Cellular Aspects of DNA Mismatch Repair Mechanism, Regulation, and Contributions to Tumorigenesis

Date of Completion

January 2010

Keywords

Biology, Molecular|Biology, Cell|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The DNA mismatch repair (MMR) system is highly conserved and vital for preserving genomic integrity. Loss of DNA MMR underlies the hereditary cancer syndrome, hereditary nonpolyposis colorectal cancer (HNPCC). However, how the loss of DNA MMR contributes to tumorigenesis remains an important question. In the first aim of this study, we extended upon previous work done in vitro to address several fundamental questions regarding the mechanism of the DNA MMR pathway in the context of cellular DNA replication. We demonstrated in vivo that the MMR proteins hMSH2, hMLH1, and PCNA localize and accumulate on the chromatin during S-phase. We showed that hMLH1 localization to the chromatin is hMSH2-dependent and that hMSH2/hMLH1/PCNA proteins, when associated with the chromatin, form a complex that is greatly enhanced by DNA damage. Furthermore, we showed that PCNA differentially interacts with the hMSH2/hMLH1 complex in a cell cycle dependent manner. Finally, we found that a population of the hMSH2-hMSH6 heterodimer localizes to the nucleolus in a DNA damage-dependent manner. From these data we gained insight into the initial steps of the MMR pathway in response to DNA damage in the context of an actively replicating cell. Based on these observations we next examined defects in these initial MMR steps as well as other MMR functions conferred by a subset of HNPCC-associated missense variants. Importantly, 18% of germline mutations in hMSH2 are missense variants; however, the consequences are not straightforward, as these alterations could simply represent benign polymorphisms. Therefore, in the second aim of this study we generated human cell lines stably expressing a subset of HNPCC-associated hMSH2 missense variants and tested both repair and checkpoint response functions. We demonstrated that two variants caused significant defects in MMR function and most likely underlie the HNPCC-phenotype. In contrast, two variants were MMR-proficient, suggesting that these may be polymorphisms. Taken together, this work provides the foundation for the future study of cancer-associated MMR gene mutations, which in turn may further our understanding of DNA MMR and tumor suppressive mechanisms, and potentially the development of novel therapeutic strategies to aid in the treatment of CRC patients. ^

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