Structure of the endonuclease domain of MutL: unlicensed to cut

Abstract

DNA mismatch repair corrects errors that have escaped polymerase proofreading, increasing replication fidelity 100- to 1000-fold in organisms ranging from bacteria to humans. The MutL protein plays a central role in mismatch repair by coordinating multiple protein-protein interactions that signal strand removal upon mismatch recognition by MutS. Here we report the crystal structure of the endonuclease domain of Bacillus subtilis MutL. The structure is organized in dimerization and regulatory subdomains connected by a helical lever spanning the conserved endonuclease motif. Additional conserved motifs cluster around the lever and define a Zn(2+)-binding site that is critical for MutL function in vivo. The structure unveils a powerful inhibitory mechanism to prevent undesired nicking of newly replicated DNA and allows us to propose a model describing how the interaction with MutS and the processivity clamp could license the endonuclease activity of MutL. The structure also provides a molecular framework to propose and test additional roles of MutL in mismatch repair.

Figure 1. Crystal structure of BsMutL-CTD

(A) Ribbon diagram of the BsMutL-CTD monomer. Secondary structure motifs are labeled and colored blue (helices) and yellow (strands) with the connecting loops in light green. The endonuclease and the endonuclease-associated motifs are shown in purple, while the additional conserved motifs are shown in orange. (B) Ribbon diagram of the BsMutL-CTD dimer with one protomer shown as in (a) and the other one as grey ribbons. (C) Sequence alignment of the C-terminal regions of BsMutL, hPMS2 and EcMutL. Secondary structure elements of BsMutL-CTD are shown as arrows (strands) and cylinders (helices). The five conserved motifs are highlighted in purple and underlined. Conserved hydrophobic residues are highlighted in yellow. The conserved ⁴⁸⁷QEMIVP motif is highlighted in orange. See also Figure S1.

Figure 2. Endonuclease activity of BsMutL

(A) Nicking activity of BsMutL (left) and BsMutL-CTD (center) in the presence of Mg²⁺, Zn²⁺, Mn²⁺ or Cd²⁺ as indicated. Comparison of the nicking activity of BsMutL and BsMutL-D462N in the presence of Mn²⁺ (right). Migration of supercoiled (SC), nicked (N) and linear (L) DNA is indicated. (B) Endonuclease activity of BsMutL in the presence of 0.5 mM (+) and 5 mM (++) nucleotide. (C) DNA binding by BsMutL (WT), BsMutL-CTD (CTD) and BsMutL variants as indicated. Data are presented as the mean of three independent measurements and the error bars correspond to the standard errors of the mean (SEM=σ/√n, where σ is the average and n the sample size). (D) Stimulation of the endonuclease activity of BsMutL (1 mM Mn²⁺) by a second divalent metal ion (1 mM).

Figure 3. Regulatory Zn²⁺-binding site in BsMutL-CTD

(A) Organization of the endonuclease site of BsMutL-CTD bound to Zn²⁺ (crystal form III). Hydrogen bonds are shown as black dashed lines with the water molecules and Zn²⁺ ions shown as red and lilac spheres, respectively. Conserved motifs are color-coded green (⁴⁶²DQHAX₂EX₄E), purple (⁶⁰⁴CPHGRP) and orange (⁵⁷²SCK). (B) Zinc-affinity profiles of BsMutL-CTD and point mutants of BsMutL-CTD as indicated. (C) Bar diagram showing the relative mutation frequency of the indicated mutL variants altered in the Zn²⁺-binding, the endonuclease site and the putative β-binding motif. Data are presented as the mean of 4 independent cultures +/- SEM. (D) Superimposition of the β-binding motif in BsMutL-CTD (green) onto those of pol II (yellow, PDB 3D1E), pol IV (cyan, PDB 1UNN) and FEN-1 (purple, PDB 1RXM) shown as a main-chain trace with the β-clamp structure (PDB 3D1E) presented as a semi-transparent electrostatic potential surface. See also Figures S2-S3 and Movie S1.

Figure 4. Model of activation of the endonuclease activity of MutL

(A) Orthogonal views of the electrostatic surface potential of the BsMutL-CTD protomer. The second protomer is shown as a ribbon diagram with the endonuclease motifs in purple. (B) Upon mismatch binding, MutS (blue) undergoes a nucleotide-dependent conformational change that triggers recruitment of MutL (green) to the mismatch site, likely aided by the β-clamp (purple). ATP binding by MutL then promotes the association of its two ATPase subunits and brings the ATPase in close proximity to the dimerization domain of the protein. Coordinated interaction of MutS and β-clamp bound to DNA (ribbon diagram) with ATP-bound MutL could thus license the latent endonuclease activity of MutL. ATP and ADP are shown as yellow and orange stars, respectively.