Tuesday, September 26, 2023
9/26/2023
Project Summary Environmental pollutants and anti-cancer chemicals induce DNA–protein crosslinks (DPCs, also known as protein adducts) of topoisomerases and other proteins that obstruct almost every chromatin transaction and lead to mutations, genome instability and cell death if not removed quickly. Cells thus possess multiple mechanisms to remove DPCs: homologous recombination, nucleotide excision repair and proteolytic repair that involves proteolysis of bound proteins and nucleolytic cleavage of peptide-DNA bonds. Among these, proteolytic DPC repair is poorly defined and there are many questions about this unique collaboration between protease and nuclease and their substrate recognition. Using genetic and biochemical approaches, we have discovered that Apn2, the evolutionarily conserved back-up AP endonuclease, possesses an enzymatic activity unblocking DNA ends with various 3’ adducts including 2’,3’-cyclic phosphate, monophosphate and tyrosine DNA conjugates. We also found that Apn2 removes DNA-topoisomerase I crosslinks, the flagship DPC at 3' termini. The results might explain several puzzling symptoms in mice deleted for APE2, the metazoan Apn2 homolog, including dys-lymphopoiesis and growth retardation. The results also underscore the importance of 3’ blocked termini processing for genome maintenance and reveal unique challenges in removing these toxic DNA lesions in a tractable model system. Nevertheless, our results also raise a new set of questions about the regulation of Apn2 and APE2 in 3' adduct repair. For instance, we aim to determine how Apn2 recognizes a wide range of 3’ termini with different structural features and how such activities are regulated under unique cellular and functional contexts. We also want to elucidate the biological significance of Apn2 and human APE2-dependent Top1cc repair in cellular physology exposed to chemotherapeutics. Furthermore, we plan to define the roles of Apn2 and its interacting protease(s) in the removal of toxic DNA protein complexes (DPCs) that form via environmental pollutants and chemicals. The current proposal will address these important questions using innovative genetic and biochemical assays and shed light on the fundamental mechanisms of 3’ DNA adduct repair, mutagenesis and maintenance of chromosomal integrity. Emerging evidence suggests that accumulated DPCs cause aging, neurodegeneration and liver cancers. The human homolog of Apn2 is also essential for the viability of BRCA-deficient breast and ovarian cancer cells. The functional conservation in Top1cc and DPC repair between yeast and human further underscores the value of the proposed research in setting the foundation for analyzing the equivalent processes in human. Information gained from our proposed research plan will not only solve one of the fundamental questions in biological processes associated with mutagenesis, DNA repair, and genome maintenance, but will also impinge directly on the management and treatment of patients suffering from several devastating diseases with no clear treatment option.
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