Inhibition of Msh6 ATPase Activity by Mispaired DNA Induces a Msh2(ATP)-Msh6(ATP) State Capable of Hydrolysis-Independent Movement along DNA  Dan J. Mazur, Marc L. Mendillo, Richard D. Kolodner  Molecular Cell  Volume 22, Issue 1, Pages 39-49 (April 2006) DOI: 10.1016/j.molcel.2006.02.010 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 The Msh2 and Msh6 Subunits Both Bind ATP, but with Different Affinities Standard crosslinking reactions (20 μl) contained 0.4 pmol (A–C) or 4 pmol (D and E) of wild-type Msh2-Msh6 and labeled ATP (A and D), ATPγS (B and E), or AMP-PNP (C) at the indicated concentrations. Dissociation constants (Kd) are presented in Table 1. Molecular Cell 2006 22, 39-49DOI: (10.1016/j.molcel.2006.02.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 Analysis of the Effect of ATP Binding Mutants and DNA on ATP Binding by Msh2 and Msh6 Standard crosslinking reactions contained the ATP binding mutants Msh2(K694M)-Msh6 (A and B), Msh2-Msh6(K988M) (C and D), or wild-type Msh2-Msh6 containing no DNA, paired DNA, or mispair-containing DNA (E). The apparent Kd of nucleotide binding determined for wild-type and mutant Msh2 and Msh6 is displayed in Table I. Pulse-chase experiments (F) contained Msh2-Msh6 and [γ-32P]ATP at indicated concentrations. Molecular Cell 2006 22, 39-49DOI: (10.1016/j.molcel.2006.02.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Nucleotide Binding Characteristics of Msh2-Msh6 Standard crosslinking reactions containing different concentrations of [α-32P]ATP with Mg2+ as indicated (A and B), [α-32P]ADP (C), 5 μM [α-32P]ADP and unlabeled ATPγS (D), different concentrations of [α-32P]ADP in the presence of 5 μM ATPγS (E), preincubation of 5 μM ADP followed by the addition of [35S]ATPγS at concentrations indicated (F), and 5 μM [α-32P]ADP with unlabeled AMP-PNP at concentrations indicated (G). The asterisk indicates nucleotide containing the label [32P]ATP or [35S]ATPγS. The apparent Kd measurements determined for all reactions are displayed in Table I. Molecular Cell 2006 22, 39-49DOI: (10.1016/j.molcel.2006.02.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 Mispaired DNA Inhibits ATP Hydrolysis by Msh6, which Causes Reduced Binding of ADP by Msh2 (A) Standard crosslinking reactions containing Msh2-Msh6 (4 pmol) and ATP (10 μM) were prepared. The presence of α- or γ-labeled ATP, Mg2+, and mispaired or paired DNA is indicated. (B) Crosslinking reactions with Msh2-Msh6 (4 pmol) and [α-32P]ADP (5 μM) were prepared. The presence of ATP (0, 0.5, 1.0, 2.0 μM) and mispaired or fully paired DNA (20 pmol) is indicated. ADP binding was set at 100% for the level seen in the absence of ATP. Molecular Cell 2006 22, 39-49DOI: (10.1016/j.molcel.2006.02.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 5 Sliding of Msh2-Msh6 Requires Nucleotide Binding to Both Subunits but Does Not Require Hydrolysis End-dependent dissociation of the Msh2-Msh6 complex from an unblocked, mispaired DNA substrate was determined by Biosensor analysis. Dissociation data generated with a LacI blocked substrate were subtracted from data generated with an unblocked substrate. Dissociation of Msh2-Msh6 from a DNA substrate after addition of ATP (A) or ATPγS (B) at the concentrations indicated. A plot of Koff versus ATP (C) or ATPγS (D) concentration was performed, yielding an apparent Kd for sliding of 17.9 + 1.4 μM and 81.5 ± 10.2 μM, respectively. Molecular Cell 2006 22, 39-49DOI: (10.1016/j.molcel.2006.02.010) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 6 Model of ATP Binding and Hydrolysis, which Increases Mispair-Specific Binding of Msh2-Msh6 (A) Mispaired DNA causes a shift to the doubly occupied state that is required for downstream MMR events. Msh2-Msh6, in the empty or Msh2(ADP) states, binds with high affinity to mispaired DNA, which then stabilizes ATP bound by Msh6. This stably bound ATP allows release of ADP by Msh2, which then binds ATP to form the dual ATP occupied state (∗) that allows end-dependent dissociation (sliding) and possibly other downstream MMR events. (B) The absence of mispaired DNA shifts Msh2-Msh6 to an empty or Msh2(ADP) state. The nucleotide binding model for non-DNA bound Msh2-Msh6 is identical to the paired DNA model. Unbound Msh2-Msh6 is shifted to the empty or Msh2(ADP) states, which are shown binding DNA and entering the cycles in (A) and (B). Although end-dependent dissociation (sliding) has not been detected on paired DNA, it could occur at a low level. This greatly reduced level of sliding in the absence of mispaired DNA is partially caused by rapid hydrolysis of ATP bound by Msh6, which in turn causes a decrease in the dual ATP occupancy state required for sliding. End-independent dissociation from paired DNA likely occurs for all Msh2-Msh6 states (for a discussion, see Mendillo et al. [2005]), also resulting in a decrease in the dual ATP occupancy state on paired DNA. Bound ATP and ADP are indicated by “T” and “D,” respectively. Molecular Cell 2006 22, 39-49DOI: (10.1016/j.molcel.2006.02.010) Copyright © 2006 Elsevier Inc. Terms and Conditions