Interaction of proliferating cell nuclear antigen with PMS2 is required for MutLα activation and function in mismatch repair Jochen Genschela,b,1, Lyudmila Y. Kadyrovac, Ravi R. Iyera,2, Basanta K. Dahalc, Farid A. Kadyrovc, and Paul Modricha,b,3 aDepartment of Biochemistry, Duke University Medical Center, Durham, NC 27710; bHoward Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710; and cDepartment of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Carbondale, IL 62901 Contributed by Paul Modrich, March 22, 2017 (sent for review February 15, 2017; reviewed by Josef Jiricny, Guo-Min Li, and Michael O’Donnell) Eukaryotic MutLα (mammalian MLH1–PMS2 heterodimer; MLH1– protein of this class. Like eukaryotic MutLα, B. subtilis MutL + PMS1 in yeast) functions in early steps of mismatch repair as a displays Mn2 -dependent endonuclease activity that is stimulated latent endonuclease that requires a mismatch, MutSα/β, and by the bacterial β-sliding clamp (10). As in the case of eukaryotic DNA-loaded proliferating cell nuclear antigen (PCNA) for activa- MutLα, this effect presumably depends on physical interaction of tion. We show here that human PCNA and MutLα interact specif- the two proteins. ically but weakly in solution to form a complex of approximately MutLα andPCNAhavebeenshowntointeractinbothhu- 1:1 stoichiometry that depends on PCNA interaction with the man and yeast systems (11–13). For S. cerevisiae proteins, PCNA C-terminal endonuclease domain of the MutLα PMS2 subunit. Amino interaction has been attributed to the yMLH1 subunit, and a acid substitution mutations within a PMS2 C-terminal 721QRLIAP conserved 572QIGLTDF motif within the yMLH1 CTD has motif attenuate or abolish human MutLα interaction with PCNA, as been suggested as a potential PCNA-interaction motif (11, 13). well as PCNA-dependent activation of MutLα endonuclease, PCNA- B. subtilis MutL and β-clamp have also been shown to form a and DNA-dependent activation of MutLα ATPase, and MutLα func- transient complex in solution in a manner that depends on a tion in in vitro mismatch repair. Amino acid substitution mutations QEMIVP motif within the CTD of the MutL homodimer, and within the corresponding yeast PMS1 motif (723QKLIIP) reduce or moderately conserved variants of this element are found in the BIOCHEMISTRY abolish mismatch repair in vivo. Coupling of a weak allele within CTDs of PMS2 and yPMS1 (14). We show here that integrity of 723 this motif ( AKLIIP) with an exo1Δ null mutation, which individu- this element (721QRLIAP) in the PMS2 CTD is required for ally confer only weak mutator phenotypes, inactivates mismatch normal PCNA–MutLα interaction, activation of MutLα endonu- repair in the yeast cell. clease, and in vitro MMR. Integrity of the corresponding yPMS1 723QKLIIP is required for MMR in vivo. However, amino acid DNA repair | mismatch repair | MutLalpha | proliferating cell substitution within the human MLH1 CTD motif 562QILIYDF, nuclear antigen | endonuclease which corresponds to that invoked in yMLH1–yPCNA interaction, has little effect on PCNA–MutLα interaction or PCNA-dependent utLα (mammalian MLH1–PMS2 heterodimer; MLH1– endonuclease activation, although MMR function of the mutant MPMS1 in yeast) plays an essential role during early steps of protein is impaired. eukaryotic mismatch repair (MMR) (1). Inactivation of the hu- man protein is a cause of Lynch syndrome (2, 3), and has been Significance implicated in the development of a subset of sporadic tumors (4). 2+ In physiological buffer (100 to 150 mM salt, 5 mM Mg ), α α MutL is required for initiation of eukaryotic mismatch repair. MutL functions as a latent, strand-directed endonuclease that Inactivation of human MutLα is a cause of Lynch syndrome, a depends on a mismatch, MutSα (MSH2–MSH6 heterodimer) or β – common hereditary cancer, and has also been implicated in the MutS (MSH2 MSH3 heterodimer), and the DNA-loaded form development of a subset of sporadic tumors. The proliferating of the proliferating cell nuclear antigen (PCNA) sliding clamp – cell nuclear antigen (PCNA) sliding clamp is required for acti- for activation (5 8). Strand direction is conferred by the loading vation and strand direction of the MutLα endonuclease. We orientation of the PCNA clamp (8). Although not evident in show that physical interaction of the two proteins, which form α physiological buffer, the intrinsic endonuclease activity of MutL a weak complex in solution, is required for MutLα activation, is demonstrable in the absence of other proteins provided the α 2+ 2+ and have identified a hexapeptide motif within the MutL ionic strength is low and Mn is substituted for Mg (5, 6). PMS2 (PMS1 in yeast) subunit that is required for interaction 2+ α Mn -dependent nuclease activity does not respond to MutS or with PCNA and for MutLα function in mismatch repair. These a mismatch but is stimulated by loaded PCNA, suggesting that findings clarify the mechanism of MutLα activation and es- MutLα interaction with PCNA is required for the effect (6, 8). tablish the importance of PCNA interaction in this process. (Human MutLα, MLH1, PMS2, and PCNA, the primary subjects of this paper, are referred to as such in the text. For the purpose Author contributions: J.G., L.Y.K., R.R.I., F.A.K., and P.M. designed research; J.G., L.Y.K., of distinction, yMutLα, yMLH1, yPMS1, and yPCNA are used for R.R.I., B.K.D., and F.A.K. performed research; J.G., L.Y.K., R.R.I., B.K.D., F.A.K., and P.M. specific reference to the corresponding Saccharomyces cerevisiae analyzed data; and J.G., F.A.K., and P.M. wrote the paper. proteins.) Reviewers: J.J., University of Zurich; G.-M.L., University of Southern California; and M.O., The MutLα endonuclease center resides within the het- Rockefeller University. erodimeric C-terminal domain (CTD) that is composed of The authors declare no conflict of interest. the C-terminal domains of MLH1 and PMS2 (PMS1 in yeast) Freely available online through the PNAS open access option. (9), and endonuclease function depends on the integrity of a 1Present address: Precision BioSciences, Durham, NC 27701. DQHA(X)2E(X)4E metal-binding active-site motif located within 2Present address: Discovery and Product Development, Teva Branded Pharmaceutical the PMS2/yPMS1 CTD (5, 6). The DQHA(X)2E(X)4Eendonu- Products R&D, West Chester, PA 19380. clease motif is also conserved in MutL proteins from bacteria that 3To whom correspondence should be addressed. Email: [email protected]. do not rely on d(GATC) methylation for strand direction of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MMR, with Bacillus subtilis MutL the most extensively studied 1073/pnas.1702561114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1702561114 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 0.4 by surface plasmon resonance. Although this motif is similar to the canonical PCNA-interacting peptide motif (16), the recent α 0.35 structure of the yMutL CTD (Fig. 3) has shown that this hep- tapeptide element is buried and unlikely to be involved in yPCNA interaction in the absence of a substantial conforma- 0.3 tional change (9). A second potential PCNA-interaction motif within the PMS2 CTD has been suggested by studies of the in- 0.25 teraction of B. subtilis MutL with the β-clamp, an interaction that depends on a QEMIVP motif within the CTD of the MutL (mol per mol) 0.2 homodimer (14). Moderately conserved variants of this motif are α found in the CTD of human PMS2 (721QRLIAP) and yeast PMS1 (723QKLIIP). Unlike the buried yMLH1 572QIGLTDF 0.15 element, this yPMS1 motif is exposed as a surface loop (Fig. 3). 0.1 PCNA/MutL MutLα 0.05 A WT P-Q721A P-AAA 0 PCNA++++ 0246810 kD PCNA ( M) 200 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 μL10μM 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 Kd 7.7 μM and a stoichiometry of 0.8 PCNA trimer per MutLα. Kd and stoichiometry values for mutant proteins were not estimated due to the low level of binding. 116 1 234 567 Results Human MutLα and PCNA Interact in Solution. We previously showed B that human PCNA and MutLα interact as judged by far-Western 350 blot (12), and confirm that conclusion here using equilibrium gel PCNA MLH1 PMS2 filtration (15). MutLα was injected onto Superdex 200 columns equilibrated with fixed concentrations of PCNA in physiological + 300 salt buffer containing Mg2 . MutLα-bound sliding clamp was determined from the area of the trough at the PCNA retention volume that results from sequestration of the clamp by MutLα 250 (Fig. S1A). Use of two independent preparations of MutLα and K μ PCNA yielded apparent d values of 4.2 and 7.7 M and stoi- 200 chiometries of 1.1 and 0.8 PCNA trimers per MutLα hetero- dimer (Fig. 1 and Fig. S1C). Because these experiments were done under conditions of PCNA excess, they do not rule out 150 potential multivalent interaction of the PCNA trimer with MutLα as might occur under conditions of MutLα excess, a possibility that was not addressed due to the prohibitive quantities of MutLα 100 required for the analysis. α Physical interaction of PCNA and MutL was also demonstrable 50 by cross-linking with bis(sulfosuccinimidyl)suberate (BS3;Fig.2A, polypeptide abundance (fmol) lanes 2 and 3).