Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP.
Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.
Karlsson
Karin H
KH
Division of Biomedical Radiation Sciences, Uppsala University, Uppsala, Sweden.
Radulescu
Irina
I
Rydberg
Björn
B
Stenerlöw
Bo
B
D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair / Q000528:radiation effects*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006358:Hot Temperature; D006801:Humans; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013997:Time Factors; D000076105:X-ray Repair Cross Complementing Protein 1
D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases