FUNCTIONAL REGULATION AND NON-CANONICAL ROLES OF DNA REPAIR PROTEINS IN MAMMALIAN CELLS

The Apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein contributing to genome stability, through its central role in the BER pathway of DNA lesions, caused by oxidizing and alkylating agents. It also plays a role in gene expression regulation, as a redox co-activator of several transcription factors and in RNA metabolism. Another interesting and yet poorly characterized function for this non-canonical DNA repair protein is associated to its ability to bind to negative calcium responsive elements (nCaRE) of some gene promoters, thus acting as a transcriptional regulator. Since nCaRE are conserved sequences located within ALU repeats, which are widespread throughout the whole human genome, many other functional nCaRE elements could exist and play a role in the transcriptional regulation of genes. Bioinformatic analyses has been performed systematically looking for functional nCaRE sequences within the human genome by filtering expression profile data of genes resulting deregulated upon APE1 knockdown. Among a list of 57 genes, whose expression is potentially regulated by APE1, attention was focused on deacetylase SIRT1 due to its relevant involvement in cell stress, including senescence, apoptosis, tumorigenesis and, in particular, its role in the cell response to genotoxic agents through its deacetylating activity on APE1 N-domain. Here, it has been showed that the human SIRT1 promoter has two nCaRE elements. Through a multidisciplinary approach, based on SPR, limited proteolysis, ChIP and gene reporter assays, I found that the APE1 N-terminus is required for the stable binding to nCaRE elements. APE1 was demonstrated to be part of a multi-protein complex, that includes hOGG1, Ku70 and RNA Pol II, which plays a central role in the regulatory function on the SIRT1 gene during early response to oxidative stress. These findings provide new insights in the comprehension of the role of nCaRE sequences in the transcriptional regulation of mammalian genes.