one of the factors affecting stability. Our current research investigates how sequence homology influences the stability of the junction, specifically whether the thermodynamic characteristics of an immobile junction are predictive for a mobile one. We have developed a mobile junction with 10‐bp homology in the central region. Previous research by (Voth A.R., Hays F.A., Ho P.S. (2007) Directing macromolecular conformation through halogen bonds. Proc. Natl. Acad. Sci. USA.; 104:6188–6193. doi: 10.1073/pnas.0610531104) has suggested that macromolecular conformations can be directed through the use of halogen bonds. Specific incorporation of brominated uracils in the sequence of two DNA strands of a mobile Holliday junction was shown to bias the structure to a single conformation in crystallographic experiments. Combined spectroscopic data from both the immobile and mobile junction will inform coarse‐grained modeling of the Holliday junction. Our experimental data combined with computational modeling furthers our current understanding of this DNA structure as it relates to branch migration and exchange of genetic information.

Integration host factor (IHF) is an E.Coli architectural protein that exists as a heterodimer, composed of an α and β subunit. Originally identified in the integration of bacterial phage DNA, IHF has been implicated in regulating replication, transcription, recombination and nucleoid compaction. IHF binds to consensus sequences in the genome with high specificity and induces a bend of ∼160° in the DNA upon binding. This process helps package the genome and facilitate DNA protein interactions. Previous studies performed in our laboratory have shown that IHF binds to DNA four-way Holliday Junction with tight affinity and induces the junction to adopt the open conformation. In this study, we are interested in exploring the population distribution of different conformations of the DNA-junction complex using single-molecule Förster resonance energy transfer (FRET)system methodologies. To refine our single molecule methodologies for our system, our initial experiments are performed with 34 bp duplex DNA containing the IHF consensus sequence. Consistent with earlier studies, we find that IHF binds to consensus DNA with high affinity (3-10nM) and introduces a bend in the duplex DNA structure, as measured by steady state FRET. Preliminary data also show that we can observe IHF induced DNA bending event under the total internal reflection fluorescence (TIRF) spectroscopy. Our future work will focus on optimizing our conditions to perform similar measurements with the IHF-junction complex.

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.

Histone‐like protein HU binds with high affinity (K d = 30 nM in gel and K d = 10 nM in solution) to a Holliday or Four‐Way junction (HJ), a DNA recombination intermediate. While previous Förster resonance energy transfer (FRET) studies have determined possible structural models for the HU‐junction interaction, X‐ray crystallographic data is needed to confirm these models. In our studies, we are using J20, a version of the well‐characterized J3 Holliday junction, that has been modified to have only ten base pairs per arm. Based on our anisotropy measurements, the shorter arms reduce the relative flexibility of the junction, potentially making this junction a better substrate for our crystallization studies. Our investigation aims to determine the X‐ray crystal structure of the HU‐HJ interaction by elucidating the ideal conditions for generating HU‐J20 crystals. Initial investigations of crystallization conditions have produced crystals that diffract to 20 Å resolution, therefore, our current efforts are focused on generating crystals that will diffract at higher Angstrom resolution. In order to do so, we will screen around our successful conditions and slightly vary the pH, viscosity, and protein concentrations of our crystallography solutions. Conditions for optimizing crystal generation and diffraction will be discussed.

The mismatch repair (MMR) pathway is responsible for maintaining the integrity of the genome by correcting errors such as insertions, deletions, or mismatched bases formed during DNA replication. MMR is initiated by Msh2-Msh6 (Msh26), a heterodimeric protein that recognizes single base mismatches and small insertion/deletion loops in DNA. Much is known about the structure of Msh26 and its role in MMR, but the precise mechanism for recognition of mismatched DNA remains unclear. In this study, we use the fluorescent DNA base analog 6-MI (6-methylisoxanthopterin) at or adjacent to a mismatch site to observe changes in the dynamics of single base mismatches upon interaction with Msh26. We observe a −10 nm spectral shift in 6-MI peak emission upon binding, consistent with an increase in local hydrophobicity possibly reflecting the intercalation of a Phe residue at the mismatch as observed in the Msh26 X-ray crystal structure. We also find that the amount of protein-induced bending is the same for all substrates, as measured by energy transfer experiments and does not correlate with binding affinity. Msh26 exhibits a higher affinity for DNA substrates (Kd < 25 nM) when the 6-MI probe exhibits a fast rotational correlation time (< 0.5 ns) indicative of greater mobility. Msh26 has a low affinity for poorly repaired mismatches such as T:T and T:C. These mismatches exhibit significantly slower 6-MI rotational correlation times (∼1-2ns) signifying reduced local mobility that is comparable to that of homoduplex DNA. Thus, we attribute the relatively low repair efficiency in vivo to poor recognition of the T:T and T:C mismatches associated with their reduced motion. Msh26 binding affinities for mismatched substrates linearly correlate with relative mobility at the mismatch site and we suggest this mobility influences recognition and binding affinity.

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.