Novel Mechanisms Of Human Holliday Junction Resolvase GEN1in Maintaining Genome Stability

Genome instability is a well-known hallmark of cancer. Faithful genome replication, repair of DNA damage, and the precise segregation of chromosomes in mitosis are required for maintenance of genomic stability. A lot of exogenous or endogenous factors can lead to kinds of DNA damage, DNA double strand breaks (DSBs) are the most severe lesions in eukaryotic cells. DSBs can be repaired by non-homologous end joining (NHEJ) and homologous recombination (HR) in mamalian cells. In HR, the resolution of four-way intermediates, also known as Holliday Junctions, is necessary for chromosome segregation. The protein GEN1possesses Holliday junction resolvase activity in vitro, and presumably functions in homology driven repair of DNA double strand breaks. However, the in vivo biological roles of human GEN1is currently unknown.The aim of the reseach is to explore the functions of GEN1and the mechanism of GEN1in regulating genome stability. In the present study we integrated the technologies of cell biology and molecular biology, discovered that GEN1is a novel centrosome associated protein and its deficiency leads to various phenotypes and demonstrated that GEN1regulates genome stability by affecting DNA repair and centrosome duplication. We have identified an N-terminal centrosome localization signal in GEN1, which is required and sufficient for centrosome localization. GEN1knock-down results in aberrant centrosome amplification and multiple spindle poles in mitosis, an increased proportion of cells with multiple nuclei, and a substantial elevated level of apoptotic induction and spontaneous DNA damage. Furthermore, a severely impaired HR was observed in GEN1deficient cells, suggesting that GEN1functions as a Holliday junction resolvase in vivo.In addition, the data acquired by complementation of various GEN1constructs in GEN1-deficient cells revealed that centrosome association, not catalytic activity of GEN1, is required for protecting centrosome from hyperamplification, and preventing formation of multiple mitotic spindles and multi-nucleation. Centrosome association and catalytic activity mutant of GEN1can not prevent spontaneous DNA damage in GEN1kncok down cells. Finally, we showed that ATR mRNA and protein levels in GEN1deficient cells are significantly reduced, which results in impaired ATR/Chk1-mediated checkpoint signaling in response to DNA damage. However, the centrosome function of GEN1is independent on ATR and ATM.In summary, our findings provide novel insight into the functions of GEN1, and discover GEN1can regulate genome stability by affecting centrosome stability, HR repair of DNA double strand breaks(DSBs) and ATR/Chk1mediated signaling.