Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d (GpG) adduct

UNC Libraries · 2021-07-02

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

UNC Libraries · 2020-11-09 · 1 citations

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

MutL traps MutS at a DNA mismatch

UNC Libraries · 2020-11-08 · 1 citations

DNA mismatch repair is the process by which errors generated during DNA replication are corrected. Mutations in the proteins that initiate mismatch repair, MutS and MutL, are associated with greater than 80% of hereditary nonpolyposis colorectal cancer (HNPCC) and many sporadic cancers. The assembly of MutS and MutL at a mismatch is an essential step for initiating repair; however, the nature of these interactions is poorly understood. Here, we have discovered that MutL fundamentally changes the properties of mismatch-bound MutS by preventing it from sliding away from the mismatch, which it normally does when isolated. This finding suggests a mechanism for localizing the activity of repair proteins near the mismatch.

Human MutLγ, the MLH1–MLH3 heterodimer, is an endonuclease that promotes DNA expansion

Proceedings of the National Academy of Sciences · 2020 · 85 citations

• Biology
• Cell biology
• Genetics

MutL proteins are ubiquitous and play important roles in DNA metabolism. MutLγ (MLH1-MLH3 heterodimer) is a poorly understood member of the eukaryotic family of MutL proteins that has been implicated in triplet repeat expansion, but its action in this deleterious process has remained unknown. In humans, triplet repeat expansion is the molecular basis for ∼40 neurological disorders. In addition to MutLγ, triplet repeat expansion involves the mismatch recognition factor MutSβ (MSH2-MSH3 heterodimer). We show here that human MutLγ is an endonuclease that nicks DNA. Strikingly, incision of covalently closed, relaxed loop-containing DNA by human MutLγ is promoted by MutSβ and targeted to the strand opposite the loop. The resulting strand break licenses downstream events that lead to a DNA expansion event in human cell extracts. Our data imply that the mammalian MutLγ is a unique endonuclease that can initiate triplet repeat DNA expansions.

Interactions of Human Mismatch Repair Proteins MutSα and MutLα with Proteins of the ATR-Chk1 Pathway

UNC Libraries · 2020-11-09

At clinically relevant doses, chemotherapeutic SN1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSα and MutLα. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSα and MutLα with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSα with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLα with TopBP1 and Claspin. We were unable to detect interaction of MutSα or MutLα with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSα with ATR, TopBP1, and Chk1 and of MutLα with TopBP1. MutSα-Claspin and MutLα-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLα- and MutSα-dependent fashion after N-methyl-N′-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.

MutS mediates heteroduplex loop formation by a translocation mechanism

UNC Libraries · 2020-11-09

Interaction of Escherichia coli MutS and MutL with heteroduplex DNA has been visualized by electron microscopy. In a reaction dependent on ATP hydrolysis, complexes between a MutS dimer and a DNA heteroduplex are converted to protein-stabilized, alpha-shaped loop structures with the mismatch in most cases located within the DNA loop. Loop formation depends on ATP hydrolysis and loop size increases linearly with time at a rate of 370 base pairs/min in phosphate buffer and about 10,000 base pairs/min in the HEPES buffer used for repair assay. These observations suggest a translocation mechanism in which a MutS dimer bound to a mismatch subsequently leaves this site by ATP-dependent tracking or unidimensional movement that is in most cases bidirectional from the mispair. In view of the bidirectional capability of the methyl-directed pathway, this reaction may play a role in determination of heteroduplex orientation. The rate of MutS-mediated DNA loop growth is enhanced by MutL, and when both proteins are present, both are found at the base of alpha-loop structures, and both can remain associated with excision intermediates produced in later stages of the reaction.

Direct Visualization of Asymmetric Adenine Nucleotide-Induced Conformational Changes in MutLα

UNC Libraries · 2020-11-06

MutLα, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutLα with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutSα and the activation of MutLα with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from x-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutLα belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutLα, and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle where sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutLα.

The mutagen and carcinogen cadmium is a high-affinity inhibitor of the zinc-dependent MutLα endonuclease

Proceedings of the National Academy of Sciences · 2018-06-25 · 26 citations

Significance MutLα (MLH1-PMS2 heterodimer) is an endonuclease that acts during an early step of eukaryotic mismatch repair. We show that human MutLα endonuclease copurifies with two equivalents of bound zinc, at least one of which resides within the endonuclease active site. We also show that cadmium, a known inhibitor of zinc-dependent enzymes and a potent mutagen and carcinogen, is a high-affinity inhibitor of MutLα endonuclease and that exogenous MutLα significantly reverses the mismatch repair defect in cadmium-treated human cell nuclear extract or nuclear extract prepared from cadmium-treated cells. Because the mutagenic action of cadmium is largely due to the selective inhibition of mismatch repair, these findings suggest that MutLα is a primary cadmium target for mutagenesis and presumably, carcinogenesis as well.

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

Proceedings of the National Academy of Sciences · 2017-04-24 · 62 citations

Significance MutLα is required for initiation of eukaryotic mismatch repair. Inactivation of human MutLα is a cause of Lynch syndrome, a common hereditary cancer, and has also been implicated in the development of a subset of sporadic tumors. The proliferating cell nuclear antigen (PCNA) sliding clamp is required for activation and strand direction of the MutLα endonuclease. We show that physical interaction of the two proteins, which form a weak complex in solution, is required for MutLα activation, and have identified a hexapeptide motif within the MutLα PMS2 (PMS1 in yeast) subunit that is required for interaction with PCNA and for MutLα function in mismatch repair. These findings clarify the mechanism of MutLα activation and establish the importance of PCNA interaction in this process.

Mechanismen der Fehlpaarungsreparatur in <i>E. coli</i> und im Menschen (Nobel‐Aufsatz)

Angewandte Chemie · 2016-05-20

DNA-Moleküle sind nicht völlig stabil. Sie unterliegen chemischen oder photochemischen Schädigungen und Fehlern, die bei der DNA-Replikation eingeführt werden und in fehlgepaarten Basenpaaren resultieren. Mechanistische Studien in der Arbeitsgruppe von Paul Modrich haben gezeigt, wie Replikationsfehler durch stranggerichtete Fehlpaarungsreparatur in E. coli und im Menschen korrigiert werden.