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 721 QRLIAP motif attenuate or abolish human MutLα interaction with PCNA, as well as PCNA-dependent activation of MutLα endonuclease, PCNAand DNA-dependent activation of MutLα ATPase, and MutLα function in in vitro mismatch repair. Amino acid substitution mutations within the corresponding yeast PMS1 motif ( In physiological buffer (100 to 150 mM salt, 5 mM Mg 2+ ), MutLα functions as a latent, strand-directed endonuclease that depends on a mismatch, MutSα (MSH2-MSH6 heterodimer) or MutSβ (MSH2-MSH3 heterodimer), and the DNA-loaded form of the proliferating cell nuclear antigen (PCNA) sliding clamp for activation (5-8). Strand direction is conferred by the loading orientation of the PCNA clamp (8). Although not evident in physiological buffer, the intrinsic endonuclease activity of MutLα is demonstrable in the absence of other proteins provided the ionic strength is low and Mn 2+ is substituted for Mg 2+ (5, 6). Mn 2+ -dependent nuclease activity does not respond to MutSα or a mismatch but is stimulated by loaded PCNA, suggesting that MutLα interaction with PCNA is required for the effect (6, 8).(Human MutLα, MLH1, PMS2, and PCNA, the primary subjects of this paper, are referred to as such in the text. For the purpose of distinction, yMutLα, yMLH1, yPMS1, and yPCNA are used for specific reference to the corresponding Saccharomyces cerevisiae proteins.)The MutLα endonuclease center resides within the heterodimeric C-terminal domain (CTD) that is composed of the C-terminal domains of MLH1 and PMS2 (PMS1 in yeast) (9), and endonuclease function depends on the integrity of a DQHA(X) 2 E(X) 4 E metal-binding active-site motif located within the PMS2/yPMS1 CTD (5, 6). The DQHA(X) 2 E(X) 4 E endonuclease motif is also conserved in MutL proteins from bacteria that do not rely on d(GATC) methylation for strand direction of MMR, with Bacillus subtilis MutL the most extensively studied protein of this class. Like eukaryotic MutLα, B. subtilis MutL displays Mn 2+ -dependent endonuclease activity that is stimulated by the bacterial β-sliding clamp (10). As in the case of eukaryotic MutLα, this effect presumably depends on physical interaction of the two proteins.MutLα and PCNA have been shown to interact in both human and yeast systems (11-13). For S. cerevisiae proteins, PCNA interaction has been attributed to the yMLH1 subunit, and a conserved 572 QIGLTDF motif within the yMLH1 CTD has been suggested as a potential PCNA-interaction motif (11, 13). B. subtilis MutL and β-clamp have also been...

Biochemical and structural characterization of two variants of uncertain significance in the PMS2 gene

“…MLH1 and PMS2 harbor conserved ATP‐binding domains located in the N‐termini of the enzymes and their C‐terminal domains are involved in heterodimerization, which is necessary for the stability of PMS2 (Guarne, ; Hinrichsen et al., ). The C‐terminal domain of PMS2 also possesses an endonuclease domain that preferentially nicks the discontinuous strand of mismatched DNA, an activity that appears to be stimulated via interactions with the proliferating cell nuclear antigen (PCNA) (Genschel et al., ; Kadyrova & Kadyrov, ).…”

“…mismatched DNA, an activity that appears to be stimulated via interactions with the proliferating cell nuclear antigen (PCNA) (Genschel et al, 2017;Kadyrova & Kadyrov, 2016).…”

Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex

“…A single substitution in this region, pms1-Y613A, conferred a mutator phenotype, (p-value <.00001 to WT; p-value<.00001 to pms1Δ584-634 compared by Mann-Whitney U test) ( Supplementary Figure S1, Supplementary Table S1). The pms1-Y613A substitution maps to a region of PMS1 that is disordered ( Figure 1B) and does not encode any known PCNAinteraction motifs, as identified in yeast PMS1 (721QRLIAP), human PMS1 (723QKLIIP), and B. subtilis MutL (QEMIVP) (27). Consistent with this, the endonuclease activity of MLH complexes containing the pms1Δ584-634 mutation is stimulated by PCNA, indicating that this region is not required for PCNA interactions (see below).…”

“…These results show that the two DLD complexes are impaired in ATP-dependent interactions with DNA ( Figure 1D). The ATPase activities of the WT complex are stimulated by DNA and PCNA (27,34). However, neither DLD complex exhibited such stimulation ( Figure 1E).…”

“…A latent MLH endonuclease activity then nicks the newly-synthesized DNA strand resulting in excision of the mismatch (19). This activity requires PCNA, and multiple nicks may enhance the excision step of MMR (20)(21)(22)(23)(24)(25)(26)(27).…”