Roles Of DNA Damage Repair Protein Of The "?H2AX-Chromatin Region" Domain In DNA Double Strand Break Repair

DNA double-strand break(DSBs)are the most serious type among the DNA damage,and if they are not repaired properly in time,the genome of biological organism may become unstable even lead to apoptosis.DNA repair has two main ways:homologous recombination(HR)and non-homologous end joining(NHEJ),HR pathway uses sister chromatid in these periods as template,so it also called "error-free"repair methods.It is suggested that the DNA repair can be performed in two regions:1)the "DNA domain"-consisting of free DSB ends,which is responsible for the most of the DSB repair;2)the "?H2AX-chromatin region"-composed of chromatin regons,cooperates to complete the DSB repair as an accessory pathway.Many DDR factors can be detected at the both sections.The"yH2AX-chromatin region "play an important role in the expansion of the damage response signal and the recruitment of specific repair factors,indicating the possible importance of "?H2AX" chromatin repairing DNA damage.The signal transduction of the "?H2AX-chromatin region" is thought to be due to the direct binding of yH2AX to the tandem BRCT domain of MDC1 and the subsequent phosphorylation of other domains of MDC1 to recruit more DNA damage repair-related proteins to play a repair function.We have noted when H2AX or MDC1 was knocked out,the efficiency of HR repair was greatly reduced 3-5 fold,which indicated that the important role of "?H2AX-chromatin region" in HR repair.There are a number of repair factors involved in HR regulation,but the role of a single repair factor mediated by yH2AX is not well known.In particular,genomic instablity caused by H2AX dificiency is significantly weaker that many defective traits of repair factors(such as BRCA1 and 53BP1)recruited into the chromatin region.Additionally,when the DNA damage repair protein BRCA1 is deleted alone,the HR repair levels decreases significantly and even to death;but the HR repair levels make little change when they were both knocked out,suggesting that there may be an antogonistic or promoting effect among the DNA damage repair-related proteins.Therefore,we put forward a hypothesis that:recruitment of "?H2AX" in chromatin region of many DNA damage repair factor in a specific state or cellur environment,will be released from the usual antagonictic state,participate in the specific environment of homologous recombination function.In order to test the hypothesis,we proposed to focus a single repair factor on "?H2AX-chromatin region" to study the regulation of HR repair by individual repair factors.In this study,three damage repair factors were examined:MRE11?NBS1?XRCC4.MRE11 has 3'-5 'exonuclease activity and single-stranded DNA endonuclease activity.NBS1 is a regulator of MRN complex and is responsible for the recruitment of MRE11 and RAD51.While XRCC4 connect the DNA end directly with XLF/DNA Ligase4 together.Specifically,we used CRISPR/Cas9 gene editing techniques to generate MDC1-/-mouse embryonic stem cell line,which could express the fusion factor.Then,we reconstructed the three repair proteins of MRE11,NBS1 and XRCC4.Each recombination protein was ligated to a tandem BRCT domain and a mutant of tandem BRCT domain.Finally,in order to verify that MRE11/NBS1/XRCC4-BRCT directly binds to ?H2AX,we transfected these fusion factors into U2OS cells and found MRE11-BRCT?XRCC4-BRCT could form foci after lOGy IR.We then performed a flow analysis of these plasmids in MDCl-/-cell#32 with HR reporter system,and it was found that these three repair factors have no HR repair when recruited to the DSB sites.In conclusion,MDCl gene could successfully knocked out by dual-sgRNA technique;the MRE11/XRCC4-BRCT can form foci in DNA damage sites,but the MRE11/NB S1/XRCC4-BRCT all had no homologous recombination repair function in "?H2AX-chromatin region" in this study.