Functional characterization of NCOA4 protein in Xenopus laevis egg extracts

This dissertation is focused on the functional characterization of NCOA4 protein in Xenopus laevis egg extracts. In human papillary thyroid carcinoma the rearrangement of RET tyrosine kinase (TK) domain with the 5' portion of a number of heterologous genes, generates the RET/papillary thyroid carcinoma (PTC) oncogenes. One of the most frequent variant of RET/PTC rearrangements is the RET/PTC3 oncogene, generated by the fusion of the RET gene TK domain encoding sequence with the first exons and the promoter region of a gene named NCOA4 (ELE1/ARA70/RFG). By yeast two-hybrid system, we previously isolated the Mini-Chromosome Maintenance 7 (MCM7) protein as an NCOA4 protein interactor. MCM7 is a member of the heteroexameric MCM 2-7 complex, a key component of the pre- Replication Complex (pre-RC), and the major helicase of the DNA replication forks. Exogenously added recombinant protein NUS-NCOA4 in activated Xenopus laevis egg extracts is able to inhibit DNA replication by interacting with MCM7 protein and blocking MCM2-7 complex helicase activity. Here we show the cloning and the functional characterization of Xenopus laevis orthologous of NCOA4 gene, XNCOA4? and ?. XNCOA4 and MCM7 proteins interact at the endogenous level in Xenopus laevis egg extracts. In XNCOA4 depleted extracts, DNA replication rate was approximately doubled with an increase of the single strands binding protein RPA and PCNA loading, suggesting an improvement of helicase activity, while Pre-RC (such as Cdc6 and MCM7) or Pre-IC (such as Cdc45 and GINS) components were not affected in their binding to DNA. Finally, we analyzed NCOA4 expression in thyroid cell line and human thyroid carcinomas, observing a reduction of NCOA4 level in few PTC samples and all ATC samples analyzed. Our results suggest that NCOA4 is a novel regulator of DNA replication and NCOA4/RET rearrangement might represent a novel paradigm of a cancer-associated chromosome rearrangement that directly targets a gene that controls DNA replication.