Study On The Role Of Ubiquitin-conjugating Enzyme RAD6B In UV-induced DNA Damage Repair

Objective:Solar ultraviolet(UV)radiation is a common cause of DNA damage.In normal cells,DNA damage repair system is activated after DNA damage.The damage is repaired in time,the structure of DNA in the cells recovers and continues to replicate normally.The fails of DNA damage repair will induce apoptosis,mutations,aging,and even malignant.RAD6B,an E2 ubiquitin-conjugating enzyme,plays an important role in DNA damage repair,chromatin structure and gene expression regulation.This study investigated the role of RAD6B in UV-induced DNA damage repair.Methods:The 293T cells were cultured and harvested in the logarithmic phrase of growth,RAD6B knockout cells were constructed by CRISPR/Cas9 technology and confirmed by western blotting.DNA damage was induced by UV.The experiments were divided into four groups:sgCON(negative control),sgCON-UV(negative control+UV irradiation),sgRAD6B(RAD6B knockout),sgRAD6B-UV(RAD6B knockout+UV irradiation).The position of RAD6B in UV-induced DNA damage repair pathway was observed by immunofluorescence.Western blotting was used to observe the response of damage factors and explore the substrate of RAD6B.CCK8 was performed to study the sensitivity of RAD6B-deficient cells to UV.Hoechst staining was used to observe the apoptosis of cells from morphology.Results:1.The result of western blotting showed that the expression of RAD6B was significantly decreased in LV-UBE2B-sgRNA-infected cells compared with the negative control(P<0.01).2.Immunofluorescence staining revealed that XPC andγH2AX were recruited to the nucleus after UV irradiation.The expression ofγH2AX was observed by immunoblotting at different doses and time points.As the UV dose increased,the expression ofγH2AX increased,indicating that the extent of cell damage increased with the increase of UV dose,but the expression ofγH2AX reached the highest at 15 J/m~2.And over time after UV(15 J/m~2)irradiation,γH2AX was also gradually increased,the expression ofγH2AX reached the highest at 2 h.The expression of CPD in sgCON and sgRAD6B cells at different time points after UV irradiation was detected by immunofluorescence.The results showed that CPD was recruited within 15 min after UV irradiation.However,RAD6B-deficient cells could still detect the expression of CPD 18h after UV irradiation compared with the negative control cells.3.Immunofluorescence staining showed that MDC1,BRCA1,53BP1 and the DNA damage markerγH2AX colocalized in the nucleus.The results of immunoblotting showed that bothγH2AX and ERCC1 responded after UV irradiation whether RAD6B was knocked out or not.It was also observed that ERCC1entered the nucleus from cytoplasm to repair damage.Subsequently,immunofluorescence technique was used to detect the expression of BRCA1 and 53BP1 in the nucleus.The result showed that BRCA1 and 53BP1 foci were significantly decreased in RAD6B-deficient cells compared with the control group(P<0.01).4.The ubiquitination of H2A in the control group increased(P<0.01),while decreased in the RAD6B-deficient group after UV irradiation(P<0.05).And after UV irradiation,H2B was deubiquitinated in control cells(P<0.01),while there was no significant difference in ubiquitination of H2B in RAD6B-deficient cells.5.The results of CCK8showed that knocking out RAD6B lowered the cell survival.Hoechst staining showed that the apoptotic cells of RAD6B-deficient cells increased after UV treatment.Conclusion:1.RAD6B is involved in UV-induced DNA damage repair.2.In the UV-induced DNA damage repair pathway,γH2AX and ERCC1 are upstream of RAD6B and they recruit RAD6B to the DNA damage site,which triggers the recruitment of downstream DNA damage repair factors such as BRCA1 and 53BP1.3.In UV-induced DNA damage repair,RAD6B may repair the damage by ubiquitinating histones H2A,H2B and H1.2.4.RAD6B deficient cells are more sensitive to UV and increase UV-induced apoptosis.