Chromosome Replication Dynamics in the Archaeon Sulfolobus Acidocaldarius

Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius

Iain G. Duggina,b,1, Simon A. McCalluma, and Stephen D. Bella,b,1

aMedical Research Council Cancer Cell Unit, Hutchison–Medical Research Council Research Centre, Hills Road, Cambridge, CB2 0XZ, United Kingdom; and bSir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, United Kingdom

Edited by Thomas J. Kelly, Memorial Sloan–Kettering Cancer Center, New York, NY, and approved August 15, 2008 (received for review July 2, 2008) The ‘‘baby machine’’ provides a means of generating synchronized The above parameters were established by using asynchronous cultures of minimally perturbed cells. We describe the use of this cultures, but the ability to synchronize the growth and division technique to establish the key cell-cycle parameters of hyperther- cycle of a population of cells is essential for further studies of mophilic archaea of the genus Sulfolobus. The 3 DNA replication chromosome replication and cell cycle. A synchronization origins of Sulfolobus acidocaldarius were mapped by 2D gel method for Sulfolobus acidocaldarius was previously described analysis to near 0 (oriC2), 579 (oriC1), and 1,197 kb (oriC3)onthe that involves an initial treatment of a midlog-phase culture with 2,226-kb circular genome, and we present a direct demonstration 3 mM acetate (2). Cells stop growth and accumulate with a 2N of their activity within the ﬁrst few minutes of a synchronous cell genomic content, suggesting a G2 or stationary phase-like ar- cycle. We also detected X-shaped DNA molecules at the origins in rested state (2, 6). Upon ‘‘release’’ of the cells by resuspension log-phase cells, but these were not directly associated with repli- in fresh growth medium, a fraction of the cells resume growth, cation initiation or ongoing chromosome replication in synchro- resulting in a broad wave of cell division and subsequent nized cells. Whole-genome marker frequency analyses of both chromosome replication very reminiscent of the partially syn- synchronous and log-phase cultures showed that origin utilization chronous growth out from stationary phase (8). Although such was close to 100% for all 3 origins per round of replication. ‘‘arrest and release’’ techniques are simple to do and can be of However, oriC2 was activated slightly later on average compared use for certain experimental approaches, they frequently cause with oriC1 and oriC3. The DNA replication forks moved bidirec- ambiguous or artifactual results, because a cellular response may tionally away from each origin at Ϸ88 bp per second in synchro- be the direct consequence of the treatment or an incomplete cell nous culture. Analysis of the 3 Orc1/Cdc6 initiator proteins showed cycle arrest (at the subcellular level), rather than normal cell a uniformity of cellular abundance and origin binding throughout cycle progression (see refs. 9–11). To synchronize cells at the start of G phase and to avoid the the cell cycle. In contrast, although levels of the MCM helicase were 1 problems associated with arrest and release, we adapted the constant across the cell cycle, its origin localization was regulated, membrane elution or ‘‘baby machine’’ synchronization tech- because it was strongly enriched at all 3 origins in early S phase. nique for high temperature use with Sulfolobus. In the baby ͉ ͉ machine, an asynchronously dividing culture of cells bound to the Archaea cell cycle DNA replication surface of a membrane is continually perfused with medium. As each cell divides, 1 daughter cell remains bound to the mem- uring initiation of chromosome replication, initiator pro- brane and the other is shed into the liquid medium (12). The Dteins bind to and act on DNA replication origins to control liquid eluate thus contains early-G1 synchronized cells that have DNA unwinding for new DNA synthesis. The genomes of the a very high degree of synchrony and are physiologically mini- hyperthermophilic archaeal genus Sulfolobus each encode 3 mally perturbed (see refs. 13 and 14). We have used the initiator proteins: Orc1-1, -2, and -3 (also known as Cdc6-1, -2, technique to study replication origin usage during the early and -3). Like most archaeal proteins involved in DNA replica- stages of chromosome replication in S. acidocaldarius, and we tion, the Orc1 proteins are homologous to their eukaryotic have measured the relative cellular levels of the above initiator counterparts, Orc1 and Cdc6. In Sulfolobus solfataricus, there are proteins and their binding to replication origins throughout the 3 specific origins of replication located around the Ϸ3-Mb single cell cycle. Our findings offer new insights into chromosome circular genome, and the 3 Orc1 proteins bind with distinct replication and the cell cycle in the archaea and open the door patterns and affinities to the different origins (1–3). The Orc1 to unambiguous cell cycle analyses in this domain of life. proteins contain AAAϩ domains, suggesting they probably remodel origin DNA during their ATPase cycle (4). However, Results almost nothing is known about how the origins are regulated. Cell-Cycle Synchronization of S. acidocaldarius. Each baby machine Sulfolobus genomes also contain a gene encoding a homolog experiment was initiated by filtering midlog phase cells onto a of eukaryotic MCM2–7, another AAAϩ domain protein in- membrane. The membrane was then inverted, and medium volved in the initiation and elongation stages of DNA replica- pumped through to culture the cells on the lower surface. Fig. 1A tion. MCM2–7 is thought to be the central component of the shows cell concentrations in the effluent in 3 independent helicase assembly that unwinds DNA for ongoing DNA synthe- experiments initiated with different cell numbers. Overall yields Ͼ 10 sis. Biochemical assays with purified recombinant protein have of cells in the effluent did not greatly increase when 10 demonstrated that Sulfolobus MCM forms a homo-hexamer and

has ATP-dependent helicase activity in vitro (5). However, it is Author contributions: I.G.D. and S.D.B. designed research; I.G.D. and S.A.M. performed still not known whether Sulfolobus MCM functions at replication research; I.G.D. analyzed data; and I.G.D. and S.D.B. wrote the paper. origins and acts as the replicative helicase in vivo. The authors declare no conﬂict of interest. Chromosome replication in Sulfolobus initiates very soon after This article is a PNAS Direct Submission. Յ the preceding cell division, to give a very short G1 phase of 5% 1To whom correspondence may be addressed. E-mail: [email protected] or of the generation time (6). Once chromosome replication is [email protected]. complete (normally taking Ϸ35–45% of the cell cycle), a rela- This article contains supporting information online at www.pnas.org/cgi/content/full/

tively long G2 phase persists until chromosome segregation and 0806414105/DCSupplemental. MICROBIOLOGY cytokinesis complete cell duplication (2, 6, 7). © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806414105 PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16737–16742 Downloaded by guest on September 25, 2021 A D grown at 75 °C in a rotary water bath, and the cell concentration, G2 cell size distribution and DNA content distribution were re- 50 Log phase corded from samples withdrawn at intervals (Fig. 1 C and D).

) G1 S 6 (Pre-babyby The results of these time courses were highly reproducible. The machine)e) mean cell diameter began to increase immediately after the start of the incubation, and it increased linearly throughout the 10 S-phase and the early G2 phase of the first synchronous cell cycle Cells/ml (x10 0 (Fig. 1C). The first round of DNA replication (S phase) also 5 15 started very soon after the experiment was started, with significant progression into S-phase clearly evident at the first time- 30 0 100 200 300 400 500 point (15 min), and it took Ϸ90 min to complete (Fig. 1D). At Elution time (min) 45 135 min, the first cells to divide were becoming evident, because there was both a downturn in the mean cell size of the population Asynchronous 60 B Synchronized (Fig. 1C), and a reappearance of cells with a G1 DNA-content 75 (Fig. 1D). The midpoint of the first wave of cell division of the Ϸ 90 population was 180 min. A second wave of chromosome replication was also in progress in many cells by the 180-min 105 stage, because replication initiates around the time of cytoki- 120 nesis (Fig. 1D). Flow cytometry analysis of longer-term incu- Cell number bated cultures indicated that at least 3 successively poorer quality 135 synchronous rounds of DNA replication were detectable (data 0.6 0.8 1.0 1.2 1.4 Synchronous growth time (min) 150 not shown), and the culture eventually reached a stationary Cell diameter (µm) 165 phase of normal cell density.

180 Mapping S. acidocaldarius Origins of Replication. Three replication C 7 1.06 m) origins in S. acidocaldarius were previously localized to 3 Ϸ50-kb µ 195

) 6 7 1.02 regions around the 2.2-Mb circular genome (2). To examine 5 210 timing of replication origin usage during initiation of chromo- 4 0.98 225 some replication by using baby machine synchronization, we first 3 0.94 sought to map the replication origins to a greater accuracy. The Cells/ml (x10 2 240 replication origins were located by sequence homology to the 0.90 1 Mean cell diameter ( 255 0 100 200 300 400 precisely known S. solfataricus origins (1, 3, 15). Samples of total cellular DNA extracted from a midlog phase culture embedded Synchronous growth time (min) 270 in agarose plugs were digested with restriction enzymes to give 64 128 192 fragments that contained each of the putative origins within DNA-dye fluorescence Ϸ4–7 kb fragments. DNA replication intermediates were ana- Fig. 1. Baby machine synchronization of S. acidocaldarious.(A) Cell numbers lyzed by 2D gel electrophoresis and Southern blot analysis by eluting from the baby machine were recorded with a Coulter counter. Three using probes for the putative origin loci; and for 1 control locus independent experiments were initiated with either 2.5 ϫ 109 (square), 5 ϫ near the expected terminus region (Fig. 2A). Three different 109 (filled circle), or 1.5 ϫ 1010 (triangle) midlog-phase cells. The vertical types of branched DNA structures were identified (Fig. 2A). dashed line and the gray region indicate the optimal time and acceptable time First, the classical ‘‘bubble arc’’ that is representative of DNA period, respectively, for collection. (B) Coulter cell size distributions for asynchronous log phase and synchronized cell populations. (C) Growth of a syn- fragments in various stages of DNA replication after initiation chronized culture was monitored by using a Coulter cell counter/sizer. The cell from an origin within the fragment (see ref. 16) was clearly concentration (filled square) and mean cell diameter (square) are plotted over present for the fragments located at 0 (oriC2), 579 (oriC1), and time. (D) DNA content analysis of cells in the culture in C. Samples were 1,197 kb (oriC3) but not for the control locus at 1,727 kb. Second, withdrawn at the indicated time-points (min) and analyzed by flow cytometry. all 4 Southern blots displayed the common ‘‘fork arc,’’ repre- The relative DNA content of cells in the G1, S, and G2 phases of the cell cycle are senting Y-shaped molecules with a single replication fork at indicated in the log phase prebaby machine sample (Top). various locations in the population of fragments. Third, the origin-containing fragments displayed X-shaped molecules (the ‘‘X-spike’’ on the 2D pattern), consisting of 2 unit-length DNA starting cells were used, and the purity of synchronized cells, segments linked together at various positions. containing Ϸ1 chromosome equivalent, was generally lower when compared with subsaturating experiments. During the Timing of Replication Origin Firing During Synchronous Growth. We early stages of elution the cell concentration declined (Fig. 1A), next sought to analyze replication origin activity during the cell and we observed a relatively poor, but steadily improving, cycle. Samples were withdrawn from a synchronized culture after enrichment of newborn cells (data not shown). After 60–120 0-, 30-, 60-, and 90-min incubation, and DNA was analyzed by 2D min, the cell concentration then steadily increased (Fig. 1A), and gel electrophoresis and Southern blot analysis with a probe for there was significant improvement in the selection for newborn oriC1 (Fig. 3A). There was clear origin activity at 0 min, which cells. The maximum purity of newborn cells was achieved after contains newborn cells (Fig. 1). No origin firing was detected at Ϸ180 min, as judged by analysis of cell size (Fig. 1B) and cellular 30, 60, or 90 min, which suggested that initiation of replication DNA content (Fig. 1D Upper). The proportion of newborn cells in the synchronous population had ended before 30 min, con- in the population was typically Ϸ70% (at 180 min), and the sistent with the flow cytometry in Fig. 1D. Also, the fork arc was modal fluorescence value of the sharp G1 peak in the DNA extremely faint at the latter 3 time points, and the X-spike was content distribution was 49% of that for the G2 peak derived not detectable in any of the Southern blots. Next, we examined from the smaller population of contaminating asynchronous the timing of each origin’s firing at a narrower time window very cells (Fig. 1D, 0 min). We conclude that the baby machine early in the cell cycle. Samples were collected at 0, 2, 6, and 10 produces sharply synchronized cells of S. acidocaldarius. min after starting growth of a baby machine synchronized Synchronized cells collected from the baby machine were culture, and all 3 origins were analyzed by 2D gel electrophoresis

16738 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806414105 Duggin et al. Downloaded by guest on September 25, 2021 Afl II + Nco I A (4.5 kb) A 0 min 30 min 60 min 90 min

oriC1 Afl II Afl II + Nde I (7.2 kb) (4.8 kb) oriC2 (0 kb)

0 min 2 min 6 min 10 min control S. acidocaldarius B (1727 kb) (2226 kb) oriC1 (579 kb)

2D gel: 1st oriC1 Acc I oriC3 (5.2 kb) (1197 kb) 2nd

linear oriC2 B oriC1 region Nhe I Nde I Nhe I probe Afl II Saci725

tRNA-ala RadA Orc1-1 oriC3 oriC2 region Afl II Afl II probe Nco I Afl II Orc1-3

Saci2374 Saci5 Fig. 3. Early firing of S. acidocaldarius replication origins. (A) Analysis of oriC1 activity during S phase. Baby machine synchronized cells were grown for oriC3 region the indicated times before analysis by 2D gel electrophoresis and Southern Acc I probe Acc I blot analysis with detection using an oriC1 probe. (B) Analysis of all 3 origins during the initiation stage of chromosome replication, all Southern blots were Saci1401 Whip Saci1409 generated from samples withdrawn from the same synchronized culture. Restriction enzymes used were: AflII ϩ NheI (oriC1), AflII ϩ NcoI (oriC2) and Fig. 2. Mapping of the 3 S. acidocaldarius origins of chromosome replica- AccI (oriC3). Note that there was Ϸ10-fold less total DNA loaded compared tion. (A) DNA extracted from a midlog-phase culture embedded in agarose with midlog phase samples in Fig. 2. was digested with the indicated restriction enzymes and then separated by neutral-neutral 2D gel electrophoresis followed by Southern blot analysis using probes centred on the indicated genomic locations. An arrow identifies plication of origin regions in all cells, due largely to the presence bubble arcs, where present. Inset illustrates the 2D patterns expected for the of the contaminating population of asynchronous cells that 3 different DNA structures indicated. (B) Maps of 10-kb chromosomal regions encompassing the 3 origins of S. acidocaldarius. The relevant restriction sites would contribute a much lower marker ratio (see below). and probed regions are shown, and a circle indicates the predicted locations Replication fork movement can be clearly seen when comparing of the origins, based on sequence similarity to the precisely defined S. solfa- the 30- and 60-min datasets (Fig. 4A). The average position of taricus origins. each replication fork was taken to be the center point of each transition from maximum to minimum marker ratios. The positions of the 2 forks between oriC2 and oriC3 at 30 and 60 min at each time point (see Fig. 3B). At the 0- and 2-min time points, allowed us to directly calculate the average speed of both forks: obvious bubble arcs were present for all 3 origins, but at the 88 (Ϯ2) bp per second. Two independent experiments are shown 6-and 10-min time points (Fig. 3), the bubble arc had faded very for the 90-min time point in Fig. 4A, demonstrating the repro- significantly. Close examination of the oriC2 2D results suggests ducibility of synchronization. By 90 min, chromosome replica- marginally enhanced persistence of the bubble arc in the 6- and tion was largely completed, as indicated by the essentially flat 10-min time-points. This was investigated further, as described marker ratio distribution along the majority of the chromosome, below. consistent with flow cytometry (Fig. 1D). The only region to remain incompletely replicated at 90 min was a terminus region Chromosome Replication in Synchronized S. acidocaldarius. To quan- oriC2 oriC3 tify origin usage and examine replication fork movement around between and , the 2 origins that are furthest apart the whole chromosome in synchronized culture, we used mi- (see Fig. 2A). croarray based whole-genome marker frequency analysis To investigate the relative usage of origins, we additionally (MFA), which compares the relative copy number of markers carried out a MFA of a standard asynchronous, log-phase from replicating cells with those in nonreplicating cells (from culture. This type of MFA determines the average marker stationary phase). Fig. 4A shows the marker ratios obtained for frequency in a population of cells at different stages of the cell baby-machine synchronized cultures, grown for 0, 10, 30, 60, or cycle. The result, shown in Fig. 4B, shows 3 peaks corresponding 90 min. At 0 min, significant replication was evident around the to the location of the 3 replication origins, as expected (2). The 3 origins, as indicated by the greater ratio for origin-proximal original marker frequency equation (17) was then used to markers compared with other origin-distal markers (note that simulate log-phase conditions in which each origin had either oriC2 is at the breakpoint in the genome sequence; see Fig. 2A). 100%, or a lower probability, of firing during a round of By 10 min, the marker ratio around the origins was significantly replication (Fig. 4C). This approach differs significantly from the greater, approaching 1.5, indicating that further initiation had previous S. acidocaldarius MFA formula (2), which was only occurred during the first 10 min, as expected (see Fig. 3B). The used to distinguish between general single and multiple origin 30- and 60-min time points represent mid-S phase cells, and they usage models and did not take into account the exponential age

showed equivalent maximum ratios of 1.6. This was clearly distribution of the replicating chromosomes (the fact that chro- MICROBIOLOGY significantly Ͻ2.0, which would be expected for complete du- mosomes at early stages of replication are more frequent than

Duggin et al. PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16739 Downloaded by guest on September 25, 2021 A 0 min5 30 60 90 120 150 180 210 0 min5 15 30 60 90 120 150 180 210 15 MCM Orc1-1

TBP TBP

Orc1-2 Orc1-3

TBP TBP

Fig. 5. Orc1-1, -2, -3, and MCM protein levels do not ﬂuctuate signiﬁcantly during the cell cycle. Baby machine synchronized cells were analyzed by Western blot analysis after samples were withdrawn from a culture at the indicated time-points (min), washed, and then cell density was normalized to equivalent absorbencies (600 nm). Blots were reprobed by using anti-TATA- box-binding protein (TBP) anti-serum as a loading control.

whether there were cell-cycle regulated protein level fluctua- tions of the candidate replication initiator proteins Orc1-1, -2, 1–3, and MCM. We withdrew samples from a synchronized culture at regular time intervals and analyzed the relative levels of the 4 proteins by using quantitative Western blot analysis. As can be seen in Fig. 5, the levels of these 4 proteins were uniform throughout the cell cycle and were thus proportional to culture B C biomass (as measured by A600), and not cell cycle position. Replicate experiments using cells collected from the baby machine for a 15-min period at 75 °C before incubation, rather than the normal collection on ice, gave the same results (data not shown).

Association of Initiator Proteins with Replication Origins During the Cell Cycle. To determine whether there were changes in the levels of binding of Orc1-1, -2, -3, and MCM to each of the origins during different cell-cycle phases in vivo, we carried out ChIP assays, in which affinity-purified antibodies were used to isolate Fig. 4. Whole-genome MFA of chromosome replication dynamics in S. specific protein–DNA complexes from extracts of formaldehyde- acidocaldarious. The normalized ratios of replicating to stationary phase DNA treated cells. The relative amount of each origin bound to each hybridization signals are plotted against chromosome position (kb). (A) Cells protein was then measured by quantitative PCR (qPCR). We were collected at the indicated synchronous growth times (six independent observed clear binding patterns for each of the 4 proteins to the experiments) for MFA by microarray hybridization. (B) Data obtained from different origins in asynchronous log-phase cells, as described ϭ analysis of an asynchronous midlog phase culture (A600 0.24). The difference below (Fig. 6A). MCM was clearly detected at all 3 replication in frequency between oriC2 and the other 2 origins was seen consistently in 3 origins but was detected only at very low levels at the control independent experiments. (C) Simulations of log-phase MFA data with a Ϸ fractional S phase length of 0.45 (determined from ﬂow cytometry analysis of locus, lrs14 ( 70 kb from oriC2). The amount of DNA recovered the culture used for the experiment shown in B). The dashed gray line from the MCM ChIP varied for the 3 origins, although influ- simulates 100% usage of all 3 origins, the solid black line simulates 100% encing this would be both the stoichiometry and affinity of the usage of oriC1 and oriC3 and 90% usage of oriC2, whereas the solid gray line various MCM–origin complexes and possible technical factors simulates 80% usage of all 3 origins per round of replication. such as potential differences in antibody accessibility to epitopes and protein–DNA cross-linking efficiency in the various protein– origin complexes. chromosomes at later stages because of continual cell/ The Orc1-1 ChIP reaction in Fig. 6A showed clear binding of chromosome duplication in the population). A good correspon- oriC1, but, unexpectedly, the levels of oriC2 and oriC3 recovered dence between the simulation and the log-phase data were were very low. In the Orc1-2 ChIP reaction, the amount of origin achieved when probabilities of 100% usage for oriC1 and oriC3 DNA recovered was only marginally above background for all 3 and 90% usage for oriC2 were simulated (compare Fig. 4 B with origins. Orc1-3 clearly bound oriC2 (Fig. 6A), but only very low C). Importantly, this differential usage effect could have been levels of the other 2 origins were detected. Western blot analysis caused by a slightly later average replication of oriC2, rather than experiments confirmed that all 4 proteins were efficiently re- a reduced probability of firing per round of replication as covered in these ChIP reactions, and, in a further control for the modeled in Fig. 4C, the log phase data alone do not allow such Orc1-2 ChIP, we detected significant binding around the orc1-2 a distinction to be drawn. However, a comparison of the relative gene promoter region, consistent with previous findings in S. frequency of the origin regions during synchronous growth solfataricus (S.D.B., unpublished results) and demonstrating that reveals that the frequency of oriC2 reaches that of oriC1 and Orc1-2 copurified with at least 1 expected locus. Finally, we oriC3 later in the S phase (Fig. 4A). We conclude that oriC2 has found that the mcm gene was not detected significantly above a slightly later average replication than oriC1 and oriC3, and that background in the MCM ChIP reaction, ruling out coupled it too would have close to 100% efficiency when considered at transcription/translation as a general factor contributing to DNA the level of the whole cell cycle. recovery (data not shown). We suspect that the failure to detect several expected Orc1 interactions that have been observed at Levels of Initiator Proteins Throughout the Cell Cycle. To begin to the homologous S. solfataricus origins by ChIP, footprinting, and study the regulation of origin activity, we wished to determine in a corresponding X-ray crystal structure of an oriC2/Orc1–1/

16740 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0806414105 Duggin et al. Downloaded by guest on September 25, 2021 Orc1–3 subcomplex (1, 3, 4) may reflect a detection limitation of A 0.06 the ChIP reported here. It is clear that the highest affinity Log phase interactions seen in S. solfataricus (1) were those clearly detected 0.05 in the present ChIP study of the S. acidocaldarius homologs. input 0.04 However, we cannot formally exclude the possibility that the ControlMCMOrc1-1Orc1-2Orc1-3 oriC1 0.03 origin binding patterns are significantly different in these 2 oriC2 species. Nevertheless, our aim in the present study was to 0.02 oriC3 determine whether the detected binding patterns varied 0.01 throughout the cell cycle, as described below. lrs14

DNA recovered (fraction of input) DNA 0.00 1 4 4 2 3 1 3 14 14 The results of ChIP experiments using cells collected at 0 (G1), riC s riC riC lrs14o oriC2oriC3 lrs1oriC1oriC2oriC3 lrs1oriC1oriC2oriC3 lr oriC1oriCo lrs o oriC2oriC 30 (S), and 110 min (G2) during synchronous growth are shown Control MCM Orc1-1 Orc1-2 Orc1-3 in Fig. 6B. When the binding patterns were compared between the various time points, we observed that both Orc1–1 binding B 0 min (G1/S) 30 min (mid-S) 110 min (G2) 1 at oriC1 and Orc1–3 binding at oriC2 remained bound at a input - input input ontrol rc1-1 CM rc1-2 constant level throughout the cell cycle, and the levels of binding contrMCMol Orc1Orc1-2Orc1-3 c MCMO Orc1-2Orc1-3 controlM Orc1-1O Orc1-3 were very similar to those seen in the log-phase sample. Signif- oriC1 icantly, however, MCM levels at all 3 origins were significantly oriC2 elevated at the G1 (0 min) time point compared with the oriC3 asynchronous log-phase situation (see Fig. 6B Bottom), strongly lrs14 implicating MCM function at the origins during initiation of replication. The levels of MCM dropped significantly after 0 min, 0.08 MCM ChIP with levels detected at the 30 (S) and 110 min (G2) time points 0.07 similar to the levels seen for the log-phase sample. 0.06 Discussion 0.05 In the current work, we describe the use of the baby machine as 0.04 a means of obtaining a nonchemically perturbed synchronized 0.03 culture of an archaeon. Previous attempts to synchronize ar- 0.02 chaea have relied on either DNA replication inhibitors or 0.01

treatment of cultures with acetate (2, 18, 19). The latter treat- recovered (fraction of input) DNA 0.00 0 0 0 0 0 0 3 10 30 1 30 10 30 10 1 1 1 1 ment leads to arrest of Sulfolobus cells in the G2 phase of the cell lrs14 oriC1 oriC2 oriC3 cycle. The mechanism of the G2 arrest is not yet known (although it may be due to respiration uncoupling; see ref. 20), and thus it Fig. 6. Interactions of the Orc1–1, Orc1–2, Orc1–3 and MCM proteins with is not possible to deconvolute genuine cell cycle effects from replication origins in vivo. (A) DNA recovered from the indicated chromatin acetate-induced perturbations of normal cell physiology. Such immunoprecipitates from a midlog phase cell extract was analyzed by quan- ambiguity is well illustrated by the difference between results titative PCR by using primer sets for oriC1, oriC2, oriC3, and lrs14. The quan- obtained when measuring levels of Orc1 proteins in baby ma- tiﬁed results are displayed in the right-hand panel. (B) Results of reactions using synchronized cell extracts. Quantiﬁed data for the MCM ChIP reactions chine vs. acetate treated cells. We observed in baby machine are shown in Bottom. synchronized cells that Orc1 levels remained constant across the cell cycle (Fig. 5), whereas cells arrested by acetate showed elevated levels of Orc1–2 and low levels of Orc1–1 and Orc1–3 2D gel analysis of DNA replication intermediates from syn- (1); resumption of growth after arrest resulted in elevated chronized cells demonstrated that activation of all 3 replication Orc1–1 and -3 and greatly reduced Orc1–2. Several laboratories origins occurs very early in the cell cycle, as predicted based on have reported microarray analyses of the response of the Sul- flow cytometry analysis of asynchronous cultures (6). Our anal- folobus transcriptome to a range of physiological stresses such as yses also identified important differences between the DNA UV treatment, viral infection, and heat shock (21–24). Interest- replication intermediates detected at origins in log-phase vs. ingly, these studies have revealed modulation of Orc1 gene synchronized cells. In addition to replication initiation ‘‘bubbles’’ transcript and/or protein levels; similar to that seen with mi- at origins, we clearly detected a similar abundance of Y- and croarray analysis of acetate-treated cells (25). It seems likely, X-shaped molecules in log-phase S. acidocaldarius (Fig. 2). The therefore, that the modulation of Orc1/Cdc6 levels induced by Y-shaped molecules were strongly associated with initiation of acetate treatment and subsequent resumption of growth reflects replication and were almost undetectable at oriC1 in mid and a stress response in Sulfolobus cells rather than a true cell cycle late S phase synchronized cells (Fig. 3A). The presence of effect. The origin occupancy of the Orc1/Cdc6 initiator proteins also Y-shaped molecules during initiation of replication at S. acido- did not vary across the cycle, as detected by ChIP (Fig. 6). This caldarius replication origins could be explained by a degree of is reminiscent of the situation in budding yeast where ORC heterogeneity in the timing of each replication fork’s departure remains chromatin-associated through the cell cycle and differs from the origins; the greater relative abundance of the large from higher eukaryotes where ORC1 levels and localization are Y-shaped molecules is consistent with this interpretation (Fig. controlled during the cell cycle (26). In contrast to the invariance 3). In contrast, the X-shaped molecules were not detected during of Orc1 at origins, we observe significant enrichment of MCM initiation of replication in synchronized cultures and were seen at all 3 origins in G1 and early S-phase cells, in strong agreement only in log-phase samples in which much larger quantities of total with the proposed role of archaeal MCM as the replicative DNA were analyzed (Figs. 2A and 3). Thus, the X-shaped helicase. We note that the ChIP analysis of MCM at oriC2 yields molecules seen at replication origins, which appear to be hemi- the greatest recovery of DNA. Although this may be simply due catenanes rather than Holliday junctions in S. solfataricus (3) and to technical reasons, it is also compatible with our observation budding yeast (27) are not directly associated with replication that oriC2 shows delayed kinetics of firing in a significant initiation or fork movement in synchronized S. acidocaldarius.

population of cells. Perhaps this reflects prolonged occupancy of Quantitative analyses of chromosome replication dynamics by MICROBIOLOGY this origin by the MCM protein in prereplicative complexes. MFA revealed that, in both log-phase cells and baby machine

Duggin et al. PNAS ͉ October 28, 2008 ͉ vol. 105 ͉ no. 43 ͉ 16741 Downloaded by guest on September 25, 2021 synchronized cells, a great majority of cells use all 3 origins in ethanol (70% final concentration) and then prepared as described (21). Ap- chromosome replication (Fig. 4). The kinetics of firing suggested proximately 105 cells were analyzed to generate DNA content distributions by a degree of coordination, with the majority of firing occurring using a MoFlo cell sorter (Dako), as described (3). within a few minutes of cytokinesis. This very tight temporal Neutral-Neutral 2D Gel Electrophoresis. 2D gel analysis was performed essen- association between cytokinesis and initiation of chromosome tially as described (1), with modifications described in SI Materials and replication suggests there might be a direct link between cyto- Methods. kinesis and the regulation of origin firing. The MFA of asynchronous cells revealed an underrepresentation of oriC2 se- Marker Frequency Analysis by Microarray Hybridization. Genomic DNA prep- quences compared with oriC1 and oriC3. Interestingly, this aration and labeling, and microarray hybridizations were performed by using phenomenon was apparent in a previous study but was not standard techniques, with modifications described in SI Materials and Meth- commented on (2). The underrepresentation of oriC2 sequences ods. The marker frequency equation was used to generate all marker fre- could be due to either a reduced frequency of usage of oriC2 per quency simulations (equation 4 in ref. 17), as detailed in SI Materials and round of replication or a later average firing of oriC2. Our MFA Methods. data from synchronized cells strongly support the latter possi- Analysis of Protein Levels by Western Blot Analysis. Cells from 5 mL of culture bility, because by 60 min, the relative frequency of the 3 origin were washed with 1 mL of PBS then resuspended in 0.1 mL of PBS, and then peaks had equalized. Thus, oriC2 of S. acidocaldarius can be the A600 was measured and the sample stored at Ϫ20 °C. Thawed samples were considered a somewhat later-firing origin compared with oriC1 diluted with PBS as necessary to give equivalent A600 values for all samples and and oriC3. were then analyzed by SDS/PAGE and Western blot analysis by using standard techniques and enhanced chemiluminescence (Amersham Biosciences) detec- Materials and Methods tion with preflashed autoradiography film. Baby Machine Synchronization of Sulfolobus. The baby machine and synchronization technique were similar to those described (13, 14), with modifications Chromatin Immunoprecipitation and qPCR. Formaldehyde cross-linked cell as described in supporting information (SI) Materials and Methods. extracts were prepared, and immunoprecipitation reactions were carried out essentially as described (1), with modifications described in SI Materials and Methods. Coulter Counting and Flow Cytometry. Cell counting and sizing were done by using a Multisizer 3 Coulter counter with a 20-␮m aperture tube, calibrated using 2-␮m diameter latex beads (Beckman–Coulter). Cells were diluted 1/100 ACKNOWLEDGMENTS. We are grateful to Chris Summerfield and Steve Scotcher from the Medical Research Council engineering workshop (Cam- by using Isoton II dilutent (Beckman-Coulter) that had been filtered by using bridge, U.K.) for construction of the baby machine apparatus, and Charles ␮ a 0.2- m filter unit. Volumetric cell count analysis was carried out within 10 Helmstetter, Kathryn Leigh Eward, Nicholas Robinson, Rachel Samson, Kate min of dilution to avoid any cell volume changes that occurred after extended Michie, and Neville Michie for scientific discussion. This work was funded by periods after dilution. For flow cytometry, cells were fixed with ice-cold the Medical Research Council and the Edward Penley Abraham Trust.

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