The Study Of Long Noncoding RNAs LRIK?ncRNA0301 And XPF Protein Functioning In DNA Damage Response

To maintain the stability of genome,DNA damage response forms during the evolution and is crucial for the life and activity of cells.A large number of DNA damages are produced due to external or internal factors in human cells every day.The timely and correct repair of these damages can prevent the occurrence of genomic aberrations.The unrepaired DNA lesions and incorrectly repaired DNA damages may result in mutations that can cause initiation of cancer and other diseases.Therefore,the understanding of DNA damage response pathways is regarded as one of hot topics and frontiers of life science.Traditional studies of repairing DNA damages have been focused on the role of protein factors in DNA repair pathways.However,with the development of ENCODE programs and high-throughput sequencing technologies,the functions of long noncoding RNAs(> 200 bases)have drawn increasing attention.Through their interaction with proteins,DNA or RNA,long noncoding RNAs involved in a variety of biological processes,such as recruiting protein factors,modulating immune processes,regulating gene transcription and post-transcriptional processes.In this study,we focus on the mechanisms of long noncoding RNAs and proteins functioning in DNA damage response.Several long noncoding RNAs involved in DNA damage repair were identified and investigated to further study their mechanisms:(1)Firstly,the differentially expressed long noncoding RNAs after UV and MMS treatment were detected by DNA microarray assays.The transcription of a long noncoding RNA LRIK is significantly upregulated after UV or MMS treatment.Although knockdown of LRIK increased the UV sensitivity of cells,it did not affect the removal of CPD,indicating that LRIK may play a role in repairing trace but letheal DNA lesions caused by UV.(2)DNA double-strand breaks are regarded as the most severe DNA damage type and are extremely toxic to cells.The transcriptional level of LRIK was increased in response to DSB stress.We found that LRIK interacts with Ku70 and participates in the NHEJ repair pathway.The Ku70/Ku80 heterodimer plays an important role in the DNA lesion recognition and initiation of repair via NHEJ.Knockdown of LRIK affects its binding to Ku heterodimer,and impairs the binding of Ku70/Ku80 to damaged chromatin that influences the recruitment of downstream NHEJ repair proteins such as DNA-PKcs and XRCC4.The reduced recruitment of DNA-PKcs may affect the phosphorylation of H2 AX and the consequnet formation of ?H2AX foci,thus reducing the repair efficiency of DSB,indicating that LRIK is involved in DSB recognition and signaling.(3)LRIK and Ku heterodimer function coordinately in the NHEJ pathway.Knockdown of LRIK did not affect the expression of NHEJ repair factors(Ku70,Ku80,DNA-PKcs and XRCC4).After hindering the interaction of LRIK and Ku70 by expressing LRIK antisense RNA in cells,they displayed significantly increased sensitivity to DSB inducing agent,reduced DSB repair efficiency and impaired ?H2AX foci formation.(4)In response to DNA damage,three protein kinases are able to phosphorylate H2 AX,which are ATM,ATR and DNA-PKcs.It has been found that LRIK does not affect the mRNA or protein levels of ATM and ATR and does not interact with ATM and ATR,suggesting that the recruitment of DNA-PKcs on damaged chromatin affects phosphorylation of H2 AX and the ?H2AX foci formation.(5)Nucleotide excision repair(NER)is the main pathway to repair UV induced DNA damages.The mutations of NER proteins are associated with many serious diseases.A long noncoding RNA(lncRNA)0301 was chosen to investigate whether it functions in NER,since its transcription is only upregulated under UV stress.Further investigation revealed that lncRNA 0301 interacts with MAT1 and CDK7,which constitute subunits of the CAK complex,a subcomplex of TFIIH.Whilst TFIIH plays a dispensible role in NER,hence,knockdown of lncRNA 0301 affects the repair of CPD and the cell cycle after UV treatment.(6)Silver nanoparticles(AgNPs)are widely used routinely and medical treatment due to their antibacterial activity.The uptaken of AgNPs,which are able to generate reactive oxygen species(ROS),can cause genotoxicity to human cells.The XPF knockdown cells were found extremely sensitive to AgNPs by using the colony formation assays.Further studies showed that the dysfunction of XPF induced the accumulation of DNA damage could further activate p53 and its downstream response pathway,indicating that XPF could relieve the DNA damage stress caused by AgNPs.