The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies.
Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.
Mansour
Wael Y
WY
Laboratory of Radiobiology & Experimental Radiation Oncology, Department of Radiotherapy and Radiation Oncology, University Medical School Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany. wmansour@uke.uni-hamburg.de
Rhein
Tim
T
Dahm-Daphi
Jochen
J
D000818:Animals; D034961:Antigens, Nuclear; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000172:deficiency; D000072200:Ku Autoantigen; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000502:physiology*; D011839:Radiation, Ionizing; D012689:Sequence Homology, Nucleic Acid
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D000072200:Ku Autoantigen