Studies indicate that significant inter-individual variation exists at the amino acid sequence level of BER proteins, and that variability exists in the BER capacity within the population. We have initiated efforts to delineate the impact of amino acid variants found in BER proteins and to develop assays to determine the extent of inter-individual variation in specific steps of BER. For example, we are currently investigating whether variants found in the major human abasic endonuclease (APE1), either within the normal population or that are tumor-associated, affect protein function and may therefore contribute to disease formation and/or progression. In addition, using established biochemical assays, we have begun to evaluate whether inter-individual variation in BER associates with disease susceptibility and/or clinical agent responsiveness;we are also assessing for age-dependent or gender-specific variation. Current biochemical methods have allowed us to interrogate the central steps of BER. Our results indicate that for AP site incision, the twenty-three individuals examined thus far exhibit an 1.9-fold inter-individual variation in repair capacity. For gap-filling and nick ligation, we observed an 1.3-fold and 3.4-fold inter-individual variation, respectively. The greater disparity in nick ligation stems at least in part from the low overall activity and the corresponding limited sensitivity of the assay. Like the AP endonuclease profile, the average repair capacity of each individual for gap-filling and nick sealing generally fell within the experimental variability of the population, suggesting a comparatively similar BER efficiency among this small group of samples. We are presently designing a high throughput BER pathway assay to more robustly evaluate the relationship of repair capacity with disease susceptibility. Current strategies to eradicate cancer cells typically employ agents that generate DNA lesions that induce cell death by blocking replication of rapidly dividing cells. Thus, a goal has been to regulate strategically the repair capacity of cancer and/or normal cells to improve the efficacy of specific therapeutic paradigms. In particular, inhibiting the DNA repair capacity of cancerous cells has been an area of promising interest. Our recent results indicate that APE1, and BER more generally, is a reasonable target for inactivation in anti-cancer treatment paradigms involving select alkylating drugs (e.g., temozolomide) and antimetabolites (e.g., 5-fluorouracil). We have recently described the development of a panel of complementary and improved miniaturized high-throughput screening and profiling assays, which will have a broad appeal to other research investigators, particularly those in the field of DNA repair. These assays have permitted the identification of novel, small molecule APE1-targeted bioactive inhibitors, which we are working to optimize with the long term goal of creating high affinity, selective inhibitors with therapeutic value. The establishment of small molecule probes will provide a platform for more extensive investigations on the therapeutic benefits of regulating cellular DNA repair capacity.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAAG000750-05
Application #
8552461
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$422,697
Indirect Cost
Name
National Institute on Aging
Department
Type
DUNS #
City
State
Country
Zip Code
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