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Coordinating DNA Replication Initiation with Cell Growth

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Coordinating DNA Replication Initiation with Cell Growth Proc. Natl. Acad. Sci. USA Vol. 93, pp. 12206-12211, October 1996 Biochemistry Coordinating DNA replication initiation with cell growth: Differential roles for DnaA and SeqA proteins (Escherichia coli/initiation mass) ERIK BOYE*t, TROND STOKKE*, NANCY KLECKNERt, AND KIRSTEN SKARSTAD* *Departments of Biophysics and Cell Biology, Institute for Cancer Research, Montebello, 0310 Oslo, Norway; and tDepartment of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Contributed by Nancy Kleckner, July 25, 1996 ABSTRACT We describe here the development of a new DnaA recognizes oriC specifically and, together with accessory approach to the analysis of Escherichia coli replication con- proteins, promotes the formation of an open complex which trol. Cells were grown at low growth rates, in which case the permits loading of the DnaB helicase and priming of DNA bacterial cell cycle approximates that of eukaryotic cells with replication (4). A second component, SeqA, has been identi- Gl, S, and G2 phases: cell division is followed sequentially by fied genetically as a negative modulator of the initiation a gap period without DNA replication, replication ofthe single process (5). Biochemical data suggest that SeqA mediates its chromosome, another gap period, and finally the next cell effects by binding to oriC prior to initiation (6). division. Flow cytometry of such slowly growing cells reveals The most detailed molecular models of initiation control in the timing of replication initiation as a function of cell mass. E. coli have focused on the DnaA protein. The amount of The data show that initiation is normally coupled to cell DnaA protein per origin (7), the activity of DnaA protein per physiology extremely tightly: the distribution ofindividual cell cell (8), or the concentration of DnaA (9) have been proposed masses at the time of initiation in wild-type cells is very to trigger initiation of DNA replication. Alternatively, how- narrow, with a coefficient of variation of less than 9%o. ever, genetic interactions between dnaA and seqA mutations Furthermore, a comparison between wild-type and seqA mu- have led to the proposal that DnaA and SeqA act in opposition tant cells shows that initiation occurs at a 10-20% lower mass as a homeostatic pair, together making possible the sensitive in the seqA mutant, providing direct evidence that SeqA is a response of initiation to physiological signals (5). bona fide negative regulator of replication initiation. In Once an origin has undergone initiation, a second process dnaA(Ts) mutants the opposite is found: the mass at initiation precludes the immediate occurrence of another initiation is dramatically increased and the variability in cell mass at event at that origin. After initiation, oriC is placed in a initiation is much higher than that forwild-type cells. In contrast sequestered state which effectively precludes reinitiation for a to wild-type and dnaA(Ts) cells, seqA mutant cells frequently go considerable period of time, about one-third of the cell cycle through two initiation events per cell division cycle, and all the (10-12). oriC contains a number of GATC sites which are origins present in each cell are not initiated in synchrony. The subject to methylation by Dam, the DNA adenine methyltrans- implications for the complex interplay amongst growth, cell ferase. Initiation normally occurs on fully methylated oriC, and division, and DNA replication are discussed. replication of that region converts the GATC sites to the hemi- methylated form. Sequestration requires the interaction of SeqA Normally, cells growing under steady-state conditions dupli- with these hemimethylated GATCs and serves to keep oriC cate their chromosomal complement once and only once hemimethylated and inaccessible for further initiation(s). between each cell division. In any given steady-state culture of Analysis of the coupling between replication initiation and Escherichia coli cells, initiation of DNA replication at the cell physiology in E. coli is complicated by the fact that under chromosomal origin, oriC, occurs at a specific time in the cell standard laboratory conditions-i.e.. rapid growth rates- cycle and at a specific cell mass. Two questions regarding the several rounds of chromosome replication are going on con- relationship between cell growth and initiation are of partic- currently. In the present work we have developed an alterna- ular importance. tive approach to this problem by studying cells that are growing First, how is the timing of initiation coupled to cell growth? so slowly that their cell cycle contains separate periods for An early hypothesis suggested that the initiation process prereplication, DNA replication, and postreplication (13). responds to cell mass per se (1). According to this model, These periods are temporally-analogous to the GI, S, and G2 initiation always occurs at a fixed ratio of mass to origins-i.e., phases of the eukaryotic cell cycle. With such a simple cell the initiation mass is a constant which is independent of the cycle it is possible to examine separately the control of the first growth conditions. More recent evidence suggests, however, initiation event as well as the occurrence of possible extra that the situation is more complicated, since the experimen- initiation events and to determine the relationship of each tally determined initiation mass varies significantly with process to cell mass. By using this approach, we demonstrate growth rate (2, 3). that SeqA and DnaA proteins have opposing roles in the Second, how variable is the mass at the time of initiation, control of replication initiation. when the cells grow under steady-state conditions? The extent of variation indicates the tightness of coupling between initi- MATERIALS AND METHODS ation and cell physiology, ofwhich cell mass is an indicator: the Bacterial Strains and Growth Conditions. The seqA and less the variation, the tighter the coupling. dnaA(Ts) mutants are all derivatives of the E. coli K-12 strain Appropriately controlled replication initiation requires the CM735 metE46 brp-3, his-4, thi-1 galK2 lacYl orlacZ4 mtl-l ara-9 assembly of the replication machinery at oriC. A central tsx-3 ton-i rps-8 or rps-9 supE44 A- (14). The seqAAl0 mutant component of this assembly is the initiator protein DnaA. contains an in-frame deletion which allows transcription/ The publication costs of this article were defrayed in part by page charge Abbreviation: CV, coefficient of variation. payment. This article must therefore be hereby marked "advertisement" in tTo whom reprint requests should be addressed. e-mail: eboye@ accordance with 18 U.S.C. §1734 solely to indicate this fact. labmed.uio.no. 12206 Downloaded by guest on September 26, 2021 Biochemistry: Boye et al. Proc. Natl. Acad. Sci. USA 93 (1996) 12207 translation of the downstream pgm gene (6). The different isogenic dnaA(Ts) mutants were CM740dnaA5, CM742dnaA46, CM748dnaA203, CM2555dnaA508, CM2556dnaA 167, and CM2735dnaA601 (15). Growth was in AB minimal medium (16) supplemented with thiamine, sodium acetate (0.4%), and the required amino acids, Met (20 ,ug/ml), His (22 ,ug/ml), and Trp 1 (20 ,ug/ml). Mass growth was monitored by measuring optical density at 450 nm. ~2 Flow Cytometry. The cells were fixed in ethanol, stained E with ethidium bromide and mithramycin, and analyzed in an Argus flow cytometer (Skatron, Lier, Norway) as described (17). Determinations of the frequency of cells in the different cell cycle phases were performed with a standard program for 0 1 2 analysis of the mammalian cell cycle (Skatron). Cell sorting DNA, genome equivalents was performed in a FACStar flow cytometer (Becton Dick- FIG. 1. A schematic representation of a DNA versus cell mass inson). (scattered light) histogram of slowly-growing E. coli cells. The closed The Coefficient ofVariation (CV) of Cell Mass at Initiation. curve contains all the cells in the measured population, and the dotted line goes through the average values (DNA or scattered light) of the We have taken mass to be represented by scattered light, as distribution. The CV is derived from the width of the mass distribution measured by flow cytometry. The cells were grown in the same and is measured at a given DNA content. The initiation mass (Mi) is medium and have the same shape, and under such conditions determined as the average mass where one-chromosome cell starts to scattered light is a good measure of cell mass (3). increase their DNA content. The distribution of cell mass was determined for each DNA content (i.e., each fluorescence channel) in each histogram. distribution of initiating cells. (iii) The occurrence of an extra The width of the distribution was expressed as the CV, which initiation event in a cell results in a cell with more than two is the standard deviation of a normal distribution divided by its genome equivalents of DNA, and the frequency of such cells mean. Thus, a CV of 10% means that the standard deviation is revealed in a DNA histogram. of the cell mass distribution is 10% of the average cell mass of Initiation Control in Wild-Type Cells. The wild-type strain the distribution. In each histogram, the CV of the mass CM735 was grown in acetate minimal medium at 37°C with a distribution was almost constant, and also lowest, in the region doubling time of 190 min and subjected to flow cytometry. between one and two chromosomes. Cells containing exactly Samples were taken at different times and compared to assure one or two chromosomes were excluded from consideration, that the cultures were growing at steady state. The DNA since such cells are not replicating their DNA but will have histogram demonstrates that the cells contained either one or varying masses due to cell growth. The CV thus obtained was two complete chromosomes or were in the process of repli- taken as an upper estimate of the CV at the time of initiation cating their single chromosome (Fig.
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