The endonuclease domain of MutL interacts with the β sliding clamp

Abstract

Mismatch repair corrects errors that have escaped polymerase proofreading enhancing replication fidelity by at least two orders of magnitude. The β and PCNA sliding clamps increase the polymerase processivity during DNA replication and are important at several stages of mismatch repair. Both MutS and MutL, the two proteins that initiate the mismatch repair response, interact with β. Binding of MutS to β is important to recruit MutS and MutL to foci. Moreover, the endonuclease activity of human and yeast MutLα is stimulated by PCNA. However, the concrete functions of the processivity clamp in the repair steps preceding DNA resynthesis remain obscure. Here, we demonstrate that the C-terminal domain of MutL encompasses a bona fide β-binding motif that mediates a weak, yet specific, interaction between the two proteins. Mutation of this conserved motif correlates with defects in mismatch repair, demonstrating that the direct interaction with β is important for MutL function. The interaction between the C-terminal domain of MutL and β is conserved in both Bacillus subtilis and Escherichia coli, but the repair defects associated with mutation of this β-binding motif are more severe in the former, suggesting that this interaction may have a more prominent role in methyl-independent than methyl-directed mismatch repair systems. Together with previously published data, our work strongly suggests that β may stimulate the endonuclease activity of MutL through its direct interaction with the C-terminal domain of MutL.

Figure 1. Conserved putative β-binding motif in MutL

(A) Sequence alignment of the putative β-binding motif (highlighted in purple) found in MutL and its location with respect to the endonuclease motif when present (highlighted in green). The top group includes Bacillus subtilis MutL (BsMutL) and Streptococcus pneumoniae HexB (SpHexB) that contain the conserved endonuclease motif, as well as, Escherichia coli MutL (EcMutL) and Salmonella typhimurium MutL (StMutL) that do not have endonuclease activity. The bottom group includes the eukaryotic Homo sapiens PMS2 (hPMS2), Mus musculus PMS2 (mPMS2), Saccharomyces cerevisiae PMS1 (yPMS1), as well as, eukaryotic MutL homologues that do not encompass an endonuclease motif: Homo sapiens MLH1 (hMLH1), Mus musculus MLH1 (mMLH1), Saccharomyces cerevisiae MLH1 (yMLH1). (B) Ribbon diagram of the C-terminal domain of Bacillus subtilis MutL (PDB ID: 3KDK) with the endonuclease motif shown in green and the β-binding motif shown in purple. The structural Zn²⁺ metal ion found at the endonuclease site is depicted as a green sphere. The N- and C-termini, the β-binding motif and the dimerization and regulatory subdomains are labeled for clarity. (C) Ribbon diagram of the C-terminal domain of Escherichia coli MutL (PDB ID: 1X9Z) shown as in (B).

Figure 2. Structural comparison of the β-binding motifs found in MutL-CTD and other clamp-binding proteins

(A) Superimposition of the regulatory subdomains of B. subtilis MutL (blue) and E. coli MutL (yellow) shown as ribbon diagram. The N- and C-terminal domain boundaries are labeled. (B) Superimposition of the β-binding motif from polIV/DinB (green, PDB ID: 1UNN) and the PIP-box from FEN-1 (tan, PDB ID: 1RXM). Dotted lines depict the polar (left) and hydrophobic (right) pockets occupied by the conserved Gln and Leu residues. (C) Superimposition of the β-binding motifs of B. subtilis MutL (blue) and E. coli MutL (yellow) onto the structure of FEN-1 bound the PCNA (tan, PDB ID: 1RXM). Conserved residues are labeled.

Figure 3. Complex formation between the endonuclease domain of B. subtilis MutL and β

Interaction between the C-terminal domain of BsMutL (BsMutL-CTD, top), the regulatory domain of BsMutL (BsMutL-RGD, center) or the C-terminal domain of BsMutL encompassing a mutated β-binding motif (BsMutL-CTD*, ⁴⁸⁷QEMIVP-AEMAAP, bottom) with the B. subtilis β (β). The proteins were incubated in the presence/absence of BS3 and the reaction products were resolved by SDS-PAGE. From left to right, the gels show molecular weight markers (MW), mixtures of MutL (0.02 mM) and β (0.01 mM) incubated with decreasing concentrations of BS3, Bsβ incubated in the presence (+) or absence (−) of BS3, BsMutL variant (as indicated) incubated in the presence (+) or absence (−) of BS3. Monomers and dimers of BsMutL are indicated with one or two asterisks, and monomers and dimers of the β are indicated with one of two arrowheads, respectively. The presence of crosslinked products corresponding to the interaction of BsMutL with β is indicated with a white dot. Incubation of BsMutL-CTD* and β in the presence of BS3 does not result in the formation of this crosslinked product, indicating that the integrity of the β-binding motif is necessary to maintain the interaction.

Figure 4. Complex formation between the C-terminal region of E. coli MutL and β

(A) 4-15% SDS-PAG showing the BS3-crosslinked products of the C-terminal domain of EcMutL (EcMutL-CTD). White asterisks indicate the migration of EcMutL-CTD oligomers. From left to right, the gel shows molecular weight markers (MW), EcMutL-CTD (0.04 mM) incubated with decreasing concentrations of BS3 (1.2, 1.0, 0.8, 0.5, 0.4, 0.3 0.2 and 0.1 mM) and EcMutL-CTD alone (−). (B) Alternative dimer interface found in the EcMutL structure (PDB ID: 1X9Z). The crystallographic dimer is maintained by the interaction of the regulatory subdomains, whereas the physiological dimer shown in Figure 1 is maintained by the interaction of the dimerization subdomains. (C) Mixtures of EcMutL-CTD and E. coli β were incubated in the presence or absence of BS3 and the reaction products were resolved by gradient SDS-PAGE. The protein and crosslinker concentrations, the gel layout and the labels of the bands are the same as Figure 3. The inset corresponds to the same experiment performed with excess EcMutL-CTD (0.2 mM, #5) and shows that some of the oligomeric forms of EcMutL-CTD (trimers #2 and dimers #4) migrate similarly to the β monomer (#3) and the crosslinked EcMutL-CTD/β product (#1). (D) Crosslinking products between the regulatory subdomain of EcMutL (EcMutL-RGD) and Ecβ obtained in the presence of BS3. The gel is shown and labeled as panel (C). (E) Comparison of the crosslinked products obtained with EcMutL-CTD and EcMutL-CTD* encompassing a mutated β-binding motif (⁴⁸²QPLLIP-⁴⁸²ASAAAP) and β. From left to right, the gels show molecular weight markers (MW), mixtures of EcMutL-CTD* and Ecβ, EcMutL-CTD and Ecβ, Ecβ, EcMutL-CTD* and EcMutL-CTD incubated in the presence (+) or absence (−) of BS3. For all reactions containing BS3 a final concentration of 1.2 mM was added.

Figure 5. Interaction between heterologous MutL-CTD and β

Interaction between BsMutL-CTD and Ecβ (top) and EcMutL-CTD and Bsβ (bottom). From left to right, the gels show: molecular weight markers (MW), mixtures of MutL-CTD and β as indicated incubated with decreasing concentrations of BS3, β and MutL-CTD in the presence (+) or absence (−) of BS3.