The Function Of RMI1 In DNA Damage Response And Its Mechanism Study

Objective Preservation of DNA integrity is crucial for the viability and fitness of all living cells and organisms.Maintaining genomic stability is a major challenge,as DNA is subject to incessant attacks from both environmental agents and endogenous metabolic processes.Environmental DNA damage can be produced by physical or chemical sources.Examples of physical genotoxic agents are ionizing radiation(IR)and ultraviolet(UV)light from sunlight which can also induce up to 105 DNA lesions(pyrimidine dimers and photoproducts)per cell per day.IR(from e.g.cosmic radiation and medical treatments employing X-rays or radiotherapy)can induce oxidation of DNA bases and generate single-strand and double-strand DNA breaks(SSBs and DSBs,respectively).The DNA damage response and DNA repair induced by ionizing radiation are complex,and there are a huge number of complex mechanisms to explore.For example,we know little about the molecular mechanisms of RMI1 and BTR(BLM-TopoIIIa-RMI1)complex underlying the DNA damage repair induced by ionizing radiation.In this study,we researched the molecular mechanisms and regulatory role of RMI1 in DNA damage response and DNA damage repair induced by IR.In addition,radiation therapy is an important method of tumor therapy,and radiation resistance is also a difficult problem.Therefore,the molecular regulation mechanism of this study can provide a theoretical basis for finding new targets for tumor radiation sensitizationMethods1.The sensitivity of RMI1 knockdown cells to IR.We utilized lentivirus-mediated shRNA to stably reduce endogenous RMI1,then we compared the colony formation efficiency of Control and RMI1-depleted cells pretreated with increasing dose of IR(0,2,4,6 and 8 Gy).To determine the cells depleted of RMI1 are more sensitive to IR whether be caused by inducing more apoptotic cells.We analyzed the apoptotic cell after exposed to IR by Annexin V-PI staining via FACS.2.The DNA damage repair of RMI1-depleted cells to IR.The level of DNA strand breaks were measured by comet assays at single cell level.To determine the role of RMI1 in DNA damage repair,RMI1 knockdown and control cells were treated withy-rays,and then analyzed the DNA damage at the different time after IR.3.The level of yH2AX and 53BPlfoci in RMI1 depleted cells.We treated control and RMI1 depleted cells with IR or not before visualization of yH2AX and 53BP1 nuclear foci by fluorescent microscope.4.The genome stability of RMI1 depleted cells after exposed to IR.The genome stability was determined by micronucleus test and chromosome aberration assay.We treated control and RMI1 depleted cells with IR.or not,and then analyzed the micronucleus rate and aberrant chromosomes by micronucleus test and chromosome aberration assay,respectively.5.The efficiency of homologous recombination(HR)repair and non-homologous end joining(NHEJ)was investigated in RMI1 depleted cells after exposed to IR.The efficiency of HR was investigated using a U20S direct-repeat green fluorescence protein(DR-GFP),and the efficiency of NHEJ was assessed using U20S EJ5 cells.Cleavage of the gene via I-SceI and subsequent repair via HR or NHEJ results in production of GFP that can be monitored by flow cytometry.6.The DNA damage response of RMI1 knockdown cells to IR.To directly measure the activation of DDR,we analyzed key DDR proteins by Western blot.To further determine whether RMI1 effected the cell cycle distribution,RMI1 depleted cells were treated with IR or not,and then the DNA content was analyzed by FACS.Results1.RMI1 depletion sensitizes cells to IR and induces more apoptosis after IR treatment.2.IR treatment induces accumulates excess DNA damage in RMI1-depleted cells3.IR treatment induces more yH2AX foci and accumulates excess 53BP1 foci in RMI1?depleted cells.4.RMI1 prevents genomic instability after exposed to IR.5.RMI1 knockdown reduces HR efficiency,but the efficiency of NHEJ has no significant effect in RMI1 depleted cells.6.RMI1 depletion activates the cell cycle checkpoint and causes more G2/M delay after IR treatment.Conclusion RMI1 knockdown reduces HR efficiency,impairs DNA damage repair,activates the cell cycle checkpoint and causes more G2/M delay after IR treatment.Data presented in our study indicates that RMI1 plays an important role in DNA damage response induce by IR.Objective Maintenance of genome integrity is critical for both faithful propagation of genetic information and prevention of mutagenesis induced by various DNA damage events.Maintaining genomic stability is a major challenge,as DNA is subject to incessant attacks from both environmental agents and endogenous metabolic processes.Chemical agents used in cancer chemotherapy can cause a variety of DNA lesions:alkylating agents,such as methyl-methane sulfonate(MMS)and temozolomide,attach alkyl groups to DNA bases,while crosslinking agents,such as mitomycin C(MMC),cisplatin,psoralen and nitrogen mustard,introduce covalent links between bases of the same DNA strand(intrastrand crosslinks)or of different DNA strands(interstrand crosslinks or ICLs).Other chemical agents,such as the topoisomerase inhibitors camptothecin(CPT)and etoposide,which inhibit topoisomerase I or II,respectively,induce the formation of SSBs or DSBs by trapping topoisomerase-DNA covalent complexes.RMI1(RecQ-mediated genome instability protein 1)forms a conserved BTR complex with BLM,Topo ??,and RMI2,which is crucial for maintaining genome stability.There are little known about the molecular mechanisms of RMI1 underlying the DNA damage induced by CPT.In our study we analyzed the function and mechanism of RMI1 in regulating DNA damage response to CPT.Methods1.The sensitivity of RMI1 knockdown cells to genotoxic agents.We utilized lentivirus-mediated shRNA to stably reduce endogenous RMI1 then we compared the colony formation efficiency of Control and RMI1-depleted cells pretreated with increasing concentration of MMS or CPT.2.The DNA damage repair of RMI1-depleted cells to CPT.The level of DNA strand breaks were measured by comet assays at single cell level.To determine the role of RMI1 in DNA damage repair,RMI1 knockdown and control cells were treated with CPT,and then analyzed the DNA damage at the different time after CPT treatment.3.The level of yH2AX and 53BPlfoci in RMI1 depleted cells after exposed to CPT.The level of yH2AX and 53BP1 nuclear foci was measured by immunofluorescence.We treated control and RMI1 depleted cells with CPT or not before visualization of yH2AX and 53BP1 nuclear foci by fluorescent microscope.4.The DNA damage response of RMI1 knockdown cells to CPT.To measure the activation of DNA damage response,we analyzed key DNA damage response proteins by Western blot.To further determine whether RMI1 effected the cell cycle distribution,RMI1 depleted cells were treated with CPT or not,and then the DNA content was analyzed by FACS.5.The RAD51 loading after CPT treatment in RMI1 depletion cells.We examined the RAD51 foci formation in RMI1 depletion cells before or after CPT treatment by immunofluorescence,and then analyzed whether the RAD51 could co-localizes with RMI1 in HeLa cells after CPT treatment.In addition,the endogenous RMI1 associates with RAD51 was determined by co-immunoprecipitation.6.The function of RMI 1 in response to the damaged DNA induced by CPT.To further explore the idea that RMI1 plays a role in DNA repair,we exposed HeLa cells to CPT or not.We analyzed the RMI1 foci using immunofluorescence,and measured the protein level of RMI 1 using western blot.7.The ability of RMI 1 to promote DSBs repair whether is dependent of BTR complex.To test whether BTR is required for the ability of RMI 1 promoting DSBs repair,we measured the level of yH2AX foci in HeLa shControl or shRMI1 cells after siRNA-mediated silencing of BLM or Topo ??.Results1.RMI1-depleted HeLa cells are hypersensitive to genotoxic agents.2.CPT treatment induces accumulates excess damaged DNA in RMI 1-depleted cells.3.CPT treatment induces more yH2AX and 53BP1 foci in RMI1-depleted cells.4.RMI1 depletion activates more DNA damge response and causes more G2/M delay after CPT treatment.5.Localization of RAD51 to damaged DNA is defective in RMI1 depleted cells.6.RMI1 forms nuclear foci and protein expression increases in response to DNA damage induced by CPT.7.The ability of RMI 1 to promote DSBs repair is likely to partly dependent of the BTR complex.Conclusion Here we report RMI1 depletion increased hypersensitivity to genotoxic agents,elevated genotoxic stress-induced DSBs,exhibited a stronger activation of DNA damage response,and causes more G2/M delay after camptothecin treatment.On DNA damage,RMI1 forms nuclear foci at the damaged regions,interacts with Rad51 and facilitates the recruitment of RAD51 to initiate homologous recombination.These data reveals the importance of RMI1 in response to DSBs,also provide a basis for the molecular mechanisms of RMI1 to maintain genome stability.Objective RMI1(RecQ-mediated genome instability protein 1)forms a conserved BTR complex with BLM,Topo ??,and RMI2,which is crucial for maintaining genome stability.The absence of functional RMI1 protein causes genome instability,including elevated levels of sister chromatid exchanges.Previous finding has revealed RMI1 localizes to nuclear foci with BLM and Topo ?? in response to replication stress,and RMI1 functions downstream of BLM in promoting replication elongation.Hydroxyurea results in the depletion of deoxynucleoside triphosphates(dNTPs),inhibiting the activity of replicative polymerases on DNAs,uncoupling replicative helicase from polymerases,and then generates excess single stranded DNA(ssDNA).In this study,we analyzed the role of RMI1 in replication stress induced by HU.Methods1.The function of RMI1 in response to DNA replication fork stress.We firstly analyzed the cell cycle distribution of HeLa cells that exposed to hydroxyurea,and then examined the formation of RMI1 nuclear foci in HeLa cells after HU treatment by immunofluorescence.2.The sensitivity of RMI1 knockdown cell to HU.We utilized lentivirus-mediated shRNA to stably knockdown endogenous RMI1,then we compared the colony formation efficiency of Control and RMI1-depleted cells pretreated with increasing concentration of HU.To determine the cells depleted of RMI1 are more sensitive to HU whether be caused by inducing more apoptotic cell.We analyzed the apoptotic cells by Annexin V-PI staining via FACS.3.The DNA damage repair of RMI1-depleted cells to HU.The level of DNA strand breaks were measured by comet assays at single cell level.RMI1 knockdown and control cells were treated with HU,and then analyzed the DNA damage at the different time after HU treatment,to determine the role of RMI1 in DNA damage repair after exposed to HU.4.The DNA damage response of RMI1 knockdown cells to HU.To determine whether RMI1 effected the cell cycle distribution,RMI1 depleted cells were treated with HU or not,and then the DNA content was analyzed by FACS.To further measure the activation of DNA damage response,we analyzed key DNA damage response proteins by Western blot.5.The recruitment of RAD51 to damaged replication fork in RMI1 depleted cells.To further confirm the localization of RAD51 to damaged forks is defective in RMI1 depleted cells,we examined whether RAD51 localizes normally at yH2AX marked damage in RMI1 depleted cells.And then we measured localization of RPA in cells before or after HU treatment with the method of immunofluorescence.Results1.RMI1 forms nuclear foci in response to replication fork stress.2.RMI1-depleted HeLa cells are hypersensitive to HU.3.RMI1-depleted cells accumulate DNA damage during long replication blocks induced by HU.4.RMI1 depletion causes more G2/M delay after HU treatment and exacerbated checkpoint activation.5.RMI1 depletion impaires RAD51 loading to replication forks,accumulates excess RPA foci.Conclusion Here we report that RMI1-depleted cells are hypersensitive to hydroxyurea(HU).Using comet assay,we show that RMI1-depleted cells exhibit accumulation of broken DNAs after release from HU treatment.Moreover,loss of RMI1 results in a failure of RAD51 loading onto DNA damage sites.Finally,we demonstrate that RMI1 facilitates the recovery from activated checkpoint and resuming the cell cycle after replicative stress.These findings reveal the importance of RMI1 in response to replication stress,which could explain the molecular basis for its function in maintaining genome integrity.