Control of DNA replication by NCOA4 protein in HeLa cells

This dissertation is focused on the functional characterization of NCOA4 protein, and in particular on its role in controlling DNA replication in HeLa cells. In human papillary thyroid carcinoma the rearrangement of RET protooncogene, in particular of its tyrosine kinase domain, with the 5ࢠportion of a number of heterologous genes, generates the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent and aggressive variants of these recombination events is the fusion of the intracellular kinase-encoding domain of RET to the first 238 amino acids of a gene named NCOA4 (ELE1/ARA70/RFG), generating the RET/PTC3 oncogene. We previously isolated the Mini-Chromosome Maintenance 7 (MCM7) protein as an interactor of NCOA4. MCM7 is a component of an heteroexameric complex named MCM2-7, considered a master regulator of DNA replication, acting both during DNA replication origins licensing, as part of the Pre-Replication Complex (Pre-RC), and during nascent strand elongation, as the major helicase of the fork. In Xenopus laevis egg extracts NCOA4 blocks DNA replication. Hindrance of DNA replication is mediated by block of MCM2-7 helicase function, leaving untouched origin licensing and activation. Here we show that NCOA4 and MCM7 proteins interact at the endogenous level in HeLa cells and this interaction is confined in the nucleus and takes place preferentially on chromatin. Moreover, in HeLa cells, NCOA4 binds canonical DNA replication origins, such as those prospicient to the c-Myc or to the Lamin B2 gene. RNAi-mediated depletion of NCOA4 accelerates the onset of DNA replication, whereas adoptive NCOA4 overexpression decreases cell growth and DNA synthesis. Our results suggest that NCOA4/RET rearrangement might represent a novel paradigm of a cancer-associated chromosome rearrangement that directly targets a gene that controls cell proliferation.