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J. Mol. Biol. (1963) 6, 208-213 The Bacterial Chromosome and its Manner of Replication as seen by Autoradiography JOHN CAIRNS Department of Microbiology, Australian National University, Canberra, A.C.T., Australia (Received 19 November 1962) In order to determine the form of replicating DNA, E. coli B3 and K12 Hfr were labelled for various periods with [3H]thymidine. Their DNA was then extracted gently and observed by autoradiography. The results and conclusions can be summarized as follows. (I) The chromosome of E. coli consists of a single piece of two-stranded DNA, 700 to 900 p.long. (2) This DNA duplicates by forming a fork. The new (daughter) limbs of the fork each contain one strand of new material and one strand of old material. (3) Each chromosome length of DNA is probably duplicated by one fork. Thus, when the bacterial generation time is 30 min, 20 to 30 p. of DNA is duplicated each minute. (4) Totally unexpected was the finding that the distal ends of the two daughter molecules appear to be joined during the period of replication. The reason for this is obscure. Conceivably the mechanism that, in vivo, winds the daughter molecules lies at the point of their union rather than, as commonly supposed, in the fork itself. (5) The chromosomes of both B3 (F-) and Kl2 (Hfr) appear to exist as a circle which usually breaks during extraction. 1. Introduction The semiconservative nature of DNA replication, predicted on structural grounds by Watson & Crick (1953), was demonstrated experimentally first for bacterial DNA (Meselson & Stahl, 1958), but how the two strands of the double helix come to separate during replication is not known. There must be formidable complexities to any process which, however feasible energetically (Levinthal & Crane, 1956), at once unwinds one molecule and winds two others. So a study was undertaken of the shape and form of replicating DNA. Bacteria, despite their complexity, promised to be the most accessible source of replicating DNA. Each bacterium in an exponentially growing culture of E. coli makes DNA for more than 80% of the generation time (McFall & Stent, 1959; Schaechter, Bentzon & Maal0e, 1959); if, as seems likely, the bacterial chromosome is a single molecule of DNA, this molecule must be engaged in replication most of the time. A method had already been devised for extracting this DNA with little degradation (Cairns, 1962a) and it seemed probable that, with more care, the chromosome could be isolated intact and, caught in the act of replication, its DNA be displayed by autoradiography. 208 AUTORADIOGRAPHY OF BACTERIAL CHROMOSOMES 209 2. Materials and Methods Bacteria. Since the chromosomes of F- and Hfr bacteria differ in the type of their genetic linkage (Jacoh & Wollman, 1958) and in the manner of their duplication (Nagata, . 1962), two strains of E. coli were used, B3 (F-) (Brenner) and K12 3000 thy- B 1 - (Hfr). Both strains require thymine or thymidine. Medium. The A medium of Meselson & Weigle (1961) was used. To this was added 3 mg /rnl. casein hydrolysate, which had first been largely freed of thymine by steaming with charcoal. In this medium, supplemented with 2 fLgjml. TDR, t both strains have a generation time of 30 min. Preparation of labelled bacteria for autoradiography. The bacteria were grown with aeration to 1o8jml., centrifuged and resuspended in an equal volume of medium containing 2 pgjml. [3H]TDR (9 ejm.mole). In pulse-labelling experiments, incorporation of label was stopped by diluting the bacteria either 50-fold into medium containing 20 fLgjml. TDR or 250-fold into cold 0,15 ~1-NaCI containing 0-01 M·KCN and 0·002% bovine serum albumin. In long-term experiments, the bacteria were labelled for two generations (1 hr) so that roughly half of the DNA would be fully labelled and half would be a hybrid of labelled and unlabelled strands. Lysis of bacteria. Only in a few minor respects has the procedure been altered from that already published (Cairns, 1962a). Labelled bacteria are lysed after dilution to a final concentration of about 1Q4jml. Since it was important in certain experiments to be sure that DNA synthesis did not continue beyond the time the bacteria were sampled, 0·01 M-KCN was added to the lysis medium (1,5 M-sucrose, 0·05 M-NaCI, 0·01 M-EDTA). Various types of cold carrier DNA (4-7 fLgjml. calf thymus, E . coli or T2 DNA) were used at various times without apparently influencing the results. As before, lysis was obtained by dialysis against 1% Duponol C (Dupont, Wilmington, Delaware, U.S.A.) in lysis medium for 2 hr at 37°C. The Duponol was then removed by dialysis for 18 to 24 hr against repeated changes of 0·05 M·NaCl, 0·005 M-EDTA. As before, the DNA was collected on the dialysis membrane (VM Millipore filter, Millipore Filter Corporation, Bedford, Mass., U.S.A.). Autoradiography. As before, Kodak ARlO stripping film was used and the exposure was about 2 months. Thymidine incorporation experiments. To determine whether incorporation was delayed following transfer to a medium containing [3H]TDR, bacteria were grown in cold medium to 5 x 10 Bjml., centrifuged and resuspended in medium containing 2 fLgjml. [3H]TDR (1 ejm-mole). Samples were then removed into cold 5% TCA and washed on Oxoid membrane filters (average pore diameter 0·5 to 1·0 p, Oxo Ltd, London, England) with cold TCA and finally with 1 % acetic acid. The filters were dried, placed in scintillator fluid (0'4% 2,5-diphenyloxazole, 0'01% 1,4-bis-2[5-phenyloxazolyl]-benzene in toluene) and counted in a scintillation counter. 3. Results In interpreting autoradiographs of extracted DNA certain assumptions are necessary. These can be stated at the outset. (1) It is not clear why some molecules of bacterial DNA choose to untangle whereas others do not. However, the few that do are assumed to be a fairly representative sample; specifically we assume that they do not belong to some special class that is being duplicated in some special way. (2) The ratio of mass to length for this untangled DNA is taken to be at least that of DNA in the B configuration, namely 2 x 106 daltonsju (Langridge, Wilson, Hooper, Wilkins & Hamilton, 1960). We assume that single-stranded DNA will not be found in an extended state and so, even if present, will not contribute to the tally of untangled DNA. t Abbreviation used: TDR stands for thymidine. 210 J. CAIRNS (3) The density of grains along these labelled molecules is assumed to be proportional to the amount of incorporated label. Specifically we assume that if one piece of DNA has twice the grain density of another this shows that it is labelled in twice as many strands. (a) Pulse labelling experiments Simple pulse-labelling experiments could tell much about the process of DNA replication. As pointed out already, DNA synthesis in E. coli is virtually continuous. If, therefore, the bacterial chromosome is truly a single piece of two-stranded DNA and if, at any moment, duplication is occurring at only a single point on this molecule, then the length of DNA labelled by a short pulse will be just that fraction of the total length of the chromosome that the duration of the pulse is a fraction of the generation time; if there are several points of simultaneous duplication on the single molecule, or several molecules which are duplicated in parallel, then the length of DNA labelled will be appropriately less. Further, from such pulse experiments it' should be possible to determine whether one or two new strands are being made in each region of replication. 6r---r--.,.---r-,------,--,...-------y----, 7 6/- 15 o x 4 .s .§. 3 :l 2 U o 2345678 Time (min) FIG. 1. Incorporation of aH into cold TCA-insoluble material, following transfer of E. coli B3 to medium containing ["H]TDR. However, first it was necessary to find out how quickly [3H]TDR gets into DNA when the bacteria are transferred from a medium containing cold TDR. The result of such an experiment with E. coli B3 is shown in Fig. 1 (see also Materials and Methods). A similar result was obtained for E. coli K12. So it seems that, under these particular conditions, the error in timing a pulse will be less than 1 minute. Autoradiographs of E. coli B3 DNA were prepared following (i) a 3 minute pulse of [3H]TDR, (ii) a 3 minute pulse, followed by 15 minutes in cold TDR, and (iii) a 6 minute pulse, followed by 15 minutes in cold TDR. Examples of what was found are shown in Plate 1. Similar results were obtained using E. coli K12. From these experiments the following conclusions can be drawn. (1) Immediately after a 3 minute pulse of label (Plate I(a), (b) and (c)) the labelled DNA consists of two pieces lying in fairly close association. Fifteen minutes later (Plate Itd), (e) and (f)) these pieces have moved apart and can be photographed separately. It seems therefore that two labelled molecules are being formed in the region of replication. : e, ,,".- ; e. ,.'/ #~"'r '·.. •· ... ... .. ," " .. . -. . .. ", ~ ' •.. 1 (0 ) ...... , 01 " . . "y. : . o' : :',.# ~ . ' .' .,. •... .. , ~ (b) J ,• • -. ' . "" ..... (e) ." '. ..... 'o.• •• 0' o • • (d) • e, ~. . " " .. .. . tII>.,. "6 " (e) • • .. • • iii> , .., - . .... .", " (f) • ,1. " . . ~ . ' ., .. ... ·t, ~ .. . '. " , (g ) , PLATE 1. Autoradiographs of E. coli B3 DNA labelled by a pulse of [3HJTDR. Exposure time was 61 days. The scale shows 501-" (a), (b), (c): Immediately after a 3 min pulse; (d), (e), (f): 15 min after a 3 min pulse; (g): 15 min after a 6 min pulse. ! l .t .., ~~, . ., - , , " ,., \ ; " • "\ i " i ·t i ! , ! I ., , : \ '. ; I ., f I' ;. I I i : . . } .- .~ , " " I .. # ~ ! \ !' i. ,, \ . J. ',' ,. } s. -, -.V' . .; i' I' (a) (b) J. (c ) (d) PLATE II. Autoradiographs of E. coli B3 (a), (b), (c) and E, coli KI2 (d) DNA following incorporation of [3HJTDR for a period of I hr (two generations). The arrows show the point of replication. Exposure time was 61 days. The scales show 100 p., .: (a) .: -, -r ' . . ..~ . ' ' .. ," . \ "- . . .. - ..':.', 0 -0"~ . .. '.~ ... ~ .. ". .~ -" ':'''''1 . • 'i. (b) d ' .~ .. . x . • t.. ' .. i ., . x· (c ) x (d) PLATE III. Autoradiographs of E. coli B3 (a), (b), (c) and E. coli K12 (d) DNA following incorporation of [3H]TDR for a period of 1 hr. In (b), (c), and (d), the postulated break is marked x - x • Exposure time 61 days. The scales show 100 p.. AUTORADIOGRAPHY OF BACTERIAL CHROMOSOMES 211 (2) There is approximately 1 grain per micron in these labelled regions. Since similarly labelled T2 and A bacteriophage DNA, both of which are known to be largely twostranded, show about twice this grain density (Cairns, 1962b), the two molecules being created at the point of duplication must each be labelled in one strand. (3) A 3 minute pulse labels two pieces of DNA each 60 to 80 p. long (Plate I(a) to (f»; a 6 minute pulse labels about twice this length (Plate I(g». In one generation time (30 minutes) the process responsible for this could cover 600 to 800 p., or slightly more than this if the duration of the pulse is being over-estimated (see Fig. 1). There is unfortunately no precise estimate for the DNA content of the E. coli chromosome. When the generation time is 1 hour, each cell contains 4 x 109 daltons DNA (Hershey & Melechen, 1957). If each cell contained only one nucleus, this value would have to be divided by 1·44 (lfln 2) to correct for continuous DNA synthesis. However, as such cells are usually multinucleate (see Schaechter, Maalee & Kjeldgaard, 1958), this corrected value of 2·8 x 109 daltons (or 1400 p. DNA) must be too high. There is therefore no marked discrepancy between the total length of DNA that has to be duplicated « 1400 p,) and the distance traversed by the replication process in one generation (at least 600 to 800 p.). This suggests that one or at the most two regions of the chromosome are being duplicated at any moment. These various conclusions are reinforced by the results reported in the next section. (b) Two-generation labelling experiments One conspicuous feature of the pulse experiments is not apparent from the photographs and was not listed. The proportion of the labelled DNA that had untangled, and could therefore be measured, was far lower immediately after the 3 minute pulse than 15 minutes later. Thus the replicating region of a DNA molecule is apparently less readily displayed than the rest. It was not surprising therefore that considerable search was necessary before untangled and replicating molecules were found in the two-generation experiments. Since the products of such searches are generally somewhat suspect, the guiding principles of this particular search will be given. As pointed out earlier there are reasons for assigning the hypothetical DNA molecule of E. coli a length of less than 1400 p,. Therefore no molecules were accepted whose length, presumably through breakage, was much less than 700 p.. No replicating (forking) molecules were accepted unless both limbs of their fork were the same length. Lastly many extended n:.olecules were excluded because of the complexity of their form; although perhaps interpretable in terms of a known scheme they could not be used to provide that scheme. Samples of what remained are shown in Plate II. They have been selected to illustrate what appear to be various stages of DNA replication. First, the rough agreement between the observed length of E. coli DNA (up to 900 p.) and the estimated DNA content of its chromosome « 1400 JL) supports the conclusion, arising from the pulse experiments, that this chromosome contains a piece of two-stranded DNA. Second, it seems clear that this DNA replicates by forking and that new material is formed along both limbs of the fork. This latter was shown by the pulse experiments and is confirmed here. In each of the forks shown, one limb plainly has about twice the grain density of the other and of the remainder of the molecule. The simplest hypothesis is that we are watching the conversion of a molecule of hybrid (hot-cold) 212 J. CAIRNS DNA into one hybrid and one fully-substituted (hot-hot) molecule. It is not surprising that, nominally after two generations of labelling, the process is seen at various stages of completion. In Plate Uta), duplication has covered about a sixth of the visible distance in what appear to be two sister molecules. In Plate II(b) and (c), duplication has gone about a third and three-quarters of the distance, respectively. In Plate II(d), duplication is almost complete, about 800 J.I. of DNA having been replicated. So these pictures support what seemed likely from the pulse experiments-namely, that the act of replication proceeds from one end of the molecule to the other. FIG. 2. The consequence of uniting the ends of the replicating fork. The arrows show the direction of rotation, as the parent molecule unwinds and the two daughter molecules are formed (modified from Delbriick & Stent, 1957). The most conspicuous and totally unexpected feature of these pictures has been left for discussion last. In the case of each replicating molecule, the ends of the fork are joined. This complication seems to have been taken one stage further in Plate II(a); here the two limbs of the fork may be joined to each other but they also appear to be joined to their opposite numbers which are being formed from the sister molecule. Conceivably such terminal union of the new double helices (which must be alike in their base sequence) is the artificial consequence of a freedom to unite that only comes with lysis ; this union may not exist inside the. bacterium. Alternatively, terminal union may be the rule during the period of replication. If so, whatever unites the two ends must have the freedom to rotate so that the new helices can rotate as they are formed (Fig . 2). This uniting structure, or swivel, could in fact be the site of the mechanism that, in vivo, spins the parent molecule and its two daughters. (c) The bacterial chromosome The primary object of this work was to determine the form of DNA when it is replicating, not the form of the entire bacterial chromosome. The pictures presented so far give little indication of the latter as they show molecules that are either broken A UTORADIOGRAPHY OF BACTERIAL CHROMOSOMES 213 (Plate II(c» or partly tangled (Plate II(a) and (h)). It seemed possible, however' that out of all the material that had been collected some model for the shape of the whole chromosome might emerge. In searching for such a model that could account for all the kinds of structure seen, the premise was adopted that since excess cold carrier DNA was invariably present these structures must be related to the model by breakage, if needs be, but not by end-to-end aggregation. Granted this premise, there seems to be only one model that Flo. 3. Two stages in the duplication of a circular chromosome. (B) and (D) mark the positions of the breaks postulated to have producedthe structures shownin Plate III(b) and (d), respectively. could generate every structure merely by breakage. This model supposes that the chromosome exists as a circle. Duplication, as in Fig. 2, proceeds by elongation of a loop at the expense of the remainder of the molecule; since, however, the distal end of the molecule is also attached to the swivel, duplication creates a figure 8 each half of which ultimately constitutes a finished daughter molecule (Fig. 3). Depending on how this structure breaks at the time of extraction, it may form a rod with a terminal loop (Plate II), a rod with a subterminal loop (Plate III(c) and (dj), or a circle (Plate III(a) and (bj}; in the case of a circle, the circumference may be up to twice the length of the chromosome. AIl structures seen, including circles of varying circumference, can be readily derived from this model whereas they do not conform to any other obvious scheme. It is, however, possible that the process of duplication may vary; that, for example, the structure shown in Plate II(a) was genuinely an exceptional case . In any event, here as elsewhere no significant difference was detected between the chromosomes of E. ooli B3 (F-) and E. coli K12 (Hfr). I am greatly indebted to Dr . A. D. Hershey and Professor Max Delbriick for helpful criticism and to Miss Rosemary Henry for able technical assistance. REFERENCES Cairns, J. (1962a). J. Mol. Biol. 4, 407. Cairns, J. (1962b). Cold Spr. Barb. Symp. Quant. Biol. 27. In the press. Delbriick, M. & Stent, G. S. (1957). In The Chemical Basi« of Heredity, ed. by W. D. McElroy & B. Glass. Baltimore: Johns Hopkins Press. Hershey, A. D. & Melechen, N. E . (1957). Virology, 3, 207. Jacob, F. & Wollman, E. L. (1958). Symp. Soc. Exp. Biol. 12, 75. Langridge, R., Wilson, H . a., Hooper, C. W., Wilkins, M.H.F. & Hamilton, L.D. (1960). J. Mol. Biol. 2, 19. Levinthal, C. & Crane, H . R. (1956). Proc. Nat. Acad. Sci., Wash. 42, 436. McFall, E. & Stent, G. S. (1959). Biochim. biophys. Acta, 34, 580. Meselson, M. & Stahl, F. W. (1958). Proc. Nat. Acad. Sc i., Wash. 44, 671. Meselson, M. & Weigle, J. J. (1961). Proc. Nat. Acad. Sci., Wash. 47, 857. Nagata, T. (1962). Biochem, Biophys. Res. Comm. 8, 348. Sehaechter, M., Bentzon, M. W. & Maal0e, O. (1959). Nature, 183, 1207. Schaechter, M., Maal0e, O. & Kjeldgaard, N. O. (1958). J. Gen. Microbiol. 19, 592. Watson, J. D. & Crick, F. H. C. (1953). Cold Spr, Harb. Symp. Quant. Biol. 18, 123.