Using molecular, biochemical and structural approaches, we have broadly contributed to defining how specific human BER proteins recognize and process target lesions, as well as coordinate with other components of the protective system. The research has centered largely on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian protein for repairing abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a non-enzymatic scaffold protein that facilitates the efficient execution of single-strand break (SSB) repair. Some of the key findings during the course of our studies include: (i) in addition to abasic sites in conventional DNA, APE1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and APE1 can endonucleolytically destroy damaged RNA; (ii) APE1 contributes to the repair of 3-modifications in DNA, such as mismatches, phosphate, phosphogycolate and tyrosyl residues; (iii) the DNA repair function of APE1 is regulated in part by S-glutathionylation; (iv) inhibition of APE1 is a potential mechanism for the co-carcinogenic effects of lead, an important environmental toxin; (v) APE1 communicates with CSB, a protein defective in the premature aging disorder, Cockayne syndrome; (vi) XRCC1 directly associates with the replication/repair protein, PCNA, establishing a novel link between DNA repair and replication factories; (vii) XRCC1 coordinates disparate responses and multi-protein repair complexes that are dependent on the context of the DNA damage; (viii) the different regions of XRCC1 play distinct roles in coordinating repair complex assembly; (ix) the interaction of XRCC1 with the DNA repair enzyme PNKP functions to retain XRCC1 at DNA damage sites and promote repair of alkylation damage; (x) XRCC1 supports an emerging pathway for uracil repair, termed replication-associated BER, through a physical association with UNG2, the major nuclear uracil DNA glycosylase; (xi) the DNA glycosylase NEIL1 recognizes interstrand crosslinks in DNA, and can obstruct the efficient removal of these lethal adducts; (xii) the flap-endonuclease FEN1 plays a role in repairing mitochondrial oxidative DNA damage through a long-patch BER pathway; (xiii) RECQL4, a human RecQ helicase mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome, regulates BER capacity both directly and indirectly; (xiv) RECQL5, another RECQ helicase family member, modulates and/or directly participates in BER of endogenous DNA damage, thereby promoting chromosome stability in normal human cells; and (xv) the multifunctional protein nucleophosmin (a.k.a., NPM1) is a modulator of BER capacity by controlling protein levels and nucleolar localization of several BER enzymes. Currently, a main focus is to establish genetically modified cell lines to dissect out the precise contribution of each proposed function of APE1 (i.e. its nuclease activity, redox regulatory role, etc.) in cell growth/viability, genome maintenance, and protection against DNA-damaging agents. Defining which of the many reported functions of APE1 are critical to normal cellular activity is a key step towards understanding the potential relationship of the protein to the aging process and disease risk. Studies indicate that significant inter-individual variation exists at the amino acid sequence level of BER proteins and in BER capacity among individuals 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 BER effectiveness. Our prior work has found that (i) of the three most common amino acid variants of the XRCC1 (i.e., R194W, R280H and R399Q), the latter two, particularly R280H, exhibit impaired recruitment to and/or retention at sites of DNA damage in live cells and (ii) variants of NEIL1 (i.e., G83D, C136R, and E181K) display altered responses to localized DNA damage in human cells. In addition, using established biochemical assays, our results indicate that for AP site incision, there exists an 1.9-fold inter-individual variation in repair capacity among the individuals examined. For gap-filling and nick ligation, an 1.3-fold and 3.4-fold inter-individual variation was observed, respectively. We are currently designing a series of more sophisticated high-throughput assays to more thoroughly evaluate the relationship of BER capacity with disease susceptibility and premature aging phenotypes. Recent work has focused on variants in APE1, and we have found that except for the endometrial cancer-associated variant R237C, the polymorphic variants Q51H, I64V and D148E, the rare population variants G241R, P311S and A317V, and the tumor-associated variant P112L exhibit largely normal structural and functional properties. The R237C mutant, conversely, displayed reduced AP-DNA complex stability, 3'-5' exonuclease activity and 3'-damage processing. Re-sequencing of the exonic regions of APE1 uncovered no novel amino acid substitutions in the 60 cancer cell lines of the NCI-60 panel, or in HeLa or T98G cancer cell lines; only the common D148E and Q51H variants were observed. Our results therefore indicate that APE1 missense mutations are rare and that the cancer-associated R237C variant may represent a reduced-function susceptibility allele. Consistent with the latter conclusion, our recent investigations have discovered that the incision activity of R237C, in comparison to the wild-type protein, was uniquely hypersensitive to nucleosome complexes in the vicinity of the AP site. These results suggest that APE1 has acquired distinct surface residues that permit efficient processing of AP sites within the context of protein-DNA complexes independent of classic chromatin remodeling mechanisms, and indicate that R237C may have contributed to carcinogenesis as an impaired-function allele. Current strategies to eradicate cancer cells commonly 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. Our 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). Moreover, we have found that BER, and APE1 in particular, are promising targets for treating cancers with a deficiency in homologous recombination via an approach that involves synergistic (or synthetic) lethality, and that FEN1 is a promising biomarker in breast and ovarian epithelial cancer. Towards the development of more effective small molecule inhibitors against enzymes in BER, we have developed a panel of complementary and improved miniaturized high-throughput screening and profiling assays. These assays have permitted the identification of novel APE1-targeted bioactive endonuclease inhibitors, which we are working to optimize with the long term goal of creating high affinity, selective inhibitors with therapeutic value. In addition, our effort has uncovered a set of compounds that impair the APE1/NPM1 protein interaction in living cells, with some of these molecules displaying anti-proliferative activity and increased cellular sensitization to therapeutically relevant genotoxins. The establishment of small molecule bioactive probes will provide a platform for more extensive investigations on the therapeutic benefits of regulating cellular DNA repair capacity.
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