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. 2010 Mar 19;285(12):9301-10.
doi: 10.1074/jbc.M109.096388. Epub 2010 Jan 20.

Interaction between the Msh2 and Msh6 nucleotide-binding sites in the Saccharomyces cerevisiae Msh2-Msh6 complex

Victoria V Hargreaves  1 , Scarlet S Shell, Dan J Mazur, Martin T Hess, Richard D Kolodner
Affiliations

Interaction between the Msh2 and Msh6 nucleotide-binding sites in the Saccharomyces cerevisiae Msh2-Msh6 complex

Victoria V Hargreaves et al. J Biol Chem. .

Abstract

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes. The formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes requires ATP binding to only the Msh6 nucleotide-binding site, whereas the formation of Msh2-Msh6 sliding clamps requires ATP binding to both the Msh2 and Msh6 nucleotide-binding sites. In addition, the properties of the different mutant complexes suggest that distinct conformational states mediated by communication between the Msh2 and Msh6 nucleotide-binding sites are required for the formation of ternary complexes and sliding clamps.

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Figures

FIGURE 1.
FIGURE 1.
Molecular modeling of the eight amino acid substitutions studied. A, ribbon diagram of human Msh2-Msh6 and bound mispaired DNA (Protein Data Bank code 2o8b) (24). Msh2 is denoted in green, Msh6 is denoted in blue, and the mispaired DNA is displayed as black and gray spheres. The box denotes the ATPase domains. B, view from underneath the ATPase domains as indicated by the vertical arrow in A. The predicted “closed” conformation of the ATPase domains is depicted (supplemental Fig. S6). The closed conformations and positions of the ATP moieties (purple) were modeled based on the ATP-bound dimeric Rad50 structure (Protein Data Bank code 1f2u) (48). The side chains of the residues altered by the eight mutations are indicated in red. Molecular images were generated with PyMOL.
FIGURE 2.
FIGURE 2.
Msh2-Msh6 undergoes a conformational change in the presence of ATPγS. One μg of Msh2-Msh6 was incubated for 1 h with the indicated quantity of trypsin in the absence and presence of 100 μm ATPγS and the presence of Mg2+. Gels were silver-stained. Full-length Msh2 is 110 kDa, whereas full-length Msh6 is 140 kDa. The 90-, 80-, and 75-kDa fragments described under “Results” are indicated. A, wild-type Msh2-Msh6; B, Msh2-Msh6-G1142D; C, Msh2-R730C-Msh6; D, Msh2-S742P-Msh6; E, Msh2-T773D-Msh6. B–E show only the portions of the gels containing fragments in the 70–100-kDa size range.
FIGURE 3.
FIGURE 3.
ATPγS-induced conformational change in Msh2-Msh6 results from filling the high affinity Msh6 nucleotide-binding site. Wild-type Msh2-Msh6 was incubated with 10 ng of trypsin in the presence of Mg2+ and the indicated concentrations of ATPγS. A, silver-stained gel; B, Western blot with an anti-Msh2 antibody to detect Msh2 proteolytic products; C, Western blot with an anti-Msh6 antibody to detect Msh6 proteolytic products.
FIGURE 4.
FIGURE 4.
Msh2-Msh6 mutants containing amino acid substitutions near the Msh6 ATP-binding site are not protected by ATP in the presence of Mg2+. Msh2-Msh6 was incubated with the indicated quantities of trypsin in the absence and presence of 100 μm ATPγS and the absence of Mg2+. Gels were silver-stained. A, wild-type Msh2-Msh6; B, Msh2-Msh6-G1142D; C, Msh2-S742P-Msh6; D, Msh2-T733D-Msh6.
FIGURE 5.
FIGURE 5.
Mutant Msh2-Msh6 complexes bind to a mispair but do not form sliding clamps. Binding of Msh2-Msh6 complexes to a 236-bp DNA containing a central GT mispair in the presence of 250 μm ATP and Mg2+ was analyzed on a Biacore T100 biosensor. The solid red lines indicate association with the DNA substrate with a LacI-blocked end. The solid green lines reflect dissociation of Msh2-Msh6 complexes from the DNA upon the addition of isopropyl β-d-thiogalactopyranoside to remove the end block. The dashed red lines indicate association with the DNA substrate in the absence of LacI. A, wild-type (WT) Msh2-Msh6; B, Msh2-R730C-Msh6; C, Msh2-S742P-Msh6; D, Msh2-T773D-Msh6; E, Msh2-G855D-Msh6; F, Msh2-Msh6-R1024C.
FIGURE 6.
FIGURE 6.
Mutant Msh2-Msh6 complexes have variable defects in formation of ternary complexes with Mlh1-Pms1. Binding of Msh2-Msh6 complexes to a 236-bp DNA containing a central GT mispair and subsequent formation of Mlh1-Pms1 ternary complexes in the presence of 250 μm ATP and Mg2+ were analyzed on a Biacore T100 biosensor. The black lines and dashed red lines indicate association of Msh2-Msh6 with an end-blocked DNA containing a central GT mispair. The solid red lines indicate increased mass binding to the DNA upon the addition of Mlh1-Pms1. The binding attributable to Mlh1-Pms1 was determined by subtracting the dashed red curve from the solid red curve (insets). A, wild-type (WT) Msh2-Msh6; B, Msh2-R730C-Msh6; C, Msh2-S742P-Msh6; D, Msh2-T773D-Msh6; E, Msh2-G855D-Msh6; F, Msh2-Msh6-R1024C.
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