The Ku-dependent non-homologous end-joining but not other repair pathway is inhibited by high linear energy transfer ionizing radiation.
Ionizing radiation (IR) induced DNA double strand breaks (DSBs) are repaired by both non-homologous end-joining (NHEJ) and homologous recombination repair (HRR) in mammalian cells. The NHEJ repair includes a Ku-dependent main pathway and a PARP-1-dependent complementary pathway. Compared with low linear energy transfer (LET) IR (X or gamma ray) at the same doses, high LET IR (high-charge particles) induces more cell death because of ineffective DNA repair. However, it remains unclear whether high LET IR inhibits all repair or specifically one repair pathway. By combining the assays of clonogenic survival, G2M checkpoint and gammaH2AX in the cell lines with deficiencies in different repair genes, we show here that high LET IR inhibits only the Ku-dependent main NHEJ pathway and does not inhibit either the HRR pathway or the PARP-1-dependent complementary NHEJ pathway. In addition, by developing an assay to detect small fragments of DSB (<400 bp) and by detecting the binding abilities of purified Ku and PARP to different sized dsDNA, we present a possible link for explaining the phenotypes. When compared with low LET IR at the same dose, high LET IR might induce similar yields of DNA DSBs in total but it might induce more small fragments of DNA DSBs (<40 base pairs) that prevent Ku binding efficiently to two ends of one DSB fragment at the same time, thus delaying Ku-dependent repair.
Wang
Hongyan
H
Department of Radiation Oncology and Kimmel Cancer Center of Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Wang
Xiang
X
Zhang
Piyan
P
Wang
Ya
Y
D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000528:radiation effects; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D053938:DNA Fragmentation / Q000528:radiation effects; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D016195:G2 Phase / Q000528:radiation effects; D006657:Histones / Q000378:metabolism; D000072200:Ku Autoantigen; D051379:Mice; D010766:Phosphorylation / Q000528:radiation effects; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics; D011839:Radiation, Ionizing
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D006657:Histones; D011065:Poly(ADP-ribose) Polymerases; C000606510:Xrcc6 protein, mouse; D000072200:Ku Autoantigen