NCOA4 inhibits initiation of DNA replication to maintain genome stability

Introduction: The Nuclear Receptor Coactivator 4 (NCOA4) gene is frequently targeted by chromosomal rearrangements in thyroid carcinoma. These events join the 5’-ter of NCOA4 to the DNA sequence encoding the tyrosine kinase (TK) domain of the receptor tyrosine kinase RET, generating the RET/PTC3 chimeric gene, whose protein product displays oncogenic activity. The NCOA4 N-ter mediates homodimerization of the RET TK domain, followed by RET kinase activation and, in turn, gain of transforming activity. Matherials: We used Xenopus laevis egg extracts to study NCOA4 role in DNA replication by performing in vitro DNA replication assays, chromatin pull down and DNA fiber stretching; Mouse embryo fibroblasts (MEFs) from NCOA4+/+ and -/- embryos to study population doublings accumulation in culture, DNA damage and DNA damage response (DDR) activation by immufluorescence staining for γH2AX and 53BP1 and DNA fiber stretching to visualize replication stalling lesions, fork speed and inter-origin distance. Results: Here we show that NCOA4 protein binds MCM7 protein, a component of the MCM2-7 complex involved in DNA replication origin licensing and functioning as the major helicase of the DNA replication fork. In Xenopus laevis egg extracts, both exogenously expressed and endogenous NCOA4 protein inhibit DNA replication by interacting with the MCM2-7 complex and restraining replication origin activation, as shown by in vitro DNA replication assays, chromatin pull-down, and DNA fiber stretching experiments. NCOA4 -/- mouse embryonic fibroblasts (MEFs) undergo premature senescence, characterized by block of cell proliferation and SA-β galactosidase positive staining. This phenotype is characterized by replication stress-associated activation of DDR. Thus, using a DNA fiber stretching assay, we show that NCOA4 depleted cells accumulate replication stalling lesions and feature increased DNA replication origin activation. Conclusions: We conclude that NCOA4 protein is involved in the maintenance of genome integrity by controlling DNA replication , possibly acting as a new genome “caretaker”. Thus RET/PTC3 rearrangement in cancer may act as genetic double-hit by causing simultaneously a gain of oncogenic RET function and a loss of care-taker NCOA4 function.