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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA167181-02
Application #
8620620
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Okano, Paul
Project Start
2013-02-15
Project End
2018-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
2
Fiscal Year
2014
Total Cost
$277,324
Indirect Cost
$90,574
Name
University of Kentucky
Department
Pharmacology
Type
Schools of Medicine
DUNS #
939017877
City
Lexington
State
KY
Country
United States
Zip Code
40506
Li, Guo-Min (2016) A Personal Tribute to 2015 Nobel Laureate Paul Modrich. DNA Repair (Amst) 37:A14-21
Guo, Jinzhen; Gu, Liya; Leffak, Michael et al. (2016) MutSβ promotes trinucleotide repeat expansion by recruiting DNA polymerase β to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis. Cell Res 26:775-86
Li, Guo-Min (2016) Celebrating the work of Nobel Laureate Paul Modrich. Sci China Life Sci 59:93-6
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
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
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
Li, Guo-Min (2014) New insights and challenges in mismatch repair: getting over the chromatin hurdle. DNA Repair (Amst) 19:48-54
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
Li, Feng; Mao, Guogen; Tong, Dan et al. (2013) The histone mark H3K36me3 regulates human DNA mismatch repair through its interaction with MutSα. Cell 153:590-600

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