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...
Background:The DNA mismatch repair (MMR) system protects humans from cancer. Results: Combining an MMR system defect (msh2⌬) with rad27⌬ causes a strong synergistic increase in the rate of 1-bp insertions, and a reconstituted MMR system removes 1-nt flaps. Conclusion: The MMR system removes 1-nt Okazaki fragment flaps. Significance: A new function of the MMR system was identified.
The DNA mismatch repair (MMR) system corrects DNA mismatches in the genome. It is also required for the cytotoxic response of O-methylguanine-DNA methyltransferase (MGMT)-deficient mammalian cells and yeast mgt1Δ rad52Δ cells to treatment with S1-type methylating agents, which produce cytotoxic O-methylguanine (O-mG) DNA lesions. Specifically, an activity of the MMR system causes degradation of irreparable O-mG-T mispair-containing DNA, triggering cell death; this process forms the basis of treatments of MGMT-deficient cancers with S1-type methylating drugs. Recent research supports the view that degradation of irreparable O-mG-T mispair-containing DNA by the MMR system and CAF-1-dependent packaging of the newly replicated DNA into nucleosomes are two concomitant processes that interact with each other. Here, we studied whether CAF-1 modulates the activity of the MMR system in the cytotoxic response to S1-type methylating agents. We found that CAF-1 suppresses the activity of the MMR system in the cytotoxic response of yeast mgt1Δ rad52Δ cells to the prototypic S1-type methylating agent N-methyl-N'-nitro-N-nitrosoguanidine. We also report evidence that in human MGMT-deficient cell-free extracts, CAF-1-dependent packaging of irreparable O-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system. Taken together, these findings suggest that CAF-1-dependent incorporation of irreparable O-mG-T mispair-containing DNA into nucleosomes suppresses its degradation by the MMR system, thereby defending the cell against killing by the S1-type methylating agent.
Heterochromatin contains a significant part of nuclear DNA. Little is known about the mechanisms that govern heterochromatic DNA stability. We show here that in the yeast Saccharomyces cerevisiae (i) DNA mismatch repair (MMR) is required for the maintenance of heterochromatic DNA stability, (ii) MutLα (Mlh1-Pms1 heterodimer), MutSα (Msh2-Msh6 heterodimer), MutSβ (Msh2-Msh3 heterodimer), and Exo1 are involved in MMR at heterochromatin, (iii) Exo1-independent MMR at heterochromatin frequently leads to the formation of Pol ζ-dependent mutations, (iv) MMR cooperates with the proofreading activity of Pol ε and the histone acetyltransferase Rtt109 in the maintenance of heterochromatic DNA stability, (v) repair of base-base mismatches at heterochromatin is less efficient than repair of base-base mismatches at euchromatin, and (vi) the efficiency of repair of 1-nt insertion/deletion loops at heterochromatin is similar to the efficiency of repair of 1-nt insertion/deletion loops at euchromatin.
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