DNA base excision repair (BER) pathway repairs damage caused by reactive oxygen species, alkylating agents and ionizing radiation, and so protects cells against the toxic effects of various endogenous and exogenous agents. The mammalian AP-endonuclease (APE1) plays a central role in the BER pathway by repairing abasic (AP) sites generated in DNA spontaneously or after removal of damaged bases by DNA glycosylases. Besides this key role in DNA repair, APE1 acts as a transcriptional regulator that binds to several transcription factors and regulates their function via both redox-dependent and -independent mechanisms;this requires its non-conserved N-terminal domain. We have shown that APE1 can be acetylated at Lys 6 and 7, which modulates its transcriptional regulatory functions. Later we showed that APE1 inactivation induces apoptosis in conditionally APE1-nullizygous cells;both its DNA repair function and acetylation sites are needed to reverse this effect. Our preliminary data show that the positive charges of these Lys are essential for APE1's interaction with all its binding partners in cells, and neutralizing the positive charges of these Lys residues by acetylation abolishes these interactions. APE1 acetylation also enhances its endonuclease activity and affects total AP-site repair in an in vitro BER assay. Our studies will thus test the hypothesis that the positive charges of the acetylable Lys residues in APE1's N-terminal domain, and their neutralization by acetylation, regulate both its essential DNA repair and transcriptional regulatory functions, and thus play a critical role in cell survival and drug resistance of tumor cells. This hypothesis will be tested in three specific aims.
Aim1 will ask how APE1's acetylation affects its interaction with other proteins or DNA to modulate repair of damaged bases or AP sites, both in vitro and in cells. Using cells stably expressing WT, nonacetylable K6R/K7R or acetylation-mimic K6Q/K7Q APE1, we will test the hypothesis that in the absence of APE1's acetylable Lys residues cells accumulate AP sites or single-strand breaks in the genome, which triggers apoptosis.
Aim2 will ask how AcAPE1 levels are increased in tumor cells after genotoxic drug treatment to alter its complex formation with partner proteins and modulate its DNA repair activity and expression of the drug-efflux transporter protein MDR1 via its transcriptional regulatory function. Using a panel of tumor cell lines we will test for a correlation between AcAPE1 levels and their resistance to chemotherapeutics dugs.
In Aim3, gene expression analysis and ChIP-on-Chip assays, which combine chromatin immunoprecipitation with DNA microarray analysis in cells stably expressing WT or nonacetylable K6R/K7R APE1, will be used to identify target genes that are directly regulated by APE1 acetylation, and the signaling pathways responsible for inducing apoptosis in the absence of APE1 acetylation. Together, our results should advance our understanding of how APE1's acetylation plays a critical role in both DNA damage repair in vivo and in regulation of transcription, which have a profound consequence for cell survival.
APE1 overexpression, often observed in tumor cells, is associated with resistance to various anticancer drugs;its downregulation sensitizes tumor cells to various anti-cancer drugs and so makes it an appropriate target for tumor therapy. Elucidating the molecular basis of how APE1's acetylation controls its essential DNA repair and transcriptional regulatory functions is extremely important from both basic science and clinical perspectives. The results of this project may ultimately lead to small molecules that affect APE1's dual essential functions by blocking its acetylation or interactions with partner proteins, and so, might serve as therapeutics to induce apoptosis in tumor cells.
|Bhakat, Kishor K; Sengupta, Shiladitya; Adeniyi, Victor F et al. (2016) Regulation of limited N-terminal proteolysis of APE1 in tumor via acetylation and its role in cell proliferation. Oncotarget 7:22590-604|
|Sengupta, Shiladitya; Mitra, Sankar; Bhakat, Kishor K (2013) Dual regulatory roles of human AP-endonuclease (APE1/Ref-1) in CDKN1A/p21 expression. PLoS One 8:e68467|
|Sengupta, Shiladitya; Chattopadhyay, Ranajoy; Mantha, Anil K et al. (2012) Regulation of mouse-renin gene by apurinic/apyrimidinic-endonuclease 1 (APE1/Ref-1) via recruitment of histone deacetylase 1 corepressor complex. J Hypertens 30:917-25|