Cell, Vol. 11, 719-727, August 1977, Copyright 0 1977 by MIT Mouse L Cell Mitochondrial DNA Molecules Are Selected Randomly for Replication throughout the Cell Cycle Daniel Bogenhagen and David A. Clayton Laboratory of Experimental Oncology Department of Pathology Stanford University School of Medicine Stanford, California 94305 Summary The number of mitochondrial DNA molecules in a cell population doubles at the same rate as the cell generation time. This could occur by a ran- dom selection of molecules for replication or by a process that ensures the replication of each indi- vidual molecule in the cell. We have investigated the rate at which mouse L cell mitochondrial DNA molecules labeled with 3H-thymidine during one round of replication are reselected for a second round of replication. Mouse L cells were labeled with 3H-thymidine for 2 hr, chased for various periods of time and then labeled with 5-bromode- oxyuridine for 4 hr immediately before mitochon- drial DNA isolation. A constant fraction of 3H- thymidine-labeled mitochondrial DNA incorpo- rated B-bromodeoxyuridine after chase intervals ranging from 1.5-22 hr. This result demonstrates that mitochondrial DNA molecules replicated in a short time interval are randomly selected for later rounds of replication, and that replication of mito- chondrial DNA continues throughout the cell cy- cle in mouse L cells. Introduction A mouse L cell contains approximately 1000 mito- chondrial DNA (mtDNA) molecules (Nass, 1969; Bo- genhagen and Clayton, 1974). Since the biosyn- thesis of an individual mtDNA molecule requires approximately 5% of the ceil generation time (Berk and Clayton, 1974; D. Bogenhagen and D. A. Clay- ton, manuscript in preparation), it is important to understand the mechanism whereby the mamma- lian cell coordinates the doubling of its mtDNA content during the cell cycle. It is conceivable that the cell may replicate each mtDNA molecule once and only once in one generation. Alternatively, a few molecules may be selected to replicate early in the cell cycle, and only their progeny may be in- volved in further rounds of replication. The latter situation could result if the complete replication apparatus were contained in only a few of a cell’s mitochondria or if some limiting factor necessary for replication were vertically transmitted to one daughter molecule. The experiments reported here determined whether recently synthesized mtDNA molecules are predisposed for or against re-repli- cation, or whether selection of molecules for repli- cation is a random process. The basic approach in these experiments, as out- lined in Figure 1, is to label mouse LA9 cell mtDNA for 2 hr with 3H-thymidine, then to chase with non- radioactive thymidine for a varied amount of time and finally to label with 5bromodeoxyuridine (BrdUrd) for 4 hr. Buoyant density analyses can then be used to determine the fraction of radioac- tive mtDNA shifted in buoyant density by a second round of replication in the presence of BrdUrd. Since the time required for biosynthesis of a mtDNA molecule is approximately 1 hr, mtDNA molecules which begin replication during the final hr of BrdUrd labeling will not have completed repli- cation before mtDNA isolation. Thus these mole- cules will not be isolated as closed circular mole- cules in the lower band of the propidium diiodide- CsCl gradient. Thus the effective BrdUrd labeling time is approximately 3 hr in this experiment. In principle, this experiment is similar to a continuous BrdUrd labeling with measurement of the produc- tion of “heavy-heavy” molecules, as performed by Flory and Vinograd (1973). The experimental de- sign used here, however, avoids toxicity associated with prolonged exposure of cells to BrdUrd (see Hakala, 1959; Simon, 1961). Moreover, as shown below, both 3H-thymidine and BrdUrd labeling can be performed discretely in LA9 cells without media changes and without interruption of exponential growth. Mathematical analysis of the results is straightforward, since the data can be fitted to a horizontal line rather than to a more complicated exponential expression. If time 0 is defined as the end of the 3H-thymidine labeling period (Figure l), the subsequent increase in the number of mtDNA molecules is given by N(t) = N(O)e”” (CY= In2/Tg; Tg = doubling time = 18 hr). During a 3 hr period of BrdUrd labeling, the in- crease of mtDNA content of the culture is AN = N(t + 3) - N(t) = ,,,(0)eKt+3,1nZ/181 - N(0)et1”2”8 = N(t)(e31nn’is - 1 ) = O.l22N(t). Thus a constant frac- tion, 12.2%, of molecules present at time t will enter replication during a fixed 3 hr period of BrdUrd labeling. If molecules replicated previously in the short period of 3H-thymidine labeling are randomly reselected from the total mtDNA popula- tion for later rounds of replication, the fraction of radioactively labeled mtDNA shifted to hybrid den- sity will be a constant 12.2% regardless of the dura- tion of the intervening chase. The results of this analysis have a direct bearing on the question of cell cycle restrictions on mtDNA synthesis. If mtDNA molecules were replicated in a discrete portion of the cell cycle, one should ob- serve a periodicity with respect to their re-replica-