146
Biochimica et Biophysica Acta, 418 (1976) 146--153
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98494 FORMATION OF NASCENT DNA MOLECULES DURING INHIBITION OF REPLICON INITIATION IN MAMMALIAN CELLS
R.B. PAINTER and B.R. YOUNG Laboratory of Radiobiology, University of California, San Francisco, Calif. 94143 (U.S.A.)
(Received August 1 lth, 1975)
Summary X-irradiation of mammalian cells with moderate doses (100--1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analyzed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant comp o n e n t of mammalian DNA.
Introduction
The possibility t h a t the inhibitory effects of moderate doses of ionizing radiation on DNA synthesis are caused by blockage of replicon initiation was proposed by Van Lancker [1] and by Weiss [2]. Watanabe [3] inferred from DNA fiber autoradiography experiments t h a t initiation of replicons is inhibited by X-radiation in L5178Y cells, and Makino and Okada [4], using velocity sedimentation techniques, obtained evidence that radiation interferes with replicon initiation in the same kind of cells. More recently, we showed [5] that 500 fads of X-radiation inhibits the initiation of replicons in mouse L cells, h u m a n HeLa cells, and Chinese hamster ovary (CHO) cells. In this communica-
147 tion we present further data on the effects of ionizing radiation on DNA replication in these three established cell lines; these data yield valuable information on the organization of the DNA-replicating system in mammalian cells. Materials and Methods
Cells and culture conditions. Mouse L, HeLa $3, and CHO cells, all from stocks used for several years in this laboratory, were grown in petri dishes with Eagle's minimal essential medium [6] supplemented with 15% fetal bovine serum in a 5% CO2/95% air atmosphere saturated with water vapor. Monthly checks assured the absence of Mycoplasma infection. All cultures were incubated for 2 4 h or more with 0.02 pCi/ml of [ 1 4 C ] t h y m i d i n e (50 Ci/mol) before irradiation to label parental DNA. Irradiation procedures. The medium was removed from all cultures and replaced with Saline G [7]. The petri dishes were then removed from the walk-in incubator and irradiated at 25 ° C with 300 kVp X-rays at a dose rate of 250 rads/min. Unirradiated cultures were treated the same as irradiated ones. The dose delivered to the cells was measured with a Victoreen electrometer and with thermoluminescent dosimeters (Eberline Instrument Co.). Immediately after irradiation, the cultures were returned to the 37°C incubator and the medium was placed back on them. In no case were the cultures o u t of the incubator for more than 10 min. Velocity sedimentation analyses. At various times after irradiation, the medium was removed from the cultures and replaced with medium containing 20 pCi/ml of [ 3H] thymidine. Incubation continued for 8 min, and then the radioactive medium was rapidly removed and ice-cold Saline G containing 242 #g/ml of unlabeled thymidine was added to the cells. The cells were washed once with this solution, scraped into it, and counted with a hemocytometer. An aliquot containing a b o u t l 0 s cells (in less than 0.5 ml) was then added directly to 0.5 ml of lysis solution (0.5 M NaOH, 0.02 M EDTA, 0.1% NP40, non-ionic detergent (Shell Oil Co.)} on top of 30 ml of preformed 5--20% alkaline sucrose gradients (0.1 M NaOH, 0.9 M NaC1, 0.01 M EDTA). Lysis of cells was allowed to proceed in the dark at 25°C for 3--6 h, and then the gradients were centrifuged in an SW27 rotor (Beckman) at 27 000 rev./min for 3 h or at 12 000 rev./min for 15 h. This gradient system has been calibrated with phage ~ DNA and phage T4 DNA as described before [8]. A hole was made in the b o t t o m of the centrifuge tube and a b o u t 25 equal-weight fractions were collected. 100 #g of calf thymus DNA were added to each fraction, and all of the DNA was precipitated by adding a solution of ice-cold 6 M HC1 plus 6% sodium pyrophosphate. Each fraction was then filtered through G F / A filters (Whatman) that had been presoaked with 4% HC104. The filters were washed sequentially with ice-cold 4% HC104, 70% alcohol, and 100% alcohol and dried. The 14C and 3H on each filter were determined by liquid scintillation spectrometry (Packard Instrument Co.). Results
Alkaline sucrose gradient profiles of DNA from mouse L cells X-irradiated with 500 or 1000 fads showed very low amounts of radioactivity in fractions
148
! ~_
.--*
Control A__n 5 0 0 Reds v " ' ~ 1000 Reds
6
Biochimica et Biophysica Acta, 418 (1976) 146--153
© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 98494 FORMATION OF NASCENT DNA MOLECULES DURING INHIBITION OF REPLICON INITIATION IN MAMMALIAN CELLS
R.B. PAINTER and B.R. YOUNG Laboratory of Radiobiology, University of California, San Francisco, Calif. 94143 (U.S.A.)
(Received August 1 lth, 1975)
Summary X-irradiation of mammalian cells with moderate doses (100--1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analyzed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant comp o n e n t of mammalian DNA.
Introduction
The possibility t h a t the inhibitory effects of moderate doses of ionizing radiation on DNA synthesis are caused by blockage of replicon initiation was proposed by Van Lancker [1] and by Weiss [2]. Watanabe [3] inferred from DNA fiber autoradiography experiments t h a t initiation of replicons is inhibited by X-radiation in L5178Y cells, and Makino and Okada [4], using velocity sedimentation techniques, obtained evidence that radiation interferes with replicon initiation in the same kind of cells. More recently, we showed [5] that 500 fads of X-radiation inhibits the initiation of replicons in mouse L cells, h u m a n HeLa cells, and Chinese hamster ovary (CHO) cells. In this communica-
147 tion we present further data on the effects of ionizing radiation on DNA replication in these three established cell lines; these data yield valuable information on the organization of the DNA-replicating system in mammalian cells. Materials and Methods
Cells and culture conditions. Mouse L, HeLa $3, and CHO cells, all from stocks used for several years in this laboratory, were grown in petri dishes with Eagle's minimal essential medium [6] supplemented with 15% fetal bovine serum in a 5% CO2/95% air atmosphere saturated with water vapor. Monthly checks assured the absence of Mycoplasma infection. All cultures were incubated for 2 4 h or more with 0.02 pCi/ml of [ 1 4 C ] t h y m i d i n e (50 Ci/mol) before irradiation to label parental DNA. Irradiation procedures. The medium was removed from all cultures and replaced with Saline G [7]. The petri dishes were then removed from the walk-in incubator and irradiated at 25 ° C with 300 kVp X-rays at a dose rate of 250 rads/min. Unirradiated cultures were treated the same as irradiated ones. The dose delivered to the cells was measured with a Victoreen electrometer and with thermoluminescent dosimeters (Eberline Instrument Co.). Immediately after irradiation, the cultures were returned to the 37°C incubator and the medium was placed back on them. In no case were the cultures o u t of the incubator for more than 10 min. Velocity sedimentation analyses. At various times after irradiation, the medium was removed from the cultures and replaced with medium containing 20 pCi/ml of [ 3H] thymidine. Incubation continued for 8 min, and then the radioactive medium was rapidly removed and ice-cold Saline G containing 242 #g/ml of unlabeled thymidine was added to the cells. The cells were washed once with this solution, scraped into it, and counted with a hemocytometer. An aliquot containing a b o u t l 0 s cells (in less than 0.5 ml) was then added directly to 0.5 ml of lysis solution (0.5 M NaOH, 0.02 M EDTA, 0.1% NP40, non-ionic detergent (Shell Oil Co.)} on top of 30 ml of preformed 5--20% alkaline sucrose gradients (0.1 M NaOH, 0.9 M NaC1, 0.01 M EDTA). Lysis of cells was allowed to proceed in the dark at 25°C for 3--6 h, and then the gradients were centrifuged in an SW27 rotor (Beckman) at 27 000 rev./min for 3 h or at 12 000 rev./min for 15 h. This gradient system has been calibrated with phage ~ DNA and phage T4 DNA as described before [8]. A hole was made in the b o t t o m of the centrifuge tube and a b o u t 25 equal-weight fractions were collected. 100 #g of calf thymus DNA were added to each fraction, and all of the DNA was precipitated by adding a solution of ice-cold 6 M HC1 plus 6% sodium pyrophosphate. Each fraction was then filtered through G F / A filters (Whatman) that had been presoaked with 4% HC104. The filters were washed sequentially with ice-cold 4% HC104, 70% alcohol, and 100% alcohol and dried. The 14C and 3H on each filter were determined by liquid scintillation spectrometry (Packard Instrument Co.). Results
Alkaline sucrose gradient profiles of DNA from mouse L cells X-irradiated with 500 or 1000 fads showed very low amounts of radioactivity in fractions
148
! ~_
.--*
Control A__n 5 0 0 Reds v " ' ~ 1000 Reds
6
