Nucleoid halo expansion indirectly measures DNA damage in single cells.
A simple test has been developed that measures how much DNA damage has occurred in a single mammalian cell. The procedure is based on the microscopic examination of "halos" of nucleoids that adhere to coverslips. Nucleoids are produced by flowing salt solutions containing detergents over the attached cells. The nucleoid halos are thought to be a tangle of loops of free DNA that emanate from the remnants of the nucleus. When visualized by staining with ethidium bromide the nucleoid halos first expand, and then contract as the concentration of ethidium increases. Exposure of nucleoids to very low levels of DNA chain-breaking treatments results in the incremental expansion of the halos to a maximum of 15 microns or more. Our assay is based upon quantitating the degree of halo expansion. If intact cells are exposed to DNA-damaging treatments, then allowed increasing periods of post-treatment growth before forming nucleoids, the DNA repair processes result first in expanded and then in contracted halos. By admixing a supercoiled plasma DNA of known length (38 kb) to nucleoids with contracted halos, the fractional halo expansion and the fraction of surviving plasmid supercoils can be measured from the same solution. Use of photodynamic DNA damage showed that the halo expansion was 11.6 times more sensitive than plasmid relaxation. Use of gamma-irradiation showed that the halo expansion was 3.6 times more sensitive than plasmid relaxation. The latter value demonstrates that one break per 137,000 bp results in the expansion of the halos to 63% of their maximal value. We estimate that this method will detect about 5000 breaks per nucleus containing 5 x 10(9) bp.
Thomas
E A
EA
Helicon Foundation Laboratories, San Diego, California 92109.
Thomas
C A
CA
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000187:drug effects / Q000528:radiation effects / Q000648:ultrastructure; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000187:drug effects / Q000528:radiation effects / Q000648:ultrastructure*; D004249:DNA Damage; D003850:Deoxyribonuclease I / Q000494:pharmacology; D004996:Ethidium / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D006367:HeLa Cells; D006801:Humans; D008297:Male
D004247:DNA; D003850:Deoxyribonuclease I; D004996:Ethidium