Function Of NME1 In DNA Damage Response

DNA double-strand break(DSB),which happens when two single strands of DNA simultaneously broken at the same or near position,is considered to be the most serious DNA damage.If the damage is unpaired in time or repaired incorrectly,it will lead to chromosome rearrangement,cell instability,tumorigenesis,and cell death.In mammalian cells,DSBs can be repaired by two mechanisms: homologous recombination(HR)and non-homologous end joining(NHEJ).NHEJ as the major pathway includes the following steps: the rapid identification and binding of ku70/80 heterodimers to DNA ends and recruits the DNA-dependent protein kinase catalytic subunit(DNA-PKcs),then Artemis,XRCC4,Ligase IV,XRCC4-like factor(XLF)together with the recent reported PAXX process and ligate DSB ends.Although a large number of important molecules that play a role in the HR or NHEJ pathway have been identified,it is unknown whether there are other key molecules or mechanisms in the DNA repair pathway.NME1(also known as Nm23-H1)was one of the most widely studied tumor metastasis suppressor gene.NME1 has three kinds of enzyme activities: nueleoside diphosphate kinase(NDPK)activity,histidine kinase(hisK)activity and 3?–5?exonuclease(3?–5? EXO)activity.The main function of NME1 is maintain the balance between NDP and NTP in cell,and it can also affects cell proliferation,cell differentiation,apoptosis,and mitosis.At present,the study on NME1 is mainly about its function on growth and migration of tumor cells,and the function of NME1 in DNA double-strand break repair has been rarely studied.In addition,NME1 can autophosphorylates itself and also phosphorylates histidine of other proteins.In recent years,antibodies have been developed to detect the phosphorylation of NME1 histidine,which will facilitate the functional study of its enzyme activity.While the detailed functions of its enzyme activity in double strand break repair,especially in NHEJ pathway is still not clear.This paper mainly studies the functional mechanism of NME1 in DNA double strand break repair.The main contents and conclusions of this paper include the following four aspects:(1)NME1 participates in DNA damage repair.When cells were treated with ionizing radiation or laser microirradiation,it was observed that both the exogenous overexpression NME1 and endogenous NME1 could be recruited to the damage sites at the early stage of damage,which suggest NME1 may participated in DNA damage repair.(2)NME1 affects the efficiency of DNA damage repair.Clonogenic survival assay showing that NME1 depletion causes cellular radiosensitivity.The Immunofluorescence of ?H2AX shown that NME1 deletion impaired DNA damage repair.Furthermore,through HR and NHEJ reporter system,it was found that NME1 affected the efficiency of DNA damage repair mainly through affecting NHEJ pathway.(3)NME1 affects the recruitment of NHEJ pathway protein LIG IV.By knocking down the critical factors in the NHEJ pathway,we found that the recruitment of NME1 was decreased suggesting its recruitment dependent on XRCC4 and its upstream proteins 53BP1,Ku80 and Ku70.Ablation of NME1 significantly downregulated the recruitment of downstream ligase protein LIG IV,indicating that NME1 mainly plays a role in NHEJ pathway through affecting the ligation at DNA break sites.(4)The 3?–5? exonuclease activity of NME1 affects the recruitment of LIG IV.By reconstituted the NME1-depleted cells with wild-type or enzyme activity-deficient mutant NME1,we found that the 3?–5? exonuclease activity of NME1 affects the recruitment of LIG IV.In all,we found that NME1 participates in the repair of DNA double strand break,and mainly affects the recruitment of NHEJ releated protein LIG IV through its 3?–5?exonuclease activity,thus affecting the repair efficiency of NHEJ pathway and ultimately affecting the repair efficiencyof DNA damage.