DNA repair pathways maintain the integrity of the genome and help prevent the onset of cancer. Conversely, DNA repair deficiencies engender tumor heterogeneity and promote the selection of highly aggressive subtypes (i.e., `mutator' phenotype). Importantly, DNA repair proteins have emerged as potential synthetic lethal targets for improving the selective response to currently available anti-cancer regimens. Key to advancing such a strategy is a detailed mechanistic understanding of the select DNA repair pathway in question, an appreciation of repair pathway redundancy & crosstalk and a molecular characterization of processes that regulate the function and stability of the proteins essential for repair. This completely revised R01 proposal builds on our recent discovery that Pol is ubiquitylated on lysines 206/244 in a manner that is dependent on the cell cycle and the type of DNA damage. Further, we reported that (i) Pol ubiquitylation may govern pathway crosstalk, (ii) select oxidized DNA lesions require/promote Pol degradation and (iii) Pol-mediated BER facilitates DNA repair pathway crosstalk that may be regulated by steady-state levels of Pol protein. Therefore, our first task was to identify the E3 ligases and de-ubiquitylation enzymes (DUBs) that may regulate Pol. High-resolution mass spectrometry proteomic analysis of Pol-interacting proteins and in response to PARP-activation allowed us to identify several E3 ligases and DUBs likely to target Pol to regulate stability and function. Our new data supports a role for the E3 ubiquitin ligase TRIP12 contributing to the regulation of Pol stability and governing Pol chromatin retention. Further, Pol ubiquitylation appears to govern BER repair complex dynamics. Most importantly, our preliminary data suggests that ubiquitylation facilitates trafficking of Pol to participate in late-phase replication associated repair (RAR) foci in response to complex DNA lesions. These RAR foci are XRCC1 dependent but devoid of DSB markers such as ?H2AX or 53BP1.
The Aims detailed in the proposal will use purified proteins, cancer cell lines, tumor stem cells, high-resolution proteomics, live-cell fluorescent imaging and mouse xenografts to address our hypothesis that ubiquitylation/de-ubiquitylation regulates Pol stability, Pol-dependent BER repair complex dynamics and facilitates a coordinated trafficking mechanism to promote Pol involvement yet suppress 53BP1 involvement at complex lesions following alkylation, cisplatin and radiation damage.

Public Health Relevance

We hypothesize that ubiquitylation/de-ubiquitylation regulates DNA polymerase (Pol) stability, Pol- dependent BER repair complex dynamics and facilitates a coordinated trafficking mechanism to promote Pol involvement yet suppress 53BP1 involvement at complex lesions following alkylation, cisplatin and radiation damage.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Okano, Paul
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University of South Alabama
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Sykora, Peter; Chiari, Ylenia; Heaton, Andrew et al. (2018) Application of the CometChip platform to assess DNA damage in field-collected blood samples from turtles. Environ Mol Mutagen 59:322-333
Sykora, Peter; Witt, Kristine L; Revanna, Pooja et al. (2018) Next generation high throughput DNA damage detection platform for genotoxic compound screening. Sci Rep 8:2771
Li, Jianfeng; Svilar, David; McClellan, Steven et al. (2018) DNA Repair Molecular Beacon assay: a platform for real-time functional analysis of cellular DNA repair capacity. Oncotarget 9:31719-31743
Golato, Tyler; Brenerman, Boris; McNeill, Daniel R et al. (2017) Development of a Cell-Based Assay for Measuring Base Excision Repair Responses. Sci Rep 7:13007
Soll, Jennifer M; Sobol, Robert W; Mosammaparast, Nima (2017) Regulation of DNA Alkylation Damage Repair: Lessons and Therapeutic Opportunities. Trends Biochem Sci 42:206-218
Simonelli, Valeria; Leuzzi, Giuseppe; Basile, Giorgia et al. (2017) Crosstalk between mismatch repair and base excision repair in human gastric cancer. Oncotarget 8:84827-84840
Bigarella, Carolina L; Li, Jianfeng; Rimmelé, Pauline et al. (2017) FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage. J Biol Chem 292:3005-3015
Nagel, Zachary D; Engelward, Bevin P; Brenner, David J et al. (2017) Towards precision prevention: Technologies for identifying healthy individuals with high risk of disease. Mutat Res 800-802:14-28
Ding, Lu; Dean-Ben, Xose Luis; Burton, Neal C et al. (2017) Constrained Inversion and Spectral Unmixing in Multispectral Optoacoustic Tomography. IEEE Trans Med Imaging 36:1676-1685
Sawant, Akshada; Floyd, Ashley M; Dangeti, Mohan et al. (2017) Differential role of base excision repair proteins in mediating cisplatin cytotoxicity. DNA Repair (Amst) 51:46-59

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