The long-term goal of this project is to study how post-translational modifications of DNA mismatch repair (MMR) proteins impact genome integrity and cancer development. MMR maintains genome stability by removing mismatches in the newly synthesized strand during DNA replication. The MMR reaction involves mismatch recognition by the initiation factors (MutS?, MutL?, and PCNA), mismatch removal by nucleases, and DNA gap-filling by DNA polymerases. The importance of MMR is under- scored by the fact that MMR defects lead to hypermutations and susceptibility to both hereditary and sporadic colorectal cancers (CRCs). Exhibiting elevated instability in simple repeats, called microsatellite instability (MSI), is a hallmark of MMR-deficient CRCs. However, only ~70% of hereditary and sporadic CRC cases that display MSI have identifiable mutations in MMR genes, suggesting that other mechanism(s) are responsible for the MSI phenotype in the remaining 30% of the cases. We recently showed that CRC cells containing high levels of PCNA tyrosine phosphorylation are defective in MMR in vitro. We therefore hypothesize that PCNA phosphorylation inhibits MMR, leading to genome instability and CRC development. To test this hypothesis, three Specific Aims are proposed.
Aim 1 is to determine how phosphorylated PCNA inhibits MMR. A well-defined in vitro MMR reaction will be conducted in both a nuclear extract system and a reconstituted system in the presence or absence of phosphorylated PCNA, and analysis of the repair products will allow determination of the specific step(s) of the reaction tht is blocked.
Aim 2 is to determine hypermutability and MMR proficiency in cells stably expressing phosphorylated PCNA mimics while suppressed for endogenous PCNA expression.
Aim 3 is to analyze tumorigenesis in transgenic mice expressing phosphorylated PCNA. A successful completion of the proposed work will establish PCNA tyrosine phosphorylation as a novel biomarker for cancer etiology and progression.

Public Health Relevance

Despite that great progress has been made in colorectal cancer (CRC) therapy, the disease is still the second leading cause of cancer deaths among adults in the United States. A major reason for this is that the factors that cause a significant fraction of CRC are not fully understood. To improve the health of Americans, this application aims to identify these factors, which will improve both CRC screening and treatment.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Okano, Paul
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University of Kentucky
Schools of Medicine
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Li, Feng; Ortega, Janice; Gu, Liya et al. (2016) Regulation of mismatch repair by histone code and posttranslational modifications in eukaryotic cells. DNA Repair (Amst) 38:68-74
Guo, Jinzhen; Gu, Liya; Leffak, Michael et al. (2016) MutS? promotes trinucleotide repeat expansion by recruiting DNA polymerase ? to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis. Cell Res 26:775-86
Li, Guo-Min (2016) A Personal Tribute to 2015 Nobel Laureate Paul Modrich. DNA Repair (Amst) 37:A14-21
Li, Guo-Min (2016) Celebrating the work of Nobel Laureate Paul Modrich. Sci China Life Sci 59:93-6
Hegde, Pavana M; Dutta, Arijit; Sengupta, Shiladitya et al. (2015) The C-terminal Domain (CTD) of Human DNA Glycosylase NEIL1 Is Required for Forming BERosome Repair Complex with DNA Replication Proteins at the Replicating Genome: DOMINANT NEGATIVE FUNCTION OF THE CTD. J Biol Chem 290:20919-33
Ortega, Janice; Li, Jessie Y; Lee, Sanghee et al. (2015) Phosphorylation of PCNA by EGFR inhibits mismatch repair and promotes misincorporation during DNA synthesis. Proc Natl Acad Sci U S A 112:5667-72
Tong, Dan; Ortega, Janice; Kim, Christine et al. (2015) Arsenic Inhibits DNA Mismatch Repair by Promoting EGFR Expression and PCNA Phosphorylation. J Biol Chem 290:14536-41
Li, Guo-Min (2014) New insights and challenges in mismatch repair: getting over the chromatin hurdle. DNA Repair (Amst) 19:48-54
Li, Guo-Min (2013) Decoding the histone code: Role of H3K36me3 in mismatch repair and implications for cancer susceptibility and therapy. Cancer Res 73:6379-83
Hegde, Muralidhar L; Hegde, Pavana M; Bellot, Larry J et al. (2013) Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins. Proc Natl Acad Sci U S A 110:E3090-9

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