Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair

Abstract

Eukaryotic MutLα (mammalian MLH1-PMS2 heterodimer; MLH1-PMS1 in yeast) functions in early steps of mismatch repair as a latent endonuclease that requires a mismatch, MutSα/β, and DNA-loaded proliferating cell nuclear antigen (PCNA) for activation. We show here that human PCNA and MutLα interact specifically but weakly in solution to form a complex of approximately 1:1 stoichiometry that depends on PCNA interaction with the C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino acid substitution mutations within a PMS2 C-terminal ⁷²¹QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNA- and DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif (⁷²³QKLIIP) reduce or abolish mismatch repair in vivo. Coupling of a weak allele within this motif (⁷²³AKLIIP) with an exo1Δ null mutation, which individually confer only weak mutator phenotypes, inactivates mismatch repair in the yeast cell.

Keywords: DNA repair; MutLalpha; endonuclease; mismatch repair; proliferating cell nuclear antigen.

Fig. 1.

PMS2 ⁷²¹QRLIA motif is required for MutLα interaction with PCNA in solution. Equilibrium gel filtration was performed (Materials and Methods) by injection of 10 μL 10 μM wild-type (circles), PMS2-Q721A (triangles), or PMS2-AAA (squares) MutLα onto a Superdex 200 column equilibrated with Hepes⋅KOH/KCl buffer and the indicated concentrations of PCNA. The curve for wild-type MutLα is a nonlinear regression fit to a multiligand binding isotherm (Materials and Methods). Best-fit parameters are K_d 7.7 μM and a stoichiometry of 0.8 PCNA trimer per MutLα. K_d and stoichiometry values for mutant proteins were not estimated due to the low level of binding.

Fig. S1.

MutLα interacts with PCNA in solution. (A and B) Examples of equilibrium gel filtration profiles after injection of 10 μL 10 μM wild-type (A) or PMS2-Q721A MutLα (B) onto a Superdex 200 column equilibrated with 10 μM PCNA as described in the legend to Fig. 1. MutLα-bound PCNA was determined from the integrated area of the trough (arrows) at the PCNA retention volume. (C) Equilibrium gel filtration (Materials and Methods) was done by injection of 10 μL 3 μM MutLα onto a Superdex 200 column equilibrated with Tris⋅HCl/MgCl₂/NaCl buffer and the indicated concentrations of PCNA. Best-fit parameters are K_d = 4.2 μM and n = 1.1.

Fig. 2.

BS³ cross-linking of MutLα and PCNA. (A) Wild-type, PMS2-Q721A, or PMS2-AAA MutLα was cross-linked with BS³ in the absence or presence of PCNA (Materials and Methods) and reaction products were resolved by SDS/PAGE and visualized with Coomassie brilliant blue. As judged by Western blot, the major cross-linked species with apparent mobility of 180 kDa corresponds to MLH1-PMS2. The cross-linked product of apparent mobility of 140 kDa (asterisk) is analyzed in B. (B) Mass spectroscopic quantification of PCNA, MLH1, and PMS2 tryptic peptides (Materials and Methods) derived from gel slices corresponding to the mobility of the 140-kDa cross-linked product produced with wild-type MutLα and PCNA, or the equivalent position in other lanes. Error bars are ±1 SD for wild-type MutLα (blue), PMS2-Q721A (green), and PMS2-AAA (red). Open and filled bars correspond to results obtained in the absence or presence of PCNA, respectively.

Fig. 3.

Putative PCNA-binding motifs within the CTD of yeast MutLα. Structure of the C-terminal domain of S. cerevisiae MutLα (MLH1-PMS1) [Protein Data Bank ID code 4fmn (9)] as visualized with PyMOL (https://www.schrodinger.com). Structural elements shown are yPMS1 residues 651 to 873 (blue), yMLH1 residues 505 to 769 (brown), the yPMS1 endonuclease motif ⁷⁰¹DQHASDEKYNFE (magenta), two bound zinc ions (yellow), and putative PCNA-binding motifs within yPMS1 (⁷²³QKLIIP; red) or MLH1 (⁵⁷²QIGLTDF; green).

Fig. S2.

BS³ cross-links MutLα MLH1-AAA and PCNA. Wild-type MutLα, MutLα MLH1-AAA, and MutLα PMS2-AAA were cross-linked with BS³ in the absence or presence of PCNA as described in the legend to Fig. 2. The asterisk indicates the cross-linked product of apparent mobility of 140 kDa.

Fig. S3.

PCNA stimulation of MutLα endonuclease activity on linear DNA depends on integrity of the PMS2 ⁷²¹QRLIAP motif. 50 ng (280 fmol) wild type MutLα, PMS2-Q721A, or PMS2-AAA were incubated with a fluorescently-labeled 49 bp oligonucleotide in the absence or presence of 50 ng (580 fmol) PCNA (Materials and Methods). Fluorescence scan of 8 M urea PAGE for IR680 (top strand label) and IR800 (bottom strand label). In both cases cleavage products have a length of about 11 nucleotides (nt). Numerical values below each lane indicate extent of incision for bottom and top strand (fmol/20 min).

Fig. 4.

PCNA- and DNA-dependent stimulation of MutLα ATPase is attenuated by amino acid substitutions within the PMS2 ⁷²¹QRLIAP motif. (A) ATP hydrolysis (Materials and Methods) by 0.68 μM wild-type MutLα (blue circles), MutLα PMS2-Q721A (green triangles), or MutLα PMS2-AAA (red squares) was determined in the absence (open symbols) or presence (closed symbols) of 1.4 μM PCNA and 2.1 μM 5′-phosphorylated 49-bp duplex DNA. ATP hydrolysis in the absence of MutLα but in the presence of 1.4 μM PCNA and 2.1 μM DNA was also determined (black diamonds). (B) As in A, but reactions contained either 0.68 μM MutLα and 2.1 μM DNA (closed symbols, broken lines) or 0.68 μM MutLα and 1.4 μM PCNA (open symbols, dotted lines). ATP hydrolysis by 1.4 μM PCNA alone is also shown (black diamonds). (C) Apparent PCNA-dependent stimulation of MutLα ATP hydrolysis was calculated as the difference between ATP hydrolysis observed in the presence of both PCNA and DNA (A, closed symbols) and that observed in the presence of DNA only (B, closed symbols). Symbols are as in A. Assays were done in triplicate, and error bars are ±1 SD. Error bars for C were computed as the square root of the quadratic sum of the individual SDs [SD_C = (SD_A² + SD_B²)^1/2].