Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU.
The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.
Garwood
D P
DP
Department of Radiation Oncology, University of California, San Francisco 94143.
Thompson
L L
LL
Dewey
W C
WC
D000818:Animals; D001973:Bromodeoxyuridine / Q000378:metabolism*; D016466:CHO Cells; D002470:Cell Survival / Q000528:radiation effects; D006224:Cricetinae; D004247:DNA / Q000032:analysis / Q000096:biosynthesis / Q000528:radiation effects*; D004249:DNA Damage; D016521:Electrophoresis, Gel, Pulsed-Field; D011836:Radiation Tolerance; D013936:Thymidine / Q000378:metabolism; D014965:X-Rays
D004247:DNA; D001973:Bromodeoxyuridine; D013936:Thymidine