The Involvement And Mechanism Of ZGRF1 In DNA Double-strand Break HR Repair And G₂/M Cycle Arrest

DNA double-strand breaks(DSBs)is one of the most lethal type of DNA damages,which is mainly repaired by non-homologous end joining(NHEJ)and homologous recombination(HR)pathways in mammalian cells.EXO1-mediated the single-strand resection on DSBs ends plays critical role in HR repair and ATR-CHK1 signaling-induced G₂/M cycle arrest,but the detailed mechanism remains to be elucidated.ZGRF1 was identified as a key helicase involved in DNA damage repair,whereas the mechanistic information is very limited regarding its biological function at present,suggesting that it is worth to extensively explore the molecular process of ZGRF1 in DNA repair.Objective:To confirm the function of ZGRF1 in the repair of DNA damages induced by ionizing radiation;Explore the DNA repair pathways and regulatory points regulated by ZGRF1,the interaction and mutual regulation of ZGRF1 with the known DNA repair proteins;Elucidate the main biological significance of ZGRF1.Methods:(1)Co-immunoprecipitation in combination of mass spectrum analysis was adopted to determine the components of ZGRF1-interacting protein complexes after radiation,and Co-IP and western blotting were applied to verify the interaction between ZGRF1 and DNA repair-related components such as BRCA1 and EXO1,etc.(2)Immunofluorescence(IF)staining was used to detect the co-localization and foci formation between:(a)ZGRF1 and?H2AX(DSBs molecular marker);(b)ZGRF1 and other DNA damage response(DDR)proteins(such as BRCA1,53BP1,MDC1,RNF8 and EXO1)in DSBs sites.(3)IF staining was utilized to observe the formation of RPA2 foci(a marker for DNA break end single-stranded DNA(3'ss DNA)formation)in ZGRF1-knockout(KO)HeLa cells.(4)DR-GFP and EJ-GFP reporter system was employed to analyze the HR and NHEJ repair efficiency of wild-type(WT)and ZGRF1knockout(KO)HeLa cells to determine the DNA repair pathway in which ZGRF1 involved;knock-down of BRCA1,EXO1,BLM separately or in combination knock-down of ZGRF1,to detect the mutual regulation relationship between ZGRF1 and the above-mentioned molecules in the HR repair pathway;(5)Colony formation assay was used to detect the sensitivity of WT and ZGRF1-KO HeLa/MDA-MB-231 cells to irradiation and PARP inhibitor(PARPi)Olarparib,and SR50 index was also used to evaluate the vulnerability of cells to PARPi.(6)MTT assay was employed to analyze the sensitivity of ZGRF1-KO HeLa/MDA-MB-231 cells to other DNA-damage agents(ADR,Cisplatin,ETO,CPT and MMC);(7)Western blotting was utilized to detect the expression level and phosphorylation level of RPA2,CHK1,CHK2 and other proteins after irradiation;(8)Flow cytometry was applied to determine the cell cycle distribution and apoptosis in WT and ZGRF1-KO cells post irradiation,and quantitative statistical analysis of G₂/M phase cells was subsequently performed.Phosphorylated histone H3 Ser10p(p H3)antibody immunostaining was used to mark the mitotic cells in WT and ZGRF1-KO cells after IR,and flow cytometry was utilized to detect the p H3 positive cells,and then statistical analysis was conducted;flow cytometry was applied to determine the proportion of mitotic cells arrested by nocodazole in WT and ZGRF1-KO cells after IR.Results:(1)The interaction of ZGRF1 with DNA repair-related proteins BRCA1 and EXO1 was identified,and their association was enhanced by IR-induced DNA damage.(2)The co-localization of ZGRF1 with?H2AX foci was observed after IR.The residual level of?H2AX foci was much higher in ZGRF1-KO cells 2h after IR as compared with WT cells,indicating that ZGRF1knockout resulted in reduction of DNA double-strand break repair efficiency.Further analysis showed that ZGRF1-knockout led to decrease of HR efficiency by 40%,and when concurrently depleted ZGRF1 and BRCA1(or ZGRF1 and EXO1),there is no significant change on HR efficacy as compared with BRCA1-or EXO1-knockdown alone,but it is slightly lower than knocking down ZGRF1.HR efficiency was decreased more significantly by double-knockdown of ZGRF1 and BLM,and is lower than the repair efficiency of either of the two knock-down cells alone,suggesting that there could be a complementary action on the helicase activity between ZGRG1 and BLM in the HR repair pathway.(3)The recruitment of ZGRF1 to DSBs sites was affected by BRCA1,MDC1 and RNF8(rather than 53BP1).Knock-out of ZGRF1 significantly decreased the formation of RPA2 focus,indicating that ZGRF1 is involved in the regulation of the single-stranded DNA resection at the broken ends of DSBs to form 3'ss DNA;(4)IR-induced phosphorylation of CHK1 and RPA2 was evidently repressed by ZGRF1-knockout,whereas there was no influence on total protein level of CHK1 and RPA2;(5)Compared with WT cells,there was no significant difference in G₂and M phase proportion 1-2h after IR in ZGRF1-KO cells.The percentage of G₂ and M phase cells was significantly enhanced at 4–8h after IR,but increase in ZGRF1-KO cells was much less than that of WT cells,and the percentage was finally recovered to the level of WT cells 12h after IR.Further analysis of M-phase cells indicated that the ratio of mitotic cells was significantly reduced 1h after IR in WT cells,but the reduction appeared at 2h after IR in ZGRF1-KO cells.There was no significant difference on mitotic cell proportion between two cell lines 4h after IR.This indicates that knockout ZGRF1 led to a delay on IR-induced G₂/M arrest;(6)ZGRF1 depression significantly increased the induction of apoptosis by irradiation,and increased the sensitivity of HeLa/MDA-MB-231 cells to DNA damage chemotherapeutics such as ionizing radiation and PARP inhibitor Olaparib,etc.Conclusion:ZGRF1 participates in regulating the homologous recombination repair pathway of DNA double-strand breaks induced by ionizing radiation.ZGRF1 cooperates with EXO1 to mediate the single-strand resection of DNA double-strand break ends to form 3'ss DNA,thereby promoting HR repair and G₂/M cycle arrest.The recruitment of ZGRF1 to DNA damage sites is dependent on BRCA1 and MDC1-RNF8pathways.ZGRF1 may be functionally complementary on the helicase activity with BLM in the HR repair pathway.Furthermore,cervical cancer and breast cancer cell lines were more vulnerable to a series of DNA-damage agents such as PARPi and IR when ZGRF1 was depressed.This research provides new information for elucidating the regulation mechanism of DNA damage repair and cellular radiosensitivity,and also provides potential new target for the treatment of clinical tumors.