| Summary: |
(CIT): DNA mismatch repair (MMR) is a highly conserved biological pathway that plays a key role in maintaining genomic stability. Defects in MMR predispose to cancers. The genome-maintenance function of the pathway includes correcting DNA biosynthetic errors, suppressing homeologous recombination, and signaling DNA damage response. This presentation reports our recent efforts to understand the molecular mechanisms of MMR in human cells and includes two lines of work: (i) identification and characterization of MMR components, and (ii) reconstitution of the human MMR reaction in a defined system. In the first part of the presentation, two biochemical mutants were generated from HeLa nuclear extracts using a fractionation approach (e.g., ammonium sulfate precipitation and column chromatography), and each mutant was used as a receptor to isolate a nuclear activity capable of restoring MMR to the biochemical mutant in a functional in vitro MMR assay. One complementing activity was identified to be replication protein A (RPA) and the other is high mobility group box 1 protein (HMGB1). Whereas HMGB1 is specifically involved in the excision reaction, RPA plays multiple roles in the MMR reaction, including protecting the nascent ssDNA generated during excision and promoting repair DNA synthesis. RPA is phosphorylated during MMR. Evidence is provided that unphosphorylated RPA, which possesses a much stronger ssDNA binding affinity than phosphrylated RPA, preferentially promotes mismatch excision, while phosphorylated RPA facilitates DNA resynthesis. Similarly, proliferating cellular nuclear antigen (PCNA) was found to be essential for MMR by participating both the MMR initiation and repair resynthesis reactions. The second part of the presentation focuses on reconstitution of the human MMR reaction using purified human proteins, including MutSa or MutSb, MutLa, RPA, EXO1, HMGB1, PCNA, RFC, DNA polymerase d, and ligase I. In this system, MutSb plays a limited role in repair of base-base mismatches, but it processes insertion/deletion mispairs much more efficiently than MutSa, which efficiently corrects both types of heteroduplexes. MutLa reduces the processivity of EXO1 and terminates EXO1-catalyzed excision upon mismatch removal. In the absence of MutLa, mismatch-provoked excision by EXO1 occurs extensively. RPA and HMGB1 play similar but complementary roles in stimulating MutSa-activated, EXO1-catalyzed excision in the presence of a mismatch, but RPA has a distinct role in facilitating MutLa-mediated excision termination past mismatch. Additionally, these reconstitution experiments show that efficient repair of a single mismatch requires multiple molecules of MutSa-MutLa complex. These data suggest a model for human MMR involving coordinated initiation and termination of mismatch-provoked excision. The DNA Repair Interest Group is concerned with all forms of DNA damage and repair. As a major defense against environmental damage to cells DNA repair is present in all organisms examined including bacteria, yeast, drosophila, fish, amphibians, rodents and humans. The members of the DNA Repair Interest Group perform research in areas including DNA repair enzymology and fine structure, mutagenesis, gene and cell cycle regulation, protein structure, and human disease. For more information, visit the DNA Repair Interest Group. |
