Cell Cycle Regulation of DNA Mismatch Repair Protein Expression and Activity at the H-ras Oncogenic Hot Spot

by Edelbrock, Michael Aaron

Abstract (Summary)
Mismatch repair (MMR) corrects mispaired bases and insertion/deletion loops and has long been regarded as a post-replicative DNA repair process. MMR proteins also are involved in DNA damage surveillance, damage-induced cell cycle arrest, apoptosis, and other DNA repair pathways. Biochemical mechanisms of MMR within human cells are not well understood and the involvement of the MMR pathway in correcting DNA mispairs at oncogenic hotspots is unknown. Additionally, cell cycle regulation of MMR proteins has not been well defined. MMR protein expression, binding activity, and repair activity were quantified using proliferating cells and discrete cell cycle populations separated by centrifugal elutriation. Specifically, the quantitative functioning of the MMR system at an oncogenic hotspot sequence located at H-ras codon12 during different cell cycle phases was investigated. We demonstrate that hMutS√° is able to recognize both codon 12G:T and 12G:A mismatches, but binding activity and accurate nick-directed repair is greater for 12G:T. Further, MMR proficient cells are able to more accurately repair mismatches at codon 12G:T versus 12G:A, while MMR deficient cells are unable to efficiently correct either mismatch. Additionally, the cell regulates MMR proteins and their activity in a cell cycle dependent manner, which may contribute to efficient MMR at hotspots. Nuclear MMR protein levels increase as the cell progresses from G1 to S phase and remain elevated during G2 phase. Importantly, this work is the first to describe the sustained increase of nuclear levels of all four major mismatch proteins: hMSH2, hMSH6, hPMS2, and hMLH1 during S to G2 cell cycle transition. Interestingly, hMutS√° binding and nick-directed repair activity is measureable in G1, increases during S phase, but contrary to protein expression this activity decreases in G2. Further, nick-directed MMR activity is greater in assays using a replication-competent plasmid versus a replication-incompetent plasmid for identical assay conditions. Collectively, we provide new insight into the complexity of human mismatch repair at the H-ras oncogenic hot spot. This work supports the hypothesis that hotspots may arise through inability of the MMR system to correct G:A mispairs at particular sequences such as the H-ras codon 12 location in resting or dividing cells.
Bibliographical Information:


School:University of Toledo Health Science Campus

School Location:USA - Ohio

Source Type:Master's Thesis

Keywords:dna repair mismatch mmr hotspot hras damage


Date of Publication:01/01/2007

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