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 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.

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.

Holliday Junctions are critical DNA intermediates central to double strand break repair and homologous recombination. The junctions can adopt two general forms: open and stacked-X, which are induced by protein or ion binding. In this work, fluorescence spectroscopy, metal ion luminescence and thermodynamic measurements are used to elucidate the ion binding site and the mechanism of junction conformational change. Förster resonance energy transfer measurements of end-labeled junctions monitored junction conformation and ion binding affinity, and reported higher affinities for multi-valent ions. Thermodynamic measurements provided evidence for two classes of binding sites. The higher affinity ion-binding interaction is an enthalpy driven process with an apparent stoichiometry of 2.1 ± 0.2. As revealed by Eu3+ luminescence, this binding class is homogeneous, and results in slight dehydration of the ion with one direct coordination site to the junction. Luminescence resonance energy transfer experiments confirmed the presence of two ions and indicated they are 6–7 Å apart. These findings are in good agreement with previous molecular dynamics simulations, which identified two symmetrical regions of high ion density in the center of stacked junctions. These results support a model in which site-specific binding of two ions in close proximity is required for folding of DNA Holliday junctions into the stacked-X conformation.

Signal peptides are critical for the initiation of protein transport in bacteria by virtue of their recognition by the SecA ATPase motor protein followed by their transfer to the lateral gate region of the SecYEG protein-conducting channel complex. In this study, we have constructed and validated the use of signal peptide-attached SecA chimeras for conducting structural and functional studies on the initial step of SecA signal peptide interaction. We utilized this system to map the location and orientation of the bound alkaline phosphatase and KRRLamB signal peptides to a peptide-binding groove adjacent to the two-helix finger subdomain of SecA. These results support the existence of a single conserved SecA signal peptide-binding site that positions the signal peptide parallel to the two-helix finger subdomain of SecA, and they are also consistent with the proposed role of this subdomain in the transfer of the bound signal peptide from SecA into the protein-conducting channel of SecYEG protein. In addition, our work highlights the utility of this system to conveniently engineer and study the interaction of SecA with any signal peptide of interest as well as its potential use for X-ray crystallographic studies given issues with exogenous signal peptide solubility.

The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner. In eukaryotic cells, the initiation of MMR is achieved by recognition by MutS homologs (Msh). The Msh2-Msh6 heterodimer plays this important initial role in recognizing single base mismatches and small insertion/deletion loops (IDL) in MMR. Although Msh2-Msh6 recognizes mismatched DNA with high affinity, the exact mechanism by which Msh2-Msh6 distinguishes different types of mismatched base pairs from a large excess of canonical Watson-Crick base pairs is still unknown. In this study, we use the intrinsic fluorescent probe 6-methylisoxanthopterin (6-MI, guanosine analog) in the context of the ATFAA (F = 6-MI) pentamer sequence where it exhibits enhanced fluorescence, to measure the binding affinity of S. cerevisiae Msh2-Msh6 to different single base pair mismatches. Fluorescence anisotropy measurements reveal the following order for Msh2-Msh6 mismatch bp binding affinity: A:A ≈ A:G > A:C ≈ G:T ≈ +T > T:T ≈ G:G > T:C ≈ G:C. Fluorescence intensity measurements suggest a greater degree of DNA distortion accompanies binding to well-recognized mismatches. We employ Förster resonance energy transfer to measure the bending angle and compare with that observed in MutS/Msh2-Msh6 co-crystal structures. We have also investigated DNA dynamics upon Msh2-Msh6 binding using time-resolved fluorescence spectroscopy. Specific placement of the probe at the mismatch site or adjacent to it reveals significant local motion prior to protein binding. We observe that high affinity binding is associated with those mismatches that exhibit the greatest amount of motion. Protein binding stabilizes mismatch local motion, which is consistent with Phe intercalation at the site, as observed in Msh2-Msh6-DNA co-crystal structures.

The DNA mismatch repair (MMR) system guards the integrity of genetic material by scanning and correcting errors in a post-replicative manner and thus improves the fidelity of DNA replication by several orders of magnitude. In eukaryotic cells, the initiation of MMR is achieved by recognition of biosynthetic errors by Msh proteins (MutS homologs). Single base mismatches and small insertion/deletion loops (IDL) are recognized by the Msh2-Msh6 heterodimer followed by recruitment of MutL homologs (Mlh/Pms) and this ATP-dependent ternary complex further activates downstream MMR events. The exact mechanism by which Msh2-Msh6 distinguishes a mismatched base pair precisely from a large excess of canonical Watson-Crick base pairs in an efficient manner is still unknown. In this study we explore the specificity of Msh2-Msh6 binding that enables discrimination between different mismatches through measurement of binding affinity and protein-induced bending using fluorescence anisotropy, gel mobility shift assays and Förster Resonance Energy Transfer (FRET). These measurements yield the following order of binding affinity: G:T > +T > G:A > G:C and are in good agreement with previous results. The FRET efficiency of free and Msh2-Msh6-bound mismatched duplex DNA suggests that Msh2-Msh6 bends both +T and G:T duplex DNA relative to G:C homoduplex DNA, which appears to remain relatively straight in the bound form. We have also monitored local DNA base pair dynamics with time-resolved fluorescence intensity and anisotropy spectroscopy measurements. By using a fluorescent nucleoside analog, 6-methylisoxanthopterin incorporated into mismatched DNA, we can explicitly investigate single base pair dynamics. These experiments reveal that protein binding stabilizes the probe placed at the mismatch or located adjacent to the unpaired thymine. This stabilization is relatively local to the mismatch site and is not propagated down the helix.

Holliday or DNA four-way junctions are important intermediates in recombination and repair processes. These structures can change conformation rapidly in solution interconverting from an open, four-fold symmetrical structure, with no central base stacking to one in which coaxial stacking of the helical arms has been observed. The open structure, which is capable of branch migration, is functionally relevant; however, many proteins have been observed to bind to and stabilize the stacked form. Our investigations have focused on elucidating the conformational changes induced by the binding of ions and the architectural proteins, HU and IHF, to improve understanding of the stacked form of the junction. Using fluorescence methods, we have examined the ion-binding site and have investigated the coordination of ions in the central region of the junction. FÅrster resonance energy transfer (FRET) experiments have revealed that the degree of stacking or interduplex angle (IDA) of the junction is modulated by ion size, where larger ionic radii lead to larger IDAs. Ion luminescence measurements indicate that minimally two ions are needed to induce junction stacking. The proteins, HU and IHF, stabilize the junction in the stacked conformation and induce a greater degree of stacking upon binding. In contrast, the repair protein, Msh2-Msh6, induces the junction to adopt an open conformation. All three proteins recognize and bind to the junction structure with nanomolar affinity and interestingly, Msh2-Msh6 binds the junction with higher affinity than a mismatch site. A FRET-mapping approach has been employed to determine the location of HU on the junction and indicates that HU binds to the central region of the junction. We have utilized molecular dynamics simulations coupled with the fluorescence information to generate several models for the protein-junction complex and propose a novel structural model for the interaction.