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A new model of DNA replication
J. Theoret. Biol. (1966) 12, 251-259
A New Model of DNA Replication J. DROBN~K AND V. VONDREJS Division of Biophysics, Faculty of Science, Charles University, Prague, Czechoslovakia (Received 16 August 1965, and in revisedform 15 November 1965) The “speedometercable” model of DNA replication is a widely used hypothesis.However, it hasnever beenproven. On the contrary, experimental facts have beenaccumulatingwhich makethis modellessand less likely. Particularly, the postulated rotation of the entire replicating molecule is highly improbable when the bacterial chromosomeis considered.We have suggestedanother hypothesiswhich doesnot involve the rotation of the moleculeand seemsto be consistentwith known experimental observations.It consistsof enzymatic disassembling of one strand which is then transferred piece by pieceand rebuilt in its original shape as a part of a daughter molecule.The other strand remainsavailable for RNA replication. The speedometercable model is presently the most popular theory as to how DNA replicates. The model was formulated on the basisof someexperimental results and several theoretical implications. We shall try to follow the steps leading to this model: (1) The structure suggestedby Watson & Crick (1953a) has been verified by many direct crystallographic measurementsof isolated DNA in fibres (Langridge et al., 1960a,b; Hamilton et a/., 1959) as well as in solution (Luzzati, 1963)and in complexes with proteins (Wilkins, 1956). It is believed that such structure exists also in vivo (Hamilton et al., 1959). (2) The semiconservative distribution of the components of DNA during replication was demonstrated with the use of heavy nitrogen by Meselson & Stahl (1958) and confirmed by other techniques including autoradiography (Cairns, 1963). (3) It has been shown in vitro that the double-stranded structure can be “melted”, i.e., unwound into single strands by various physical treatments (temperature, ionic strength, pH) and that this is reversible (Marmur, Rownd & Shildkraut, 1963). (4) The autoradiographic pattern of replicating circular chromosomes of bacteria showsY-like splitting (Cairns, 1963). 251
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(5) An enzymatic system was isolated which catalyses the formation of high molecular weight DNA from all four nucleoside 5’-triphosphates in the presence of DNA “primer” and Mgzf (Kornberg, 1957). Both strands of the primer are extensively copied during this reaction. These are the principal experimental starting points for the evolution of any model of replication. The speedometer cable model is the logical result of a group of assumptions and implications. (a) The winding observed irz vitro during the physical denaturation and renaturation of double-stranded polynucleotides is believed to be involved also in the enzymatic replication in z~ico. (b) It is also believed that the integrity of both strands must be preserved during the replication because of the semiconservative nature of that process (Baldwin, 1964). (c) If the preceding conditions are granted the Watson & Crick (19536) structure implies the speedometer cable type of rotation because of topological reasons. Some theoretical calculations have indicated that: (d) There is enough energy available to drive the rotation of a rod-like cylinder with dimensions similar to a DNA molecule in a medium of cytoplasmic viscosity (Levinthal & Crane, 1956) (see, however, further discussion). (e) It is possible to derive a physico-chemical model of this type of replication which would be able to complete the chromosome duplication within the time period observed in viao (Fong, 1964a,b). One other assumption with indirect consequences to this problem should be mentioned : (f) The specifity of hydrogen bonding is high enough to ensure proper mating of purines and pyrimidines. No specific aid from a third componente.g., enzyme-is needed for this purpose (Watson & Crick, 1953b). There are, how’ever, some serious arguments against the speedometer cable model. (i) The rotating body is not limited to the naked molecule of DNA: counterions, the hydration shell, the RNA replicase system, including newly formed RNA, repressors, etc., must rotate with the DNA. (ii) The driving motor must be situated at the point of replication because there is a source of energy (triphosphates) and because of the necessity for synchronizing the speed of rotation and the rate of synthesis. (iii) It has been established that the circular chromosome of at least some bacteria (chromosome length 1 to 2 mm) is replicated from one point in one direction within the cell (cell size about a few ~1)(Cairns, 1963; Bonhoeffer & Gierer, 1963; Nagata, 1963a,b).
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(iv) Further, the situation is even more complicated after thymine starvation when several growing points are present on one chromosome (Hayes, 1965). (v) Considering the first and the third points, we come to the conclusion that it is questionable whether there is enough energy to support the rotation of the chromosome. An energy of l-2 x lo-” Cal/rev. was derived (Levinthal 8c Crane, 1956) using the total length of 20 p, radius 10 A, viscosity 1 CP and the speed 60 rev./set. Using more realistic values for the chromosome of Escherichiu coli, i.e., length of 1000 p viscosity of 4 cP, radius of 10 A (which is underestimated) and speed of 200 rev./set, we obtain about 10-l’ Cal/rev. This figure is the lowest estimate because the effective radius is greater (see point (i)) and the multiply folded “speedometer cable” needs more energy to rotate than a simple cylinder-like body rotating around its own axis. In principle there are two ways to avoid these difficulties and construct an alternative model of replication which would still be consistent with known experimental results. One can assume that: (A) The speedometer cable type of replication is limited to subunits of the chromosome: the chromosome dissociates into several parts which are duplicated separately and then aggregate again (Sarkar, Mukundan & Devi, 1963); there are some single bonds inserted into the double-stranded molecule which provide for free rotation of shorter segments (Crothers, 1964). Either of these mechanisms must involve some break, unusual linkage, or nonnucleotide element (Bendich & Rosenkranz, 1963). The explanation of how such a “swivel” is replicated provides serious problems. Or, one can assume that: (B) Semiconservative distribution is the result of highly organized enzymatic uncoupling, transfer and rejoining of single nucleotides or groups thereof, within the “old” strand rather than the result of continous preservation of the strand integrity. We present a model which demonstrates the possibility of an enzymatic disassembling and recombining of the deoxyribosephosphate backbone of one strand as a mechanism for DNA replication without unwinding. The Principle of Proposed Mechanism We propose an enzymatic system (reduplicase) attached to one strand of each individual DNA molecule-for example, to the (+) strand (Fig. 1). This strand is disassembled during the replication process and its building blocks-nucleotides-are transferred piece by piece to the daughter molecule where they are rejoined, forming the “old” or “conservative” part of it. The opposite strand of the parent molecule (-) is not involved in the duphcaT.B. 17
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FIG. 1. The possible mechanism of DNA replication model (see text). S, deoxyribose; P, phosphate.
according to the “split-rejoin”
tion process (except, directing the specifity of complementary bases) and preserves its integrity all the time, remaining free for RNA replication, repression, etc. This is consistent with the fact that replication of RNA does not affect the DNA reduplication in any regard; this is, the reprimed parts of DNA are reduplicated with the same rate as the most active ones. The opposite is also true: there is no indication that the RNA replication is obstructed in the region of the growing point of the chromosome. This (-) strand forms the “old” part of the other daughter molecule. The “new” strands of both daughter molecules are polymerized as complements of old strands. All three parts-both daughter molecules and parent molecule--are fixed in space; only the single nucleotide being transferred rotates. The result of such reduplication are two daughter molecules lying parallel to each other. Therefore they can be easily separated even if the parent molecule had a rather complicated tertiary structure. It is possible to suggest many details of this general mechanism. Each has some advantages and weak points. Because no direct experimental data are available we cannot specify the mechanism in more detail. Most probably
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the hydrogen bonds of the C-c pair (Fig. 1) are released under the influence of the enzyme which allows the base c to rotate out of the helix. It is then bonded to the 5’-triphosphate of the complementary base C’. This new C’ nucleotide is attached to the growing new part of the daughter molecule, releasing PPi (this step is shown in Fig. 1). The c-nucleotide is liberated from the parent molecule by pyrophosphorolysis with the formation of a 5’triphosphate end. The base pair c-C’ rotates into the proper position, the c-nucleotide is rejoined with the “old” part of the daughter molecule and PPi is released. Alternatively, the transfer of the c-nucleotide may be accomplished by the enzyme alone without involvement of the C’ complementary nucleotide. The latter may be added later or not at all as in the case of single-stranded phages. The removed c-nucleotide is then replaced by new c’-5’-triphosphate which is linked to the new part of the molecule. The pyrophosphate necessary for the pyrophosphorolysis is probably attached to the enzyme or protected by it from hydrolysis by pyrophosphatase. We cannot specify at present the polarity of the strands. Thus, if the residue X in Fig. 1 is a 3’-OH group, Y signifies 5’-triphosphate and vice versa. We would prefer the former alternative. Then the additon of new triphosphate to the 5’ end would be accomplished in such situation (base C’ bonded to c linking in this way both parts of the molecule) which cannot be at present simulated in vitro. This might be the reason why the addition to the 5’ end of the primer strand has never been observed in vitro. This alternative was chosen for Fig. 2 where the geometry of the transfer is shown from the top view. Discussion It is very difficult to suggest an experimental proof of this mechanism. According to our model, there should be only two to three reduplicase complexes in the growing microbial cell, firmly attached to the growing point of the chromosome and simultaneously to the mesosomes and cytoplasmatic membrane (Jacob, Brenner & Cuzin, 1963). Each “purification” of such a complex means its degradation into individual subunits. The results of Hanawalt & Ray (1964) show that the growing point of the bacterial chromosome makes a complex with a protein (or lipoprotein) which is resistant to normal deproteinization procedure. Therefore the enzymes obtained by means of typical purification procedures are probably the subunits and/or the “repair” enzymes as known from the “cut-and-patch” dark reactivation processes (Setlow, 1964; Howard-Flanders, 1965; Stacey, 1965). On the other hand, these enzymes active in repair might be the building blocks from which the reduplicase complex is made. There are some data which support our model:
I.
I
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FIG. 2. The section in the growing point in the plane of the c--C base pair (see Fig. 1). The co-ordinates of the atoms are taken from model 3 of DNA proposed by Langridge cr al. (19606). I, Parent double-stranded molecule; II, daughter double-stranded molecule. O,, O’,, The points of rotation (which coincide approximately with the P-O,,, bonds) in the replication of the ith base pair. Starting point: (heavy lines). Original position of the base pair c-C in the parent molecule. 1st step: rotation of the base c around the point 0,. 2nd step (light lines): addition of the complementary triphosphate of the base C’ and substitution of the C’ nucleotide into the daughter molecule with the release of PPi. 3rd step (dotted lines): pyrophosphorolysis of the bond between parent molecule and the c nucleotide followed by the rotation of the c-C’ base pair around the point O’, to the final position in the daughter molecule.
(a) An exonuclease activity accompanies the DNA polymerase through many steps of purification in various systems (Oleson & Koerner, 1964; Korn & Weissbach, 1964; Baldwin, 1964). This might reflect the stepwise dissociation of the complex. (b) The pyrophosphorolytic activity of the DNA polymerase was demonstrated in vitro (Bessman, Lehman, Simms & Kornberg, 1958) where it needs a high concentration of free inorganic pyrophosphate. Assuming that the pyrophosphate is attached in uiro to the reduplicasecomplex, we can imagine that its effective concentration in the proper place is raised enough to cause the pyrophosphorolysis of the proper band. If phosphorolysis is alternatively suggested,the transfer mechanismshould deal with the diphosphates instead of triphosphates.
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(c) The recombination in viral and bacterial chromosomes might be easily explained on the basis of our model: it involves the very close attachment of two double-stranded molecules of DNA. This contact should be very specific to bring corresponding markers together. Further, the high negative interference known in phages can be understood if the contact lasts for a time sufficient for the replication of several markers to be accomplished. The London forces as suggested by Jehle, Parke, Shirven & Aein (1964) might provide a physical explanation on such a contact in our model but can hardly Speedometer
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a
I 8 G, + 4 G,, 12 12A,
:6Ap
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9G,+3GeU 12
6G,+6G,,
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FIG. 3. The alternative responses of the isolated growing point of a bacterial chromosome to the enzymatic digestion of proteins in the experiments described by Hanawalt & Ray (1964). Light lines represent the DNA strands labelled by thymine-[3H]thymine, heavy lines the strands doubly labelled by 5-bromouracil and 3zP. The density and activity of fragments are given at the bottom assuming that the average lengths of a, b and c are the same. The density is calculated from the density of light parts (GT) and heavy parts (Ga”). The ratio of 3H and 32P activity is given as A, and A,, respectively.
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be applied to polymer molecules rotating at several hundred revolutions per second. Once the contact occurs, the transfer of building blocks to the wrong molecule might readily occur. The analysis of the DNA in its replicative form gives only a small chance to discriminate the speedometer cable model from the “split-rejoin” mechanism suggested above. There exists, however, a small difference: in the speedometer cable model the intergrity of both strands is preserved so that both daughter molecules are covalently bonded to the parent molecule. In our split-rejoin model only one daughter molecule is directly covalently bonded; and the other is attached to the rest by means of hydrogen bonds and/or the reduplicase complex itself. Thus the type of experiment described by Hanawalt & Ray (1964) is of great interest. The growing point is obtained in the form shown in Fig. 3. Let us suppose that by random breaks the segments a, b and c are, on the average, of equal length. Thus, in the case of the split-rejoin mechanism two new fractions should occur after deproteinization by enzymes: one heavy with 1 : 1 labelling with 32P and 3H, the other of intermediate density with 1 : 3 labelling. The examination of Hanawalt’s results shows that fractions can be detected (Figs 5(b) and 6(a) in Hanawalt & Ray, 1964) which would correspond to the latter. Nevertheless, this result is not straightforward because the maxima of density are too close together, some traces of proteins still attached to the DNA might change the density, and, even when the experiments are very carefully made, a small amount of shearing force may cause preferential breakage at the branching point even in the case of the speedometer cable type of replication. Therefore, more experimental data are necessary. REFERENCES BALDWIN, R. L. (1964). The Bacteria, vol. V, p. 327. New York: Academic Press. BENDICH,A. & ROSENKRANZ,H. S. (1963). Progress in Nucleic Acid Research, vol. 1, p. 219. New York: Academic Press. BESSMAN,M. J., LEHMAN, 1. R., SIMMS, E. S. & KORNBERG, A. (1958). J. Eiol. Gem. 233, 171. BONHOEFFER, F. & GIERER, A. (1963). J. molec. Biol. 7, 534. CAIRNS, J. (1963). J. molec. Biol. 6, 208. CAIRNS, J. (1963). Cold Spring Harb. Symp. quant. Biol. 28, 43. CRICK, P. H. C. (1963). Progress in Nucleic Acid Research, vol. 1, p. 164. New York: Academic Press. CROIXERS, D. M. (1964). J. molec. Biol. 9, 712. FONG, P. (19644. Proc..natn. Acad. Sci. U.S.A. 52, 239. FONG. P. (1964b). Proc. natn. Aead. Sci. U.S.A. 52. 641. HAMILTON;
L. k,
BARCLAY,
R. K.,
WILKINS,
M. H.
P., BROWN,
G. L., WILSON,
H. R.,
MARVIN, D. A., EPHRUW-TEYLOR, H. & SIMMONS, N. S. (1959). J. biophys. biochem. Cytol. 5, 397. HANAWALT, P. C. & RAY, D. S. (1964). Proc. nafn. Acad. Sci. U.S.A. 52, 125.
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HAYES, W. (1965). Symp. Sot. Gem Microbial. XV, 294. Cambridge University Press. HOWARD-FLANDERS, P. Private communication to STACEY, K. A. (1965). Symp. Sot. Gen. Microbial. XV, 159. Cambridge University Press. JACOB, F., BRENNER, S. & CUZIN, F. (1963). Cold Spring Harb. Symp. quant. Biol. 28,329. JEHLE, H., PARKE, W. C., SHIRVEN, R. M. & AEIN, D. (1964). Biopolymers, Symp. 1, 209. KORN, D. & WEISSBACH,A. (1964). J. biol. Chem. 239, 3849. KORNBERG, A. (1957). Adv. Enzymol. 18, 191. LANGRIDGE, R., WILSON, H. R., HOOPER, C. W., WILKINS, Ma H. F. & HAMILTON, L. D. (1960~). J. molec. Biol. 2, 19. LANGRIDGE, R., MARVIN, D. A., SEEDS,W. E., WILSON, H. R., HOOPER, C. W., WILKINS, M. H. F. & HAMILTON, L. D. (19606). J. molec. Biol. 2, 38. LEVINTHAL, C. & CRANE, H. R. (1956). Proc. natn. Acad. Sci. U.S.A. 42,436. LUZZA~, V. (1963). Progress in Nucleic Acid Research, vol. 1, p. 347. New York : Academic Press. MARMUR, J., ROWND, R. & SCHILDKRAUT, C. L. (1963). Progress in Nucleic Acid Research, vol. 1, p. 232. New York: Academic Press. MESELSON, M. & STAHL, F. W. (1958). Proc. Natn. Acad. Sci. U.S.A. 44, 671. NAGATA, T. (1963a). Proc. Natn. Acad. Sci. U.S.A. 49, 551. NAGATA, T. (19636). Cold Spring Harb. Symp. quant. Biol. 28, 55. OLESON, A. E. & KOERNER, J. F. (1964). J. biol. Chem. 239,2935. SARKAR, N. K., MUKUNDAN, M. A. & DEVI, A. (1963). Nature, Land. 200, 1205. Smow, R. B. (1964). .I. Cell. camp. Physiol. Suppl. 1, 64, 51. STACEY, K. A. (1965). Symp. Sot. Gen. Microbial. 15, 159. Cambridge University Press. WATSON, J. D. & CRICK, F. H. C. (1953a). Nature, Lond. 171,737. WATSON, J. D. & CRICK, F. H. C. (19536) Cold Spring Harb. Symp. quant. Biol. 28, 123 WILKINS, M. H. F. (1956). Cold Spring Harb. Symp. quant. Biol. 21, 75.
A New Model of DNA Replication J. DROBN~K AND V. VONDREJS Division of Biophysics, Faculty of Science, Charles University, Prague, Czechoslovakia (Received 16 August 1965, and in revisedform 15 November 1965) The “speedometercable” model of DNA replication is a widely used hypothesis.However, it hasnever beenproven. On the contrary, experimental facts have beenaccumulatingwhich makethis modellessand less likely. Particularly, the postulated rotation of the entire replicating molecule is highly improbable when the bacterial chromosomeis considered.We have suggestedanother hypothesiswhich doesnot involve the rotation of the moleculeand seemsto be consistentwith known experimental observations.It consistsof enzymatic disassembling of one strand which is then transferred piece by pieceand rebuilt in its original shape as a part of a daughter molecule.The other strand remainsavailable for RNA replication. The speedometercable model is presently the most popular theory as to how DNA replicates. The model was formulated on the basisof someexperimental results and several theoretical implications. We shall try to follow the steps leading to this model: (1) The structure suggestedby Watson & Crick (1953a) has been verified by many direct crystallographic measurementsof isolated DNA in fibres (Langridge et al., 1960a,b; Hamilton et a/., 1959) as well as in solution (Luzzati, 1963)and in complexes with proteins (Wilkins, 1956). It is believed that such structure exists also in vivo (Hamilton et al., 1959). (2) The semiconservative distribution of the components of DNA during replication was demonstrated with the use of heavy nitrogen by Meselson & Stahl (1958) and confirmed by other techniques including autoradiography (Cairns, 1963). (3) It has been shown in vitro that the double-stranded structure can be “melted”, i.e., unwound into single strands by various physical treatments (temperature, ionic strength, pH) and that this is reversible (Marmur, Rownd & Shildkraut, 1963). (4) The autoradiographic pattern of replicating circular chromosomes of bacteria showsY-like splitting (Cairns, 1963). 251
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(5) An enzymatic system was isolated which catalyses the formation of high molecular weight DNA from all four nucleoside 5’-triphosphates in the presence of DNA “primer” and Mgzf (Kornberg, 1957). Both strands of the primer are extensively copied during this reaction. These are the principal experimental starting points for the evolution of any model of replication. The speedometer cable model is the logical result of a group of assumptions and implications. (a) The winding observed irz vitro during the physical denaturation and renaturation of double-stranded polynucleotides is believed to be involved also in the enzymatic replication in z~ico. (b) It is also believed that the integrity of both strands must be preserved during the replication because of the semiconservative nature of that process (Baldwin, 1964). (c) If the preceding conditions are granted the Watson & Crick (19536) structure implies the speedometer cable type of rotation because of topological reasons. Some theoretical calculations have indicated that: (d) There is enough energy available to drive the rotation of a rod-like cylinder with dimensions similar to a DNA molecule in a medium of cytoplasmic viscosity (Levinthal & Crane, 1956) (see, however, further discussion). (e) It is possible to derive a physico-chemical model of this type of replication which would be able to complete the chromosome duplication within the time period observed in viao (Fong, 1964a,b). One other assumption with indirect consequences to this problem should be mentioned : (f) The specifity of hydrogen bonding is high enough to ensure proper mating of purines and pyrimidines. No specific aid from a third componente.g., enzyme-is needed for this purpose (Watson & Crick, 1953b). There are, how’ever, some serious arguments against the speedometer cable model. (i) The rotating body is not limited to the naked molecule of DNA: counterions, the hydration shell, the RNA replicase system, including newly formed RNA, repressors, etc., must rotate with the DNA. (ii) The driving motor must be situated at the point of replication because there is a source of energy (triphosphates) and because of the necessity for synchronizing the speed of rotation and the rate of synthesis. (iii) It has been established that the circular chromosome of at least some bacteria (chromosome length 1 to 2 mm) is replicated from one point in one direction within the cell (cell size about a few ~1)(Cairns, 1963; Bonhoeffer & Gierer, 1963; Nagata, 1963a,b).
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(iv) Further, the situation is even more complicated after thymine starvation when several growing points are present on one chromosome (Hayes, 1965). (v) Considering the first and the third points, we come to the conclusion that it is questionable whether there is enough energy to support the rotation of the chromosome. An energy of l-2 x lo-” Cal/rev. was derived (Levinthal 8c Crane, 1956) using the total length of 20 p, radius 10 A, viscosity 1 CP and the speed 60 rev./set. Using more realistic values for the chromosome of Escherichiu coli, i.e., length of 1000 p viscosity of 4 cP, radius of 10 A (which is underestimated) and speed of 200 rev./set, we obtain about 10-l’ Cal/rev. This figure is the lowest estimate because the effective radius is greater (see point (i)) and the multiply folded “speedometer cable” needs more energy to rotate than a simple cylinder-like body rotating around its own axis. In principle there are two ways to avoid these difficulties and construct an alternative model of replication which would still be consistent with known experimental results. One can assume that: (A) The speedometer cable type of replication is limited to subunits of the chromosome: the chromosome dissociates into several parts which are duplicated separately and then aggregate again (Sarkar, Mukundan & Devi, 1963); there are some single bonds inserted into the double-stranded molecule which provide for free rotation of shorter segments (Crothers, 1964). Either of these mechanisms must involve some break, unusual linkage, or nonnucleotide element (Bendich & Rosenkranz, 1963). The explanation of how such a “swivel” is replicated provides serious problems. Or, one can assume that: (B) Semiconservative distribution is the result of highly organized enzymatic uncoupling, transfer and rejoining of single nucleotides or groups thereof, within the “old” strand rather than the result of continous preservation of the strand integrity. We present a model which demonstrates the possibility of an enzymatic disassembling and recombining of the deoxyribosephosphate backbone of one strand as a mechanism for DNA replication without unwinding. The Principle of Proposed Mechanism We propose an enzymatic system (reduplicase) attached to one strand of each individual DNA molecule-for example, to the (+) strand (Fig. 1). This strand is disassembled during the replication process and its building blocks-nucleotides-are transferred piece by piece to the daughter molecule where they are rejoined, forming the “old” or “conservative” part of it. The opposite strand of the parent molecule (-) is not involved in the duphcaT.B. 17
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FIG. 1. The possible mechanism of DNA replication model (see text). S, deoxyribose; P, phosphate.
according to the “split-rejoin”
tion process (except, directing the specifity of complementary bases) and preserves its integrity all the time, remaining free for RNA replication, repression, etc. This is consistent with the fact that replication of RNA does not affect the DNA reduplication in any regard; this is, the reprimed parts of DNA are reduplicated with the same rate as the most active ones. The opposite is also true: there is no indication that the RNA replication is obstructed in the region of the growing point of the chromosome. This (-) strand forms the “old” part of the other daughter molecule. The “new” strands of both daughter molecules are polymerized as complements of old strands. All three parts-both daughter molecules and parent molecule--are fixed in space; only the single nucleotide being transferred rotates. The result of such reduplication are two daughter molecules lying parallel to each other. Therefore they can be easily separated even if the parent molecule had a rather complicated tertiary structure. It is possible to suggest many details of this general mechanism. Each has some advantages and weak points. Because no direct experimental data are available we cannot specify the mechanism in more detail. Most probably
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the hydrogen bonds of the C-c pair (Fig. 1) are released under the influence of the enzyme which allows the base c to rotate out of the helix. It is then bonded to the 5’-triphosphate of the complementary base C’. This new C’ nucleotide is attached to the growing new part of the daughter molecule, releasing PPi (this step is shown in Fig. 1). The c-nucleotide is liberated from the parent molecule by pyrophosphorolysis with the formation of a 5’triphosphate end. The base pair c-C’ rotates into the proper position, the c-nucleotide is rejoined with the “old” part of the daughter molecule and PPi is released. Alternatively, the transfer of the c-nucleotide may be accomplished by the enzyme alone without involvement of the C’ complementary nucleotide. The latter may be added later or not at all as in the case of single-stranded phages. The removed c-nucleotide is then replaced by new c’-5’-triphosphate which is linked to the new part of the molecule. The pyrophosphate necessary for the pyrophosphorolysis is probably attached to the enzyme or protected by it from hydrolysis by pyrophosphatase. We cannot specify at present the polarity of the strands. Thus, if the residue X in Fig. 1 is a 3’-OH group, Y signifies 5’-triphosphate and vice versa. We would prefer the former alternative. Then the additon of new triphosphate to the 5’ end would be accomplished in such situation (base C’ bonded to c linking in this way both parts of the molecule) which cannot be at present simulated in vitro. This might be the reason why the addition to the 5’ end of the primer strand has never been observed in vitro. This alternative was chosen for Fig. 2 where the geometry of the transfer is shown from the top view. Discussion It is very difficult to suggest an experimental proof of this mechanism. According to our model, there should be only two to three reduplicase complexes in the growing microbial cell, firmly attached to the growing point of the chromosome and simultaneously to the mesosomes and cytoplasmatic membrane (Jacob, Brenner & Cuzin, 1963). Each “purification” of such a complex means its degradation into individual subunits. The results of Hanawalt & Ray (1964) show that the growing point of the bacterial chromosome makes a complex with a protein (or lipoprotein) which is resistant to normal deproteinization procedure. Therefore the enzymes obtained by means of typical purification procedures are probably the subunits and/or the “repair” enzymes as known from the “cut-and-patch” dark reactivation processes (Setlow, 1964; Howard-Flanders, 1965; Stacey, 1965). On the other hand, these enzymes active in repair might be the building blocks from which the reduplicase complex is made. There are some data which support our model:
I.
I
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FIG. 2. The section in the growing point in the plane of the c--C base pair (see Fig. 1). The co-ordinates of the atoms are taken from model 3 of DNA proposed by Langridge cr al. (19606). I, Parent double-stranded molecule; II, daughter double-stranded molecule. O,, O’,, The points of rotation (which coincide approximately with the P-O,,, bonds) in the replication of the ith base pair. Starting point: (heavy lines). Original position of the base pair c-C in the parent molecule. 1st step: rotation of the base c around the point 0,. 2nd step (light lines): addition of the complementary triphosphate of the base C’ and substitution of the C’ nucleotide into the daughter molecule with the release of PPi. 3rd step (dotted lines): pyrophosphorolysis of the bond between parent molecule and the c nucleotide followed by the rotation of the c-C’ base pair around the point O’, to the final position in the daughter molecule.
(a) An exonuclease activity accompanies the DNA polymerase through many steps of purification in various systems (Oleson & Koerner, 1964; Korn & Weissbach, 1964; Baldwin, 1964). This might reflect the stepwise dissociation of the complex. (b) The pyrophosphorolytic activity of the DNA polymerase was demonstrated in vitro (Bessman, Lehman, Simms & Kornberg, 1958) where it needs a high concentration of free inorganic pyrophosphate. Assuming that the pyrophosphate is attached in uiro to the reduplicasecomplex, we can imagine that its effective concentration in the proper place is raised enough to cause the pyrophosphorolysis of the proper band. If phosphorolysis is alternatively suggested,the transfer mechanismshould deal with the diphosphates instead of triphosphates.
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(c) The recombination in viral and bacterial chromosomes might be easily explained on the basis of our model: it involves the very close attachment of two double-stranded molecules of DNA. This contact should be very specific to bring corresponding markers together. Further, the high negative interference known in phages can be understood if the contact lasts for a time sufficient for the replication of several markers to be accomplished. The London forces as suggested by Jehle, Parke, Shirven & Aein (1964) might provide a physical explanation on such a contact in our model but can hardly Speedometer
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bI
-0
i
a
I 8 G, + 4 G,, 12 12A,
:6Ap
density label
9G,+3GeU 12
6G,+6G,,
12A,.4A,
12AT:12Ap
12
FIG. 3. The alternative responses of the isolated growing point of a bacterial chromosome to the enzymatic digestion of proteins in the experiments described by Hanawalt & Ray (1964). Light lines represent the DNA strands labelled by thymine-[3H]thymine, heavy lines the strands doubly labelled by 5-bromouracil and 3zP. The density and activity of fragments are given at the bottom assuming that the average lengths of a, b and c are the same. The density is calculated from the density of light parts (GT) and heavy parts (Ga”). The ratio of 3H and 32P activity is given as A, and A,, respectively.
258
J.
DROBNiK
AND
V.
VONDREJS
be applied to polymer molecules rotating at several hundred revolutions per second. Once the contact occurs, the transfer of building blocks to the wrong molecule might readily occur. The analysis of the DNA in its replicative form gives only a small chance to discriminate the speedometer cable model from the “split-rejoin” mechanism suggested above. There exists, however, a small difference: in the speedometer cable model the intergrity of both strands is preserved so that both daughter molecules are covalently bonded to the parent molecule. In our split-rejoin model only one daughter molecule is directly covalently bonded; and the other is attached to the rest by means of hydrogen bonds and/or the reduplicase complex itself. Thus the type of experiment described by Hanawalt & Ray (1964) is of great interest. The growing point is obtained in the form shown in Fig. 3. Let us suppose that by random breaks the segments a, b and c are, on the average, of equal length. Thus, in the case of the split-rejoin mechanism two new fractions should occur after deproteinization by enzymes: one heavy with 1 : 1 labelling with 32P and 3H, the other of intermediate density with 1 : 3 labelling. The examination of Hanawalt’s results shows that fractions can be detected (Figs 5(b) and 6(a) in Hanawalt & Ray, 1964) which would correspond to the latter. Nevertheless, this result is not straightforward because the maxima of density are too close together, some traces of proteins still attached to the DNA might change the density, and, even when the experiments are very carefully made, a small amount of shearing force may cause preferential breakage at the branching point even in the case of the speedometer cable type of replication. Therefore, more experimental data are necessary. REFERENCES BALDWIN, R. L. (1964). The Bacteria, vol. V, p. 327. New York: Academic Press. BENDICH,A. & ROSENKRANZ,H. S. (1963). Progress in Nucleic Acid Research, vol. 1, p. 219. New York: Academic Press. BESSMAN,M. J., LEHMAN, 1. R., SIMMS, E. S. & KORNBERG, A. (1958). J. Eiol. Gem. 233, 171. BONHOEFFER, F. & GIERER, A. (1963). J. molec. Biol. 7, 534. CAIRNS, J. (1963). J. molec. Biol. 6, 208. CAIRNS, J. (1963). Cold Spring Harb. Symp. quant. Biol. 28, 43. CRICK, P. H. C. (1963). Progress in Nucleic Acid Research, vol. 1, p. 164. New York: Academic Press. CROIXERS, D. M. (1964). J. molec. Biol. 9, 712. FONG, P. (19644. Proc..natn. Acad. Sci. U.S.A. 52, 239. FONG. P. (1964b). Proc. natn. Aead. Sci. U.S.A. 52. 641. HAMILTON;
L. k,
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MARVIN, D. A., EPHRUW-TEYLOR, H. & SIMMONS, N. S. (1959). J. biophys. biochem. Cytol. 5, 397. HANAWALT, P. C. & RAY, D. S. (1964). Proc. nafn. Acad. Sci. U.S.A. 52, 125.
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HAYES, W. (1965). Symp. Sot. Gem Microbial. XV, 294. Cambridge University Press. HOWARD-FLANDERS, P. Private communication to STACEY, K. A. (1965). Symp. Sot. Gen. Microbial. XV, 159. Cambridge University Press. JACOB, F., BRENNER, S. & CUZIN, F. (1963). Cold Spring Harb. Symp. quant. Biol. 28,329. JEHLE, H., PARKE, W. C., SHIRVEN, R. M. & AEIN, D. (1964). Biopolymers, Symp. 1, 209. KORN, D. & WEISSBACH,A. (1964). J. biol. Chem. 239, 3849. KORNBERG, A. (1957). Adv. Enzymol. 18, 191. LANGRIDGE, R., WILSON, H. R., HOOPER, C. W., WILKINS, Ma H. F. & HAMILTON, L. D. (1960~). J. molec. Biol. 2, 19. LANGRIDGE, R., MARVIN, D. A., SEEDS,W. E., WILSON, H. R., HOOPER, C. W., WILKINS, M. H. F. & HAMILTON, L. D. (19606). J. molec. Biol. 2, 38. LEVINTHAL, C. & CRANE, H. R. (1956). Proc. natn. Acad. Sci. U.S.A. 42,436. LUZZA~, V. (1963). Progress in Nucleic Acid Research, vol. 1, p. 347. New York : Academic Press. MARMUR, J., ROWND, R. & SCHILDKRAUT, C. L. (1963). Progress in Nucleic Acid Research, vol. 1, p. 232. New York: Academic Press. MESELSON, M. & STAHL, F. W. (1958). Proc. Natn. Acad. Sci. U.S.A. 44, 671. NAGATA, T. (1963a). Proc. Natn. Acad. Sci. U.S.A. 49, 551. NAGATA, T. (19636). Cold Spring Harb. Symp. quant. Biol. 28, 55. OLESON, A. E. & KOERNER, J. F. (1964). J. biol. Chem. 239,2935. SARKAR, N. K., MUKUNDAN, M. A. & DEVI, A. (1963). Nature, Land. 200, 1205. Smow, R. B. (1964). .I. Cell. camp. Physiol. Suppl. 1, 64, 51. STACEY, K. A. (1965). Symp. Sot. Gen. Microbial. 15, 159. Cambridge University Press. WATSON, J. D. & CRICK, F. H. C. (1953a). Nature, Lond. 171,737. WATSON, J. D. & CRICK, F. H. C. (19536) Cold Spring Harb. Symp. quant. Biol. 28, 123 WILKINS, M. H. F. (1956). Cold Spring Harb. Symp. quant. Biol. 21, 75.