The Function And Mechanism Analysis Of USP19in Regulating DNA Damage Response

Cell cycle regulation, especially for the complex and sophisticated regulation duringmitosis, is the key factor to ensure the genome stability. DNA damage is ubiquitousthroughout the cell cycle. When DNA damage occurs, checkpoint can be activated in normalcells to stop the mitosis and start the DNA repair process in normal cells, and mitosis won’trestart until DNA repair complete. If there are cells with checkpoint function or DNA repairdefect, DNA damage will be brought to the daughter cells, with mutation accumulation asmitosis continues, which at last lead to chromosomal instability and tumorigenesis.DNA double strand break is the most crucial form of DNA damage, DNA double strandbreak with incorrect repair may lead to tumorigenesis. There are two repair pathways inresponse to double-strand breaks: homologous recombination(HR) and non-homologous endjoining(NHEJ), the two work together to maintain genomic stability. Inducing DNA damageis often used as an anti-cancer tactics, so DNA repair is essential in cancer treatment. Thedefect in DNA repair make tumor cells sensitive to DNA damage, while when DNA repair isoveractivatied, tumor cells may develop the drug resistant. Meanwhile, the ones with DNArepair defect often get kinds of genetic diseases, and they are apt to suffer from cancer as theygrow older. Above all, targeting DNA damage is an important plot as cancer treatment.The work start from a high-throughput siRNA screening platform with a time-lapse livecell imaging system. The purpose is to find new regulator of mitosis. From the screeningresults we found a deubiquitinating enzyme called USP19involved in mitosis regulation.Knocking down USP19expression leads to a increasing rate of chromosome mis-segregationand “chromosome bridge” formation during anaphase, which reveals disorders in DNAdamage response and DNA repair. Meanwhile, the high rate of chromosome mis-segregationis reported to be an important factor in chromosome instability and tumorigenesis.USP19is a deubiquitinating enzyme which belongs to USP(ubiquitin-specific processingenzyme), it play important roles in hypoxia, ER stress, muscle atrophy, cell proliferation.Currently there is few reports studying on USP19functions, and whether it functions in DNAdamage response or DNA repair has never be concerned. Therefore, study on the function and molecular mechanisms of USP19involving DDR and DNA repair seems to be of greatsignificance.To find out how USP19functions in maintain chromosome stability, we acquired someresults as follow:1We detected the protein level of USP19post IR and found an increase, which indicatedthe participation of USP19in DDR;2It was reported that USP19contains SQ motifs which is preferred sites ofphosphorylation by ATM, ATR. So we tested whether USP19can be phosphorylated post IR.Flag-USP19was transfected and immunoprecipitated with anti-Flag beads, andimmunoblotted with anti-phospho-SQ/TQ antibody. The results indicated USP19can bephosphorylated post IR indeed, what’s more, the phosphorylation can be blocked by PIKKsinhibitor wortmannin, suggesting that the kinase was PIKK.3Silencing USP19leads to a down-regulated of the phosphorylation of γ-H2AX， chk1,chk2and kap-1post IR, along with a G2/M arrest delay. These findings support theinvolvement of USP19in DNA damage response.4Silencing USP19has a direct influence on DNA double strand break repair. Wedetected the effect through HR and NHEJ repair reporter system and found both HR andNHEJ were up-regulated, especially for the NHEJ repair pathway. This finding suggestedUSP19be involved in regulating DNA double strand break repair. One of the causes of“Chromosome bridge” is chromosome ends fusion through NHEJ pathway, so theup-regulated NHEJ repair pathway is consistent with the formation of “chromosome bridge”.Meanwhile, the fact that DNA repair was up-regulated may be related to the mechanism ofdrug resistant of tumors.5We found strong exogenous interactions between USP19and HDAC1or HDAC2,since HDAC1and HDAC2were reported to function on DNA double strand break repair, theinteractions may indicated one of the possible mechanisms of USP19regulating DNA repair.6An bioinformatics analysis revealed that there was a loss of USP19copy numbers insqua cancers and ovian cancers, indicated the functions in tumorigenesis.The work reveals a new function that USP19regulate DNA damage response and DSBrepair, on one hand, USP19may regulate DDR through modification suppression, on theother hand, USP19may mediate DNA repair by regulating HDAC1and HDAC2.USP19is a protein with lots of unknown functions. Our results provide a new insightinto USP19’s role in DNA damage and repair signaling, and indicate new trail intumorigenesis and cancer drug resistant.