Binding of MSH2/MSH3 and MSH2/MSH6 to their preferred repair substrates increases FRET efficiency. The dynamics of different substrate molecules in the presence of or in the absence of added MSH2/MSH3. (A) smFRET efficiencies for MSH2/MSH3 binding to the (CA)₄ loop substrate without (top) or with (bottom) MSH2/MSH3. The schematic of the labeled substrates: green ball is Cy3 label; red ball is Cy5 label (bottom); blue ball is biotin label. The protein concentration is indicated. (B) The time traces of representative donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5). The black line indicates the time of the (CA)₄ loop in the high FRET state. Time of acceptor photobleaching is indicated by black arrow. (upper) Blue traces are the corresponding FRET efficiencies. (C, D) Same as (A, B) for homoduplex substrate. (E, F) Same as (A, B) for binding of MSH2/MSH6 to a G/T mismatched substrate.

Repair-resistant (CAG)₁₃ template traps nucleotide-bound MSH2/MSH3 in the low FRET state. (A) A fold back loop of CAG DNA forms with A/A mispaired base every three nucleotides. Green ball is Cy3 label; Red ball is Cy5 label (bottom). (B) FRET efficiency for MSH2/MSH3 binding to the (CAG)₁₃ hairpin in the absence of nucleotides. The FRET efficiency histograms indicate that MSH2/MSH3 binding induces a high FRET state and a low FRET state relative to the substrate alone. Individual time traces of the low (C) and high (D) FRET states. The time traces of representative donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5). Black bars indicate the binding event, and the arrows indicate photo bleaching of the acceptor dye. (E) Addition of ATP to (B) strongly reduces the relative abundance of the high FRET state compared to the low FRET state. (F) The individual time traces indicate that the low FRET state remains stable and (G) the time in the high FRET state is shorter in the presence of ATP relative to its absence (D).

The (CAG)₁₃ loop intrinsically adopts more than one conformational state in the absence of protein. (A) The FRET efficiency histograms of the (CAG)₁₃ substrate in the absence of protein. Increasing MgCl₂ concentration resolves the presence of two FRET states. (B) Few transitions are observed within the time resolution for concentrations below 5 mM MgCl₂, but become more apparent at 20 mM MgCl₂. (C) Blue trace is the corresponding FRET efficiency. (D) A schematic of the sequence changes at the junction of the AT(CAG)₉ template. The two A-A mismatches closest to the junction in the (CAG)₁₃ substrate have been replaced by A-T pairs. (E) Conformation of AT-(CAG)₉ substrate at different MgCl₂ concentrations. In the absence of protein, the substrate exists in a single conformation. Addition of MgCl₂ increases the shift towards higher FRET values, (F, G) but the individual time traces are not dynamic. (F) The time traces of representative donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5), and (G) the blue traces are the corresponding efficiencies.

Proposed model for conformational regulation of loop repair by MSH2/MSH3 at three-way DNA junctions. The conformational flexibility of the substrate determines the possible binding modes of MSH2/MSH3. (i) Binding of MSH2/MSH3 to a (CA)₄ (repair-competent) loop stabilizes a high FRET (bent) state. In the presence of nucleotides, MSH2/MSH3 undergoes binding-dissociation transitions and is able to leave the lesion to initiate downstream signaling to mismatch repair machinery. (ii) Binding of MSH2/MSH3 to a (CAG)₁₃ (repair-resistant) hairpin junction results in two conformational states. Nucleotide binding to MSH2/MSH3 results in dissociation from a bent state, while the straightened (CAG)₁₃ hairpin junction traps nucleotide-bound MSH2/MSH3 in a nonproductive complex, which cannot leave the lesion to initiate efficient repair by the mismatch repair pathway. (iii) Binding of MSH2/MSH3 to a rigid AT-(CAG)₉ hairpin junction results in single straightened conformation, from which MSH2/MSH3 cannot dissociate whether or not ATP is bound. MSH2/MSH3 cannot leave the lesion or initiate repair. Circles with 2 and 3 represent the MSH2/MSH3 heterodimer.

Conformational Integrity of Mismatch Recognition Complexes and DNA templates. (A) Schematic structure of the three-way junction DNA templates. The (CA)₄ loop and CAG hairpins are centrally located in the upper unlabeled strand. The duplex base comprising eighteen base pairs, which were labeled at the 5′ end with Cy3 and at the 5′ end with Cy5 for smFRET experiments, or with a 5′ fluorescein for the FA experiments. (B) The purified templates resolved on native polyacrylamide gels and visualized by ethidium bromide staining. SS is the 18nuc single strand DNA that is the complementary strand for the looped templates, DS is the 18 bp homoduplex DNA, and the heteroduplex looped substrates are as labeled. (C) Resolution of purified human MSH2/MSH3 (middle lane) and MSH2/MSH6 (right lane) proteins by SDS-PAGE. The size markers (right) indicate the molecular weights. (D) SAXS structure of MSH2/MSH3 protein alone (top, left) or in the presence of the (CA)₄ loop (top, right) overlaid on the crystal structure of human MSH2/MSH6 bound to a G-T mispaired base (11). View of the MSH2/MSH3-(CA)₄ loop complex from front (bottom, right) and side (bottom, left).

ATP increases dissociation of MSH2/MSH3 from a (CA)₄ loop substrate. FRET efficiency histograms for MSH2/MSH3 binding to the (CA)₄ substrate at different protein concentrations in the presence of 100 μM ATP (A) without MgCl₂ and (B) with 5 mM MgCl₂. The MSH2/MSH3 concentration is indicated. (C) The dynamics of the (CA)₄ substrate upon MSH2/MSH3 binding in (B). The time traces of representative donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5). The black line indicates the time of the (CA)₄ loop in the high FRET state. Time of acceptor photobleaching is indicated by black arrow. (upper) Blue traces are the corresponding FRET efficiencies. (D) Time traces like the one shown in (C) analyzed in a two state system using a Hidden Markov Model (36) to determine the average transition rates from initial to high and high to initial FRET states. The transition rates from the initial to high FRET states depends on protein concentration (gray balls), while the transition rates from high to initial FRET state were independent of the protein concentration (black balls). (E) Model for conformational dynamics observed for MSH2/MSH3 binding to (CA)₄ loop substrate: MSH2/MSH3 binds to the a high FRET state of the (CA)₄ loop, while addition of ATP or ADP under hydrolytic conditions increases dissociation of MSH2/MSH3 from the (CA)₄ loop. The high FRET state is indicated as a bent structure.

A (CAG)₁₃ hairpin binds only one nucleotide-bound MSH2/MSH3 complex, but displays both high and low FRET states. (A) Schematic diagram of two possible MSH2/MSH3-(CAG)₁₃ hairpin complexes with nucleotide bound in either the MSH2 or MSH3 subunit. (B) Sequences of the mutant MSH2 and MSH3 subunits aligned with the canonical Walker A box sequence motif of the wt MSH2/MSH3. The conserved Lysine residue has been replaced with a methionine in the mutant proteins to destroy the ATP binding pocket. (C) Resolution of the wild-type and mutant MSH2/MSH3 on denaturing gels. Wt, wild-type MSH2/MSH3 subunits, sgl2, Walker A box mutations in the MSH2 subunit only, sgl3, Walker A box in the MSH3 subunit only, dbl, Walker A motif mutations in both subunits. (D) Binding of [α-³²P]-ATP to wild-type and mutant MSH2/MSH3 proteins analyzed by UV-cross-linking followed by resolution on denaturing gel. Only intact nucleotide binding sites bind ATP efficiently. (E) Fluorescence anisotropy measurements of Bodipy-labeled ATP binding to both wild-type and mutant MSH2/MSH3-(CAG)₁₃ hairpin complexes. (F) Mutation of the Walker (A) box in the MSH2 subunit only inhibits binding of Bodipy labeled ADP to a MSH2/MSH3-(CAG)₁₃ hairpin complex. (G) Association of nucleotide-bound wild-type and mutant MSH2/MSH3 to the (CAG)₁₃ templates result in high and low FRET states. ATP is retained poorly in the MSH3 subunit when bound to DNA (18). Thus, sgl3 and wild type in the presence of nucleotides are similar, and sgl2 in the presence of nucleotides is the same as wt without bound nucleotides. ATP is 100 μM.

The junction of the AT-(CAG)₉ hairpin adopts only one stable three-way junction, from which MSH2/MSH3 does not dissociate. (A) The FRET efficiencies for binding of MSH2/MSH3 to the AT-(CAG)₉ substrate in the presence of (+MgCl₂) results in a single low-FRET population. (B) The FRET efficiency histograms for AT-(CAG)₉ in the presence of ATP under hydrolytic conditions. The affinity of nucleotide-bound MSH2/MSH3 for the AT-(CAG)₉ substrate (+MgCl₂) is reduced, but binding results in the same low FRET state as observed in the absence of nucleotides. (C) The individual time traces of the AT-(CAG)₉ substrate alone (top), AT-(CAG)₉ bound to MSH2/MSH3 (middle), and AT-(CAG)₉ bound to MSH2/MSH3 at the indicated concentrations in the presence of ATP (+Mg) (100 μM) (bottom). All traces are similar and display no dynamics. (C) The time traces of representative donor fluorescence (green, Cy3) and acceptor fluorescence (red, Cy5), and (D) the blue traces are the corresponding FRET efficiencies. (E) Proposed model for conformational regulation of loop repair by MSH2/MSH3 at three-way DNA junctions. The conformational flexibility of the substrate determines the possible binding modes of MSH2/MSH3. (F) Binding of MSH2/MSH3 to a (CA)₄ loop binds, bends DNA. Upon downstream nucleotide hydrolysis and exchange, MSH2/MSH3 adopts a doubly bound form which is verify and to leave the lesion to signal downstream repair by the MMR machinery. (G) The straightened (CAG)₁₃ hairpin junction traps nucleotide-bound MSH2/MSH3 in a nonproductive complex, which cannot leave the lesion to initiate efficient repair by the MMR pathway. Successful mismatch repair couples DNA binding and ATP hydrolysis. Trapping does not allow processing of ATP in the MSH3 subunit, and prevent ADP/ATP exchange needed to leave the site. Circles with 2 and 3 represent the MSH2/MSH3 heterodimer. Red ball are ADP and blue balls are ATP.