Small polydisperse circular DNA of HeLa cells

Abstract

Small, polydisperse closed-circular DNA has been studied in HeLa cells and has also been found in several other animal cells in culture. The DNA is recovered from ethidium bromide/CsCl gradients from whole-cell extracts or from homogenates from which nuclei and mitochondria have been removed; it is absent from purified nuclei and mitochondria. Exponentially growing cells contain 50 to 200 circles. Cycloheximide treatment results in a 20- to 30-fold increase. A 10- to 20-fold increase is seen in cells which remain at saturation density for two to five days. The size distribution obtained by electron microscopy ranges from 0.1 μm to more than 2 μm, with a mean length of 0.32 μm and a weight-average molecular weight ( M ̄ w ) of 1.0 × 10 6. The analytical band sedimentation patterns were consistent with the electron microscopy size distribution. The weight-average sedimentation coefficients, 14.8 s at pH 8 and 26.5 s at pH 12.5, also indicate an M ̄ w of 1.0 × 10 6. The neutral buoyant density, 1.692 g/ml., indicates a G + C content of 38 mole %. At pH 12.5, the closed DNA exhibits the expected buoyant shift, and is resistant to the first alkali-induced nick for at least 24 hours. The complementary strands are not detectably different in buoyant density at pH 12.5. The average superhelix density of spc-DNA § § Abbreviation used: spc-DNA, small polydisperse circular DNA. Except in Results (Section (g)) this abbreviation is used to designate spc-DNA isolated as closed-circular molecules. is slightly higher than that of mitochondrial DNA of these cells. The genetic complexity of spc-DNA from cycloheximide-treated cells rules out the possibility that circles are composed of subunits having the size of the smaller circles in the population. Cycloheximide was used to increase the amount of spc-DNA in cells first labeled with [ 3H]thymidine and then allowed to grow one generation in unlabeled medium. The specific activities of spc-DNA and nuclear DNA were identical, suggesting that spc-DNA arises from pre-existing nuclear DNA. A possible mechanism for the formation of spc-DNA is discussed. Open circular spc-DNA was also detected in exponentially growing cells, as well as in cycloheximide-treated cells. The amounts in both cases were one-fourth to one-half of the amount in the closed form.