The functional study of negative regulators in DNA damage responses

Abstract

The DNA damage response (DDR) has a pivotal role in safeguarding genome integrity in cells, hence preventing genomic instability, one of the hallmarks of cancer. Here, the current dissertation details parts to two ongoing studies that characterise USP7, RNF169 and DYRK1A as candidate negative regulators of DNA double-strand break (DSB) repair processes. DSBs represent one class of the most deleterious types of DNA damage. Additionally, aberrant expression of USP7, RNF169 or DYRK1A has been detected in several types of cancer. DYRK1A, in particularly, contributes to pathogenesis of Down syndrome and promotes Down syndrome-related acute megakaryoblastic leukaemia (DS-AMkL). In this study, USP7 and RNF169 knockout HeLa cells were generated using the CRISPR-Cas9 approach. From reconstituted cell lines, it was observed that both USP7 and RNF169 confer cellular resistance to poly(ADP-ribose) polymerase inhibitor Olaparib. Given that cells with deficiency in DSB repair by homologous recombination (HR) are hypersensitive to Olaparib, this supports the hypothesis that USP7 and RNF169 promotes DSB repair via the high fidelity HR pathway. Moreover, inactivation of USP7 deubiquitinase (DUB) activity or disruption of the USP7-RNF169 interaction compromised HR repair, suggesting that both USP7 DUB and its interaction with RNF169 are required for HR-dependent DSB repair. Secondly, DYRK1A depletion by RNA interference led to supra-physiological capacity of DSB repair by both HR and non-homologous end joining (NHEJ), while no major change in cell cycle distribution or dynamics of irradiation induced checkpoint response was observed. The findings reflect the suppressive nature of DYRK1A on overall DSB repair, possibly as a result of DYRK1A’s ability to limit the assembly of repair factors at sites of DSBs. Together, the present work encourages further clinical studies on the association of negative DDR regulations and disease pathogenesis.