Rates of DNA synthesis during the S-phase of HeLa cells.

J.M Collins

doi:10.1016/s0021-9258(17)34330-2

Abstract

The rates of DNA synthesis were determined for each of two consecutive journeys through S-phase by highly synchronized HeLa cells. Cells at various times after release from the metabolic block were pulsed with [3H]thymidine. The amount of radioactivity in whole cells, purified DNA, and Okazaki fragments provided indexes of the rates of DNA synthesis. Measurements of the average DNA content per cell by the diphenylamine method and the individual DNA content per cell by DNA:propidium fluorescence provided better estimations of the actual rates of DNA synthesis, independent of thymidine metabolism. Unsynchronized cells that had been pulsed with [3H]thymidine were sorted into early, middle, and late S-phase preparations for estimations of the amount of radioactivity per cell. There were differences in the rates predicted by each of the various methods. Rates estimated by fluorescence measurements of DNA content per cell, or by diphenylamine measurements of average DNA content per cell exhibited a pattern of an initial burst, followed by a decreased rate then a final burst. Similar patterns were obtained for the amount of radioactivity in Okazaki fragments, and in early, middle, and late S-phase cells separated from a log-phase culture by electronic cell sorting. Rates estimated by measurements of the amount of radioactivity in whole cells, and the specific activity of purified DNA exhibited a different pattern of an initial slow rate, followed by a maximal rate then a slow rate.

Highlights

The rates of DNA synthesis were determined for each of two consecutive journeys through S-phase by highly synchronized HeLa cells
In an ongoing study of the relationships of the different cell cycle phases to the replication of DNA, we have examined the structure of parental DNA in resting and proliferating cells (Collins, 1974a) and, in various cell cycle phases (Collins, 1977; Collins et al, 1977), the ability of DNA from various phases to serve as a template for heterologous DNA polymerase (Collins, 1974133, the formation of Okazaki-like fragments (Collins, 1975), and the assembly of 4.5 S DNA intermediates into chromosomal sized DNA (Rawles and Collins, 1977)
The overall DNA distributions of each aliquot of cells analyzed by flow microfluorometry were analyzed for the proportions of cells in the G, S, and G2 + M phases of the cell cycle according to two models, one described by Fried (1975, 1977) and one developed at our institution, similar to the trial and error evaluation method described by Gray (1976)) but modified so that the coefficients of variation of Gaussian distributions are not constant, but are allowed to vary (Brunsting et al, 1978)

Summary

PROCEDURES

Shrewsbury, MA 01545) were maintained in spinner culture by daily dilutions with Joklik’s modified. The overall DNA distributions of each aliquot of cells analyzed by flow microfluorometry (described above) were analyzed for the proportions of cells in the G,, S, and G2 + M phases of the cell cycle according to two models, one described by Fried (1975, 1977) and one developed at our institution, similar to the trial and error evaluation method described by Gray (1976)) but modified so that the coefficients of variation of Gaussian distributions are not constant, but are allowed to vary (Brunsting et al, 1978) The use of these modifications in our model will be described in a separate publication. Amount of “H in Whole Cells-The relative rate of DNA synthesis per cell for a 30-min period at tl, tz t,,, was calculated as: amount of “H + number of cells x fraction of S-phase cells, at tl, TV t,. Specific Actiuity-The relative rate of DNA synthesis per cell for a 30-min period was calculated as: specific activity + number of cells x fraction of cells in S-phase, at t,, t2 t,,. Sorting of Labeled Log Phase Cells-The relative rate of DNA synthesis for a 30-min period, during various parts of S-phase, was calculated as: amount of “H in sort + number of cells in sort x fraction of S-phase cells in sort

RESULTS

13 Ii’ 21 25 29

DISCUSSION