DNA-dependent protein kinase and XRCC4-DNA ligase IV mobilization in the cell in response to DNA double strand breaks.
Repair of DNA double strand breaks (DSBs) by the non-homologous end joining (NHEJ) pathway in mammals requires at least the DNA-dependent protein kinase (DNA-PK) and the DNA ligase IV-XRCC4 protein complexes. DNA-PK comprises the Ku70/Ku80 heterodimer and the catalytic subunit DNA-PKcs. Here we report the first description of the nuclear mobilization of endogenous NHEJ proteins after exposure of human cells to double strand-breaking agents. DSB infliction specifically induced a dose- and time-dependent mobilization of Ku70/80, DNA-PKcs, XRCC4, and DNA ligase IV proteins from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. XRCC4 recruitment was accompanied by its DNA-PK-dependent phosphorylation. The recruited proteins co-immunoprecipitated, indicating that they had assembled into complexes. However, DNA-PK was attached to chromatin, whereas XRCC4-ligase IV resisted solubilization by DNase I. The rates of appearance and dissolution of NHEJ proteins paralleled that of histone variant H2AX phosphorylation and dephosphorylation. We established that under conditions of genomic DSB infliction 1) Ku recruitment was not dependent on the co-recruitment of the other NHEJ proteins, 2) DNA-PKcs was physically required for the mobilization of the XRCC4-ligase IV complex, 3) DNA ligase IV was physically necessary for stable recruitment of XRCC4, and 4) phosphorylation of either H2AX or XRCC4 was unnecessary for DNA-PK or XRCC4-ligase IV recruitment. Altogether these results offer insights into the interplay between key NHEJ proteins during this repair process in the cell.
Drouet
Jérôme
J
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, 205 route de Narbonne, 31077 Toulouse, Cedex 4, France.
Delteil
Christine
C
Lefrançois
Jacques
J
Concannon
Patrick
P
Salles
Bernard
B
Calsou
Patrick
P
D034961:Antigens, Nuclear / Q000378:metabolism; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000378:metabolism*; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D000072200:Ku Autoantigen; D046912:Multiprotein Complexes / Q000096:biosynthesis; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D021381:Protein Transport
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; C000607893:LIG4 protein, human; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C101467:XRCC4 protein, human; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen; D011088:DNA Ligases; D000072481:DNA Ligase ATP