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Genetic circularity and evolution
J. Theoret. Biol, (1968) 21, 217-228
Genetic Circularity
and,Evolution
D. C. REANNEY Department
of Biochemistry, Lincoln College, University Christchurch, New Zealand
of Canterbury,
AND
R. K. RALPH Department of Cell Biology, University of Auckland, Auckland, New Zealand (Received 29 December 1967, and in revisedform 13 June 1968) A well-documentedobservationfor which thereis at presentno satisfactory explanation is the fact that in many simple non-nucleatedsystems,the chromosomehasa ring structure. A modelis proposedherewhich directly implicatescyclic polynucleotidesin the evolutionary process.The model provides a simpleway of increasingthe informational content of DNA during evolution. It is suggestedthat primitive circular polynucleotides were copied and translated in a repetitive but highly inaccurate manner. This statistical processwould produce a variety of “templates”, allowing selectionmaximum opportunity to group the proteins thus specifiedinto biochemicalpathways or to enhancepre-existingdifferencesso as to lead to the production of apparently “unrelated” proteins. Some possible consequences of circularity and its relevanceto contemporary genomesare also discussed.
1. Introduction Recent research has revealed that the genetic material exists in the form of a closed ring in many simple biological systems. For example, the DNA of Escherichia coli, which was inferred to be circular from linkage data (Jacob & Wollman, 1958) has been shown by radio-autography to be circular (Cairns, 1963). The DNA’s of the T-even phageswhich were also thought to exist in the form of physical circles from linkage studies have since been shown to contain terminally repetitious linear DNA molecules which are cyclic permutations of the same fundamental sequence (for review see Thomas & MacHattie, 1967). It has been suggestedthat this situation may arise by random breakage of a circular phase in DNA replication which produces a collection of linear phage DNA molecules each with a different 217
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circular permutation of a common nucleotide sequence (Josse & Eigner, 1966). An alternative view envisages union and breakage of preformed phage genomes (Thomas, 1967). The DNA’s of polyoma virus (Dulbecco & Vogt, 1963; Weil & Vinograd, 1963), SV40, rabbit papilloma and human papilloma (Crawford, 1965) occur in the form of circular molecules. The linear DNA injected by the lysogenic phage lambda rapidly cyclizes to form circular molecules (Young & Sinsheimer, 1964). The genome of phage $X174 is a covalently closed ring of single-stranded DNA (Fiers & Sinsheimer, 1962) while the replicating forms of 4X174 and of phage fd are circular doublestranded DNA’s (Kleinschmidt, Burton & Sinsheimer, 1963; Pouwels & Jansz, 1964; for review see Thomas & MacHattie, 1967). Even the RNA in icosahedral viruses (e.g. TYMV) may exist in circular form (Strazielle, Benoit & Hirth, 1965) or form a “functional circle” (e.g. Q/?) by virtue of H bonding between complementary sequences (Haruna & Spiegelman, 1965). A circular replicative form of RNA has also been proposed for FMD virus (Brown & Martin, 1965). Circular DNA has been isolated from mitochondria (Sinclair & Stevens, 1966) and the evidence that chloroplasts contain bacterial-type ribosomes suggests that circular DNA may be present in chloroplasts. These subcellular organelles are thought to have evolved from symbiotic relationships between bacteria and blue-green algae, respectively, and the cells of higher organisms. Claims have also been made for the circularity of DNA from mammalian cells (Hotta & Bassel, 1965). Genetic considerations have led Callan (1967) and Whitehouse (1967) to propose a cycloid model for the chromosome to explain the existence of redundant copies of the genetic information in the DNA of many plant and animal species (Stebbins, 1966; Ritossa, Atwood & Spiegelman, 1966). The cycloid model differs from the circular DNA’s discussed above in concept if not in function. In view of the widespread occurrences of circular genomes, particularly in simple systems, it is pertinent to ask what evolutionary advantages result from the existence of circular genetic material. The purpose of the present communication is to explore the implication of circular DNA for polynucleotide evolution. 2. Primordial Templates Under primordial conditions the original polynucleotides were probably formed by random condensation of mono nucleotides following reaction with natural condensing agents such as carbodi-imides (Ponnamperuma & Peterson, 1965; Steinman, Lemmon & Calvin, 1965). Condensation of deoxyribomononucleotides may have produced the first unbranched deoxyribopolynucleotides capable of serving as templates for further polynucleotide
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synthesis (for discussion see Reanney & Ralph, 1967). It is likely that cyclization of early polynucleotides also occurred, since during the chemical synthesis of polynucleotides with carbodi-imides cyclization of the resulting polymers occurs and interferes with the synthesis of longer linear polynucleotides (Tener, Khorana, Markham & Pol, 1958; Ralph & Khorana, 1961; Ralph, Conners, Schaller & Khorana, 1963). Cyclization occurs mostly with shorter oligonucleotides and decreases as the size of the linear product increases, However, cyclization is favoured under conditions of high dilution. Thus small linear and/or circular oligonucleotides might originally have acted as templates for polynucleotide synthesis. This process could have been facilitated by periodic fluctuations in temperature (diurnal, seasonal) which probably exceeded the Tm’s of the intermediate double-stranded replicating structures. That is, double-stranded complementary polynucleotides formed by condensing mono- or oligonucleotides on original templates at lower temperatures, would have disassociated during hotter periods and then have acted as templates for the further assembly of mono- or oligonucleotides during subsequent cooler periods (cf. Naylor & Gilham, 1966; Bloom, 1967). Conceivably this may have been the first mode of “replication” before the advent of enzymic catalysis. Assembly and condensation of mono- and/or oligonucleotides on the original templates may have been facilitated by secondary stabilizing forces if the template was immobilized against a surface, for example quartz, silica or clay (Bernal, 1951). McLaughlin et a2. (1966) have confirmed that the ribosome contributes to the stability of mRNA-tRNA interaction. Possibly an inert support contributed in like manner to the stability of the intermediate complex during early template replication. The fact that replication of the circular E. coli chromosome now occurs via attachment to the cell wall may reflect the original situation wherein early templates required association with a stabilizing surface to serve their template function. Condensation of deoxyribonucleotides in the above manner could have given rise to (a) single-stranded circular polynucleotides, (b) single-stranded linear polynucleotides perhaps terminating in non-nucleotide materials, (c) single-stranded circular polynucleotides with or without adhering complete or incomplete linear polynucleotides, (d) covalently closed doublehelical circular polynucleotides. With time it seems possible that more of the latter compounds would have accumulated due to cyclization of linear structures and the enhanced stability of the intertwined closed double-helix (Vinograd & Lebowitz, 1966). Small covalently linked circular doublehelical polynucleotides might still have acted as templates for condensation of mononucleotides when “melted”, but would have displaced the new strands on “reannealing”.
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This would propose that the initial templates for nucleic acid replication were linear and/or circular single-stranded deoxyribopolynucleotides. The circular single-stranded templates proved advantageous (see later) and were eventually utilized predominantly. These in turn were eventually replaced by closed double-helical circular polynucleotides formed by covalently closing the complementary strand formed on a single-stranded circular template. This stable, double-helical material finally became the preferred material for gene replication and for messenger nucleic acid synthesis, accepting that messenger nucleic acids may also originally have been polydeoxyribonucleotides and functioned as templates. Denatured DNA “templates” can serve as “message” in in vitro systems in the presence of certain antibiotics (McCarthy, Holland & Buck, 1966). 3. Protein Synthesis The evolutionary origin of the first protein produced under genetic direction is likely to remain for some time one of the prime dilemmas of prebiological evolution. Thermal proteins of the type synthesized by Fox (Fox, 1965; Harada & Fox, 1965) may well have been produced by volcanic action. However, thermal proteins appear to be branched structures rather than linear peptides and it is tempting to conclude that these branched thermal proteins gave rise to protein coacervates, conceivably primitive precursors of cells, rather than to regeneratable enzymes. Nevertheless coacervates of this or other types (Oparin, 1965; Smith, Bellware & Silver, 1967) may have concentrated precursors from the environment and so been necessary for further evolution. A clear distinction must be made here between genetically determined enzymes and structural proteins. Although protein-like molecules “catalysing” various chemical reactions may have been produced prior to the evolution of a coding mechanism, the first enzymes required for replication processes and themselves requiring replication were undoubtedly made under genetic direction. By contrast genetically determined cell-wall proteins were probably a later phenomenon. Under the circumstances envisaged for polynucleotide synthesis, abortive polymerization of mononucleotides may frequently have terminated by reaction of the terminal nucleotide with an activated amino acid to produce an aminoacyl polynucleotide. The resulting aminoacyl polynucleotides may have been the primitive precursors of present-day adaptors (i.e. aminoacyl transfer RNA’s). Condensation of the amino acids, directed by primitive singlestranded polynucleotides might then give rise to linear primordial proteins (cf. Nirenberg, Caskey & Levin, 1967). In this circumstance some means of selection of an amino acid by a specific adaptor must eventually have operated to permit the synthesis of a
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specific polypeptide, since without this restriction any adaptor might have carried any amino acid, leading to random insertion of amino acids into polypeptides. Such a selection might originally have been extremely crude and permitted quite gross ambiguities, but achieved specificity with evolution. An intriguing possibility is that the “messenger’‘-“adaptor” complex may originally have acted to select the amino acid most suited to producing a stable intermediate for polypeptide synthesis. This would imply that amino acids originally played a role in establishing the intermediates necessary for specific protein synthesis, leading eventually to the selection of the most appropriate adaptor (anticodon?)-amino acid relationships and to the exclusion of less stable or less satisfactory alternatives. The observation that aminoacyl tRNA’s can bind to copolymers with specificity in the absence of ribosomes is compelling evidence for the argument, logical on a priori grounds, that ribosomes were absent at this stage of evolution (Nirenberg et al., 1967). Ordered base pairing of the aminoacyl polynucleotides dictated by primordial polynucleotide messengers and involving perhaps two (Jukes, 1965) or three bases could then have assembled the amino acid residues in appropriate juxtaposition to facilitate specific polypeptide formation, much as is observed under present-day conditions. The interaction of polynucleotide template or “messenger” (itself derived from a circular single-stranded or double-stranded template) and aminoacyl polynucleotide “adaptor” may have been aided by a surface (today the ribosome) to which both polymers attached (McLaughlin et al., 1966). This attachment would have provided better stabilization and stereo-specificity for the intermediate complex while additional stability, specificity and order of attachment resulted from the interaction of bases in the template with those in the adaptors. In this way specificity for the repeated synthesis of specific polypeptides resulted, much as it does today, but with simpler entities. Protein synthesis probably occurred by nucleophilic attack of the NH, group of one amino acid on the appropriately juxtaposed protonated C==O group of the adjacent aminoacylated tRNA. If any ancient geochemical niche or coacervate contained a sufficient concentration of the relevant precursors, the production of the first polynucleotide “messenger” directed protein synthesis becomes statistically possible. We propose that in time a protein which facilitated condensation of mono- or oligonucleotides on a pre-existing messenger was generated and became the primitive precursor of DNA polymerase (“duplicase”). The first proteins were undoubtedly very small. Perhaps the first “duplicase” was little more than a crude version of the “active centre” of a modern duplicase. The striking discovery that phylogenetically ancient proteins such as Ferridoxin (Eck & Dayhoff, 1966) and cytochrome c (Cantor & Jukes,
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1966) show evidence of evolution from short repetitive amino acid sequences suggests that the initial templates were small and that the earliest polypeptides were probably small. 4. Possible Advantages of Circular Templates under Primitive Conditions Until the advent of duplicase mediated replication, little or no advantage would have accrued to cyclic templates over their linear counterparts. Subsequent to this event however, cyclic nucleic acids would have been the preferred templates; owing to the fact that a circle has neither beginning nor end, a duplicase once attached could continually produce complementary copies of the template; under the conditions envisaged detaching a duplicase from its template would render inactivation likely and its re-attachment unlikely. Re-attachment to other polynucleotides might also have occurred and not produced more duplicase. The production of a duplicase would have accelerated the rate of production of new templates and eliminated the necessity for temperature variation to separate replicating polynucleotides. Figure 2 outlines the stages through which the evolution of duplicase is postulated to have progressed. If the situation envisaged in Fig. 2(b) did in fact occur, then a cyclic model could explain one of the prime dilemmas of evolution. Watson-Crick copying merely replicates a pre-existing length of DNA. It cannot of itself increase this length. However, during the course of evolution the number of bases in DNA has increased from an unknown small number (about 10’ ?) to about 2 x 10’ in the case of a human germ cell. This represents a lo’-fold increase. Elongation of templates is quite conceivable on a cyclic model since the repeated passage of a duplicase round a circular nucleic acid could produce a polynucleotide many times longer than “unit” length (Fig. 1). In modern
FIG. 1. (@) Duplicax.
translational systems this would generate a reiterative series of base sequences and identical multiple peptide sequences in proteins. At the prebiological stage large numbers of related but differing peptide sequences would have been produced owing to coding inaccuracy. For example, under the conditions envisaged for the primitive earth mutation by radiation would have occurred with far greater frequency than under contemporary conditions.
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Furthermore, heat and/or variable pH would have increased the probability of deamination of bases. Mutation coupled with frequent mispairing (Reanney & Ralph, 1967) and variable fidelity of translation (Woese, 1965) would have made both replication and translation of polynucleotides highly variable processes. However, in this context, such variation would be a tremendous evolutionary advantage. Of the polynucleotides produced a small but statistically signz&ant fraction would contain the information needed to generate the duplicase in a segment (“cistron”) of their length while the remaining base sequences were suficiently novel to generate a wide spectrum of proteins. If one of these novel proteins happened to fulfil a useful function
+ 0
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(d)
FIG. 2. The evolution of duplicase-template relationships. (a) Duplicase. (a) Nonbrought about by cycles of denaturation and subsequent conenzymic “replication” densation during temperature fluctuations. The steps between (a) and (b) are uncertain but possibly involve the introduction of an extremely primitive “duplicase” capable of facilitating condensation of mononucleotides on a pre-existing nucleic acid template but incapable per se of strand separation. (b) A possible early duplicase: this would act preferentially upon a covalently linked single-stranded nucleic acid ring from which, by displacement, serial repetitions of the complementary sequence could be produced. A likely predecessor for such a duplicase would be a less complex molecule capable of copying the parental sequence but not of displacing it so as to allow continued polynucleotide synthesis. A possible analogous mechanism still operating may be found in the-replication of FMD virus (Brown & Martin. 1965). It seems likely. however. that this staee has no functional contemporary equivalent. (c) A later duplic& now acting preferent~lly upon the more stable duplex DNA circles. The displaced DNA shown here is duplex. However, in the analogous contemporary process of transcription, only one strand is displaced. (d) A contemporary duplicase acting on duplex circular DNA as found in the E. coli genome. Elongation of the template no longer occurs.
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the combination would have (a) been selected for and (b) been potentially self-replicating. A fraction of such “multi-cistronic” polynucleotides might themselves have condensed into circular contigurations, a percentage of which would again be capable of self-replication and elongation while containing potentially novel information. It is characteristic of the bacterial genome that functionally related genes tend to occur close together. Cistrons related to one compound are often contiguous and closely mimic the reaction sequence they determine (e.g. the tryptophan operon). Others are contiguous but do not mimic the reaction sequence they determine (the histidine operon). Others are merely clustered in a small region of the chromosome while a few map in widely separated areas (the arginine operon). This type of distribution is exactly what one would expect on the basis of the statistical circularity concept presented above. It also allows for selective pressure to concentrate related genes so as to permit co-ordinate regulation of enzyme synthesis (Jacob & Monod, 1961). The selective pressure to maintain concentrated genes would have resulted in many cases from the original circumstances of their genesis. Thus the above concept has the flexibility to account for the evolutionary origin of a great number of diverse proteins together with the inbuilt constraints necessary to facilitate grouping of certain of these proteins into biosynthetic pathways. For example, the enzymes of the GAL operon all recognize galactose in diferent biochemical reactions. On the basis of the circularity hypothesis, these enzymes were generated by a process in which information was replicated, modified in varying degrees, to give a series of enzymes of related function. Whether the base sequences in the coding nucleic acid remained similar or diverged would depend upon the relative selective advantages of the proteins they specified. The presence of multiple copies of information in DNA resulting from transcription on a circular template may have infIuenced the selection of circular templates. Multiple copies of genes permit mutation to occur in one copy without loss of all function. It is likely that this would have been advantageous to the early evolutionary process, permitting a “trial and error” selection in which loss of gene function did not immediately eliminate the primitive organism. For the proposed mechanism of DNA elongation to operate plausibly, breakage and recyclization of the DNA product would be necessary (Fig. 1). The possibility of linear DNA molecules cyclizing spontaneously at high dilution has been discussed (see section 2). Under the putative primitive earth conditions selection would have favoured cyclic molecules since circular DNA molecules (e.g. circular lambda DNA) are relatively stable while long linear DNA’s are fragile and easily sheared (Smith, 1967). Thus
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cyclization would confer enhanced survival value on replicated DNA’s It would in effect “protect” information gathered during elongation. An additional advantage would arise if exposure of the earth to U.V. radiation, or other circumstances caused greatly increased numbers of breaks in DNA. Circular DNA molecules would not lose information when broken (c.f. linear DNA’s), provided cyclization rapidly occurred to “repair” the break. The above factors may have favoured the early evolution of a DNA-ligase enzyme, to catalyse (a) a rapid recyclization of elongated DNA’s and (b) immediate repair of broken DNA circles. This latter process would prevent loss of information in the event of a second break. 5. The Relevance of Circularity
in Contemporary Genomes It is probably futile to look among contemporary replicative processes for any mechanism formally analogous to that postulated to occur under conditions which may have vanished from the earth over 1.5 billion years ago. The following discussion is not meant to imply that modern circular genomes have preserved ancient mechanisms of nucleic acid replication or that such genomes have evolved specifically to meet the needs of novel situations which are evolutionary cul-de-sacs. Rather it is intended to highlight features, unique to circular nucleic acids, which may have played and may still play an important role in the selection and maintenance of genetic circularity in contemporary genomes. For primitive cells replication and translation were both statistical mechanisms (see above and Woese, 1965). As the translation apparatus became more accurate, so in proportion, would the fidelity with which the genetic information was replicated, increase. Whereas the problem of the primitive cell was to decrease variability, subsequent generations with precise modes of protein synthesis were also faced with the task of enhancing genetic variation in order to facilitate further evolution. Numerous means of promoting and storing variation have been exploited during evolution, the most effective “innovation” being the union of homologous DNA molecules within one cell and the concomitant mechanism of recombination. E. coli, normally haploid, possesses a primitive form of sexuality. Insertion of the circular F factor into the continuous bacterial chromosome enables a polarized transfer of information to take place between donor and recipient cells (Jacob & Wollman, 1961). The probability that any given marker will be transferred is a function of its distance from the F insertion site (the breakage point or origin). In assessing the mechanism of this phenomenon one basic advantage of a continuous chromosome becomes apparent. Only a circular structure can break at any point and still yield molecules of identical size. Random breakage of a linear molecule would yield fragments of varying
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size and thereby exclude many markers from transfer. For a linear molecule which did not break prior to transfer there would be an invariant gradient of transfer probability as measured from either free end. However, for a circular chromosome every marker has an equal probability of transfer provided the probability of breakage is evenly distributed around the length of the chromosome. The genomes of the different hfr strains analysed to date appear to break at different points prior to transfer (Hayes, 1964). Moreover, the fact that the polarity of transfer can be either clockwise or anticlockwise (Hayes, 1964) confers an additional plasticity. Taking the markers from azi-r to ade in the E. coli genome and assuming a break just prior to ade; if polarization were clockwise ade would have a higher probability of transfer than azi-r; assuming an identical breakage point counterclockwise polarization reverses the probability of transfer for these two markers. Thus a circular chromosome is a far more $exible instrument for promoting genetic variation while retaining the total information in the popula tion than a linear molecule of equivalent size. Circularity in the E. coli chromosome may have influenced or been influenced by selection of a circular form of the F factor which mediates
sex transfer. Circularity in the episome is economical since it ensures that the regions of homology between the F and host DNA’s are concentrated in a small area. If the episome were linear the chromosomal region of homology would presumably require separating and after breakage the broken ends of the host chromosome would have to separate appreciably to permit insertion to occur. Similar arguments apply to the reassociation of disconnected chromomeres during pachytene as envisaged by Whitehouse (1967), if in fact the cycloid model of the chromosome is correct. The modern circular genomes which seem most rigorously analogous in their function to those postulated in section 4 of this paper are those which occur in phage infected cells together with long reiterative copies of the genetic information. Brown & Martin (1965), for example, claimed to have found an RNA species apparently longer than the viral genome in baby hampster kidney cells infected with foot-and-mouth disease virus. On the basis of this finding they proposed a model for the production and extension of the viral RNA involving cyclization of the minus strand. Smith (1967) has estimated that the molecular weight of the replicating form of lambda DNA in infected cells is greater than 250 million daltons; this implies a length of many viral DNA subunits. The existence of covalently linked circular DNA in the previral nucleic acid pool is interesting and might seem to support a mechanism whereby long linear precursors of lambda DNA were generated by repeating passage of duplicase round a circular template. Covalently linked circular DNA found in infected cells could thus represent
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an obligatory but nonmaturing intermediate, prevented by virtue of its circular structure, from being broken down to mature phage DNA equivalents as occurs with the linear repetitive copies produced from it. This would account for the small quantities of cyclic material found in infected cells (Weissbach & Salzman, 1967). However, the kinetics of the appearance of the covalently closed circles and the concatenated DNA’s at present do not appear entirely consistent with a precursor relationship of the former for the latter. The recent discovery of a DNA joining enzyme or DNA ligase (Gellert, 1967; Olivera & Lehman, 1967; Goulian, Kornberg & Sinsheimer, 1967) suggests that in uiuo this enzyme may link phage DNA’s by their cohesive ends to form linear concatenated intermediates. On this basis cyclic DNA’s might result from fortuitous ring closure by DNA ligase and be by-products of no fundamental importance. Thus DNA ligase could in theory account for all the multiple length DNA’s found in infected systems, e.g. cells infected with T4 phage (Frankel, 1966) and 4X174 (Rush, KleinSchmidt, Hellmann & Warner, 1967). If any contemporary process retains analogy with the model proposed here it would not be formal replication but transcription. Unlike replication, transcription involves the displacement of a newly synthesized polynucleotide from its templating strand. This raises the interesting possibility that “transcriptase” (DNA dependent RNA polymerase) may be more closely related to the original “duplicase” than DNA polymerase itself. Transcriptase may be a close evolutionary relative of duplicase, modified to fulfil a specialized function. The third “polymerase” (DNA ligase) may also be of extreme antiquity in view of the necessity for continued cyclization of elongating DNA’s (section 4). All three enzymes have in common the ability to catalyse the formation of phosphodiester bonds. The circularity hypothesis provides an ideal mechanism for generating the three enzymes by statistical modification of a common progenitor nucleotide sequence (cf. the genesis of the GAL enzymes: section 4). Thus it will be of considerable interest to determine (a) whether the three corresponding genes are closely linked and (b) whether these enzymes retain significant homologies in primary structure. REFERENCES BERNAL, J. (1951). “The Physical Basis of Life”. London: Routledge and Kegan BUX)M, B. (1967). Perspect. Biol. Med. 10,269. BROWN, F. & MARTIN, S. (1965). Nature, Loti. 208,861. CAIRNS, J. (1963). Cold Spring Harb. Symp. quant. Biol. 28, 43. CALLAN, H. G. (1967). .I. cell Sci. 2, 1. CANTOR, C. & JUKES, T. H. (1966). Proc. narn. Acad. Sci. U.S. A. 56, 177. ~AWFORD, L. V. (1965). J. molec. Biol. 13, 362. DULBECCO, R. & VOGT, M. (1963). Proc. nutn. Acud. Sci. U.S.A. 50, 236. ECK, R. & DAYHOFF, M. (1966). Science, iV.Y. 152,363. FIERS, W. & SINSHEIMER, R. L. (1962). J. molec. Biol. 5, 424.
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FOX, S. W. (1965). In “Evolving Genes and Proteins” (V. Bryson & H. Vogel, 4~). New York: Academic Press. FRANKEL, F. R. (1966). .Z.molec. Biol. 18, 127. GELLERT, M. (1967). Proc. nutn. Acad. Sci. U.S.A. 57, 148. GOULIAN, M., KORNBERG, A. & SINSHEIMER, R. L. (1967). Proc. natn. Acad. Sci. U.S.A. 58, 2321. HARADA, K. & Fox, S. W. (1965). In “Origin of Pre-biological Systems” (S. W. FOX, ed.). New York: Academic Press. HARuNA, I. & SPIEGELMAN, S. (1965). Proc. natn. Acad. Sci. U.S.A. 54, 1189. HAYES, W. (1964). In ‘The Genetics of Bacteria and their Viruses”. Oxford: Blackwell Scientific Publications. HOTTA, Y. & BASSEL, A. (1965). Proc. natn. Acad. Sci. U.S. A. 53, 356. JACOB, F. & MONOD, J. (1961). J. molec. Biol. 3, 318. JACOB, F. & WOLLMAN, E. L. (1958). In “The Biological Replication of Macromolecules”. Sytnp. Sot. Expl. Biol. 12,15. JACOB, F. & WOLLMAN, E. L. (1961). In “Sexuality and the Genetics of Bacteria”. New York: Academic Press. JOSSE, J. & EIGN~R, J. (1966). Ann. Rev. Biochem. 35, 789. JUKES, T. H. (1965). Biochem. biophys. Res. Commun. 19, 391. KLEINSCHMIDT, A. K., BURTON, A. & SINSHEIMER, R. L. (1963). Science, N.Y. 142, 961. MCCARTHY, B. J., HOLLAND, J. J. & BUCK, C. A. (1966). Cold Spring Harb. Symp. quant. Biol. 31,683. MCLAUGHLIN, G. S., DONDON, J., GRUNBERG-MANAGO, M., MICHELSON, A. & SAUNDERS, G. (1966). Cold Spring Harb. Symp. quant. Biol. 31, 601. NAYUIR, R. & GIWAM, P. (1966). Biochemistry, N. Y. 5, 2722. NIRENBERO, M., CASKEY, T. & LEVIN, J. (1967). Abstracts. 7th Int. Con@. Biochem. Supplement I, 1068. OLIVERA, B. M. &LEHMAN, I. R. (1967). Proc. natn. Acad. Sci. U.S.A. 57, 1426. OPARIN, A. I. (1965). Adv. Enzymol. 27,347. PONNAMPERUMA, C. & PETERSON, E. (1965). Science, N. Y. 147, 1572. POUWELS, P. H. & JANSZ, H. S. (1964). Biochim. biophys. Acta, 91,177. RALPH, R. K., CONNORS, W. J., SCHALLER, H. & KHORANA, H. G. (1963). J. Am. them. Sot. 85,1983. RALPH, R. K. & KHORANA, H. G. (1961). J. Am. them. Sot. 83,2926. REANNEY, D. C. & RALPH, R. K. (1967). J. Theoret. Biol. 15,41. RITOSSA, F. M., ATWOOD, K. C. & SPIEGELMAN, S. (1966). Genetics, Princeton, 54,663. RUSH, M. G., KCEINSCHMIDT, A. K., HELLMANN, W. & WARNER, R. C. (1967). Proc. natn. Acad. Sci. U.S.A. 58,1676. SINCLAIR, J. H. & STEVENS,B. J. (1966). Proc. natn. Acad. Sci. U.S.A. 56, 508. SMITH, A. E., BELLWARE, F. T. & SILVER, J. J. (1967). Nature, Lond. 214, 1038. SMUH, M. G. (1967). IX Intemat. Congr. Microbial., Moscow, p. 483. Oxford: Pergamon Press. STEBBINS, G. L. (1966). Science, N. Y. 152, 1463. STEINMAN, G., LEMMON, R. M. & CALVIN, M. (1965). Science, N. Y. 147, 1574. STRAZIELLE, C., BENOIT, H. & HIRTH, L. (1965). J. molec. Biol. 13, 735. TENER, G. M., KHORANA, H. G., MARKHAM, R. G. & POL, E. H. (1958). J. Am. them. Sot. 80, 6223. TITOMAS, C. A. (1967). J. cell Physioi. 70, 13. THOMAS, C. A. & MACHATI’IE, L. A. (1967). Ann. Rev. Biochem. 36,485. VINOGRAD, J. & LEBOWITZ, J. (1966). J. gen. Physiol. 49, 103. WEIL, R. & VINOGRAD, J. (1963). Proc. natn. Acad. Sci. U.S.A. 50, 730. WEISSBACH, A. & SALZMAN, A. L. (1967). Proc. natn. Acad. Sci. U.S.A. 58, 1096. W~HOUSE, H. L. K. (1967). J. cell Sci. 2, 9. WOES, C. (1965). Proc. natn. Acad. Sci. U.S.A. 54, 1546. YOUNG, E. T. & SINSHEIMER, R. L. (1964). J. molec. Biol. 10, 562.
Genetic Circularity
and,Evolution
D. C. REANNEY Department
of Biochemistry, Lincoln College, University Christchurch, New Zealand
of Canterbury,
AND
R. K. RALPH Department of Cell Biology, University of Auckland, Auckland, New Zealand (Received 29 December 1967, and in revisedform 13 June 1968) A well-documentedobservationfor which thereis at presentno satisfactory explanation is the fact that in many simple non-nucleatedsystems,the chromosomehasa ring structure. A modelis proposedherewhich directly implicatescyclic polynucleotidesin the evolutionary process.The model provides a simpleway of increasingthe informational content of DNA during evolution. It is suggestedthat primitive circular polynucleotides were copied and translated in a repetitive but highly inaccurate manner. This statistical processwould produce a variety of “templates”, allowing selectionmaximum opportunity to group the proteins thus specifiedinto biochemicalpathways or to enhancepre-existingdifferencesso as to lead to the production of apparently “unrelated” proteins. Some possible consequences of circularity and its relevanceto contemporary genomesare also discussed.
1. Introduction Recent research has revealed that the genetic material exists in the form of a closed ring in many simple biological systems. For example, the DNA of Escherichia coli, which was inferred to be circular from linkage data (Jacob & Wollman, 1958) has been shown by radio-autography to be circular (Cairns, 1963). The DNA’s of the T-even phageswhich were also thought to exist in the form of physical circles from linkage studies have since been shown to contain terminally repetitious linear DNA molecules which are cyclic permutations of the same fundamental sequence (for review see Thomas & MacHattie, 1967). It has been suggestedthat this situation may arise by random breakage of a circular phase in DNA replication which produces a collection of linear phage DNA molecules each with a different 217
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circular permutation of a common nucleotide sequence (Josse & Eigner, 1966). An alternative view envisages union and breakage of preformed phage genomes (Thomas, 1967). The DNA’s of polyoma virus (Dulbecco & Vogt, 1963; Weil & Vinograd, 1963), SV40, rabbit papilloma and human papilloma (Crawford, 1965) occur in the form of circular molecules. The linear DNA injected by the lysogenic phage lambda rapidly cyclizes to form circular molecules (Young & Sinsheimer, 1964). The genome of phage $X174 is a covalently closed ring of single-stranded DNA (Fiers & Sinsheimer, 1962) while the replicating forms of 4X174 and of phage fd are circular doublestranded DNA’s (Kleinschmidt, Burton & Sinsheimer, 1963; Pouwels & Jansz, 1964; for review see Thomas & MacHattie, 1967). Even the RNA in icosahedral viruses (e.g. TYMV) may exist in circular form (Strazielle, Benoit & Hirth, 1965) or form a “functional circle” (e.g. Q/?) by virtue of H bonding between complementary sequences (Haruna & Spiegelman, 1965). A circular replicative form of RNA has also been proposed for FMD virus (Brown & Martin, 1965). Circular DNA has been isolated from mitochondria (Sinclair & Stevens, 1966) and the evidence that chloroplasts contain bacterial-type ribosomes suggests that circular DNA may be present in chloroplasts. These subcellular organelles are thought to have evolved from symbiotic relationships between bacteria and blue-green algae, respectively, and the cells of higher organisms. Claims have also been made for the circularity of DNA from mammalian cells (Hotta & Bassel, 1965). Genetic considerations have led Callan (1967) and Whitehouse (1967) to propose a cycloid model for the chromosome to explain the existence of redundant copies of the genetic information in the DNA of many plant and animal species (Stebbins, 1966; Ritossa, Atwood & Spiegelman, 1966). The cycloid model differs from the circular DNA’s discussed above in concept if not in function. In view of the widespread occurrences of circular genomes, particularly in simple systems, it is pertinent to ask what evolutionary advantages result from the existence of circular genetic material. The purpose of the present communication is to explore the implication of circular DNA for polynucleotide evolution. 2. Primordial Templates Under primordial conditions the original polynucleotides were probably formed by random condensation of mono nucleotides following reaction with natural condensing agents such as carbodi-imides (Ponnamperuma & Peterson, 1965; Steinman, Lemmon & Calvin, 1965). Condensation of deoxyribomononucleotides may have produced the first unbranched deoxyribopolynucleotides capable of serving as templates for further polynucleotide
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synthesis (for discussion see Reanney & Ralph, 1967). It is likely that cyclization of early polynucleotides also occurred, since during the chemical synthesis of polynucleotides with carbodi-imides cyclization of the resulting polymers occurs and interferes with the synthesis of longer linear polynucleotides (Tener, Khorana, Markham & Pol, 1958; Ralph & Khorana, 1961; Ralph, Conners, Schaller & Khorana, 1963). Cyclization occurs mostly with shorter oligonucleotides and decreases as the size of the linear product increases, However, cyclization is favoured under conditions of high dilution. Thus small linear and/or circular oligonucleotides might originally have acted as templates for polynucleotide synthesis. This process could have been facilitated by periodic fluctuations in temperature (diurnal, seasonal) which probably exceeded the Tm’s of the intermediate double-stranded replicating structures. That is, double-stranded complementary polynucleotides formed by condensing mono- or oligonucleotides on original templates at lower temperatures, would have disassociated during hotter periods and then have acted as templates for the further assembly of mono- or oligonucleotides during subsequent cooler periods (cf. Naylor & Gilham, 1966; Bloom, 1967). Conceivably this may have been the first mode of “replication” before the advent of enzymic catalysis. Assembly and condensation of mono- and/or oligonucleotides on the original templates may have been facilitated by secondary stabilizing forces if the template was immobilized against a surface, for example quartz, silica or clay (Bernal, 1951). McLaughlin et a2. (1966) have confirmed that the ribosome contributes to the stability of mRNA-tRNA interaction. Possibly an inert support contributed in like manner to the stability of the intermediate complex during early template replication. The fact that replication of the circular E. coli chromosome now occurs via attachment to the cell wall may reflect the original situation wherein early templates required association with a stabilizing surface to serve their template function. Condensation of deoxyribonucleotides in the above manner could have given rise to (a) single-stranded circular polynucleotides, (b) single-stranded linear polynucleotides perhaps terminating in non-nucleotide materials, (c) single-stranded circular polynucleotides with or without adhering complete or incomplete linear polynucleotides, (d) covalently closed doublehelical circular polynucleotides. With time it seems possible that more of the latter compounds would have accumulated due to cyclization of linear structures and the enhanced stability of the intertwined closed double-helix (Vinograd & Lebowitz, 1966). Small covalently linked circular doublehelical polynucleotides might still have acted as templates for condensation of mononucleotides when “melted”, but would have displaced the new strands on “reannealing”.
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This would propose that the initial templates for nucleic acid replication were linear and/or circular single-stranded deoxyribopolynucleotides. The circular single-stranded templates proved advantageous (see later) and were eventually utilized predominantly. These in turn were eventually replaced by closed double-helical circular polynucleotides formed by covalently closing the complementary strand formed on a single-stranded circular template. This stable, double-helical material finally became the preferred material for gene replication and for messenger nucleic acid synthesis, accepting that messenger nucleic acids may also originally have been polydeoxyribonucleotides and functioned as templates. Denatured DNA “templates” can serve as “message” in in vitro systems in the presence of certain antibiotics (McCarthy, Holland & Buck, 1966). 3. Protein Synthesis The evolutionary origin of the first protein produced under genetic direction is likely to remain for some time one of the prime dilemmas of prebiological evolution. Thermal proteins of the type synthesized by Fox (Fox, 1965; Harada & Fox, 1965) may well have been produced by volcanic action. However, thermal proteins appear to be branched structures rather than linear peptides and it is tempting to conclude that these branched thermal proteins gave rise to protein coacervates, conceivably primitive precursors of cells, rather than to regeneratable enzymes. Nevertheless coacervates of this or other types (Oparin, 1965; Smith, Bellware & Silver, 1967) may have concentrated precursors from the environment and so been necessary for further evolution. A clear distinction must be made here between genetically determined enzymes and structural proteins. Although protein-like molecules “catalysing” various chemical reactions may have been produced prior to the evolution of a coding mechanism, the first enzymes required for replication processes and themselves requiring replication were undoubtedly made under genetic direction. By contrast genetically determined cell-wall proteins were probably a later phenomenon. Under the circumstances envisaged for polynucleotide synthesis, abortive polymerization of mononucleotides may frequently have terminated by reaction of the terminal nucleotide with an activated amino acid to produce an aminoacyl polynucleotide. The resulting aminoacyl polynucleotides may have been the primitive precursors of present-day adaptors (i.e. aminoacyl transfer RNA’s). Condensation of the amino acids, directed by primitive singlestranded polynucleotides might then give rise to linear primordial proteins (cf. Nirenberg, Caskey & Levin, 1967). In this circumstance some means of selection of an amino acid by a specific adaptor must eventually have operated to permit the synthesis of a
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specific polypeptide, since without this restriction any adaptor might have carried any amino acid, leading to random insertion of amino acids into polypeptides. Such a selection might originally have been extremely crude and permitted quite gross ambiguities, but achieved specificity with evolution. An intriguing possibility is that the “messenger’‘-“adaptor” complex may originally have acted to select the amino acid most suited to producing a stable intermediate for polypeptide synthesis. This would imply that amino acids originally played a role in establishing the intermediates necessary for specific protein synthesis, leading eventually to the selection of the most appropriate adaptor (anticodon?)-amino acid relationships and to the exclusion of less stable or less satisfactory alternatives. The observation that aminoacyl tRNA’s can bind to copolymers with specificity in the absence of ribosomes is compelling evidence for the argument, logical on a priori grounds, that ribosomes were absent at this stage of evolution (Nirenberg et al., 1967). Ordered base pairing of the aminoacyl polynucleotides dictated by primordial polynucleotide messengers and involving perhaps two (Jukes, 1965) or three bases could then have assembled the amino acid residues in appropriate juxtaposition to facilitate specific polypeptide formation, much as is observed under present-day conditions. The interaction of polynucleotide template or “messenger” (itself derived from a circular single-stranded or double-stranded template) and aminoacyl polynucleotide “adaptor” may have been aided by a surface (today the ribosome) to which both polymers attached (McLaughlin et al., 1966). This attachment would have provided better stabilization and stereo-specificity for the intermediate complex while additional stability, specificity and order of attachment resulted from the interaction of bases in the template with those in the adaptors. In this way specificity for the repeated synthesis of specific polypeptides resulted, much as it does today, but with simpler entities. Protein synthesis probably occurred by nucleophilic attack of the NH, group of one amino acid on the appropriately juxtaposed protonated C==O group of the adjacent aminoacylated tRNA. If any ancient geochemical niche or coacervate contained a sufficient concentration of the relevant precursors, the production of the first polynucleotide “messenger” directed protein synthesis becomes statistically possible. We propose that in time a protein which facilitated condensation of mono- or oligonucleotides on a pre-existing messenger was generated and became the primitive precursor of DNA polymerase (“duplicase”). The first proteins were undoubtedly very small. Perhaps the first “duplicase” was little more than a crude version of the “active centre” of a modern duplicase. The striking discovery that phylogenetically ancient proteins such as Ferridoxin (Eck & Dayhoff, 1966) and cytochrome c (Cantor & Jukes,
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1966) show evidence of evolution from short repetitive amino acid sequences suggests that the initial templates were small and that the earliest polypeptides were probably small. 4. Possible Advantages of Circular Templates under Primitive Conditions Until the advent of duplicase mediated replication, little or no advantage would have accrued to cyclic templates over their linear counterparts. Subsequent to this event however, cyclic nucleic acids would have been the preferred templates; owing to the fact that a circle has neither beginning nor end, a duplicase once attached could continually produce complementary copies of the template; under the conditions envisaged detaching a duplicase from its template would render inactivation likely and its re-attachment unlikely. Re-attachment to other polynucleotides might also have occurred and not produced more duplicase. The production of a duplicase would have accelerated the rate of production of new templates and eliminated the necessity for temperature variation to separate replicating polynucleotides. Figure 2 outlines the stages through which the evolution of duplicase is postulated to have progressed. If the situation envisaged in Fig. 2(b) did in fact occur, then a cyclic model could explain one of the prime dilemmas of evolution. Watson-Crick copying merely replicates a pre-existing length of DNA. It cannot of itself increase this length. However, during the course of evolution the number of bases in DNA has increased from an unknown small number (about 10’ ?) to about 2 x 10’ in the case of a human germ cell. This represents a lo’-fold increase. Elongation of templates is quite conceivable on a cyclic model since the repeated passage of a duplicase round a circular nucleic acid could produce a polynucleotide many times longer than “unit” length (Fig. 1). In modern
FIG. 1. (@) Duplicax.
translational systems this would generate a reiterative series of base sequences and identical multiple peptide sequences in proteins. At the prebiological stage large numbers of related but differing peptide sequences would have been produced owing to coding inaccuracy. For example, under the conditions envisaged for the primitive earth mutation by radiation would have occurred with far greater frequency than under contemporary conditions.
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Furthermore, heat and/or variable pH would have increased the probability of deamination of bases. Mutation coupled with frequent mispairing (Reanney & Ralph, 1967) and variable fidelity of translation (Woese, 1965) would have made both replication and translation of polynucleotides highly variable processes. However, in this context, such variation would be a tremendous evolutionary advantage. Of the polynucleotides produced a small but statistically signz&ant fraction would contain the information needed to generate the duplicase in a segment (“cistron”) of their length while the remaining base sequences were suficiently novel to generate a wide spectrum of proteins. If one of these novel proteins happened to fulfil a useful function
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FIG. 2. The evolution of duplicase-template relationships. (a) Duplicase. (a) Nonbrought about by cycles of denaturation and subsequent conenzymic “replication” densation during temperature fluctuations. The steps between (a) and (b) are uncertain but possibly involve the introduction of an extremely primitive “duplicase” capable of facilitating condensation of mononucleotides on a pre-existing nucleic acid template but incapable per se of strand separation. (b) A possible early duplicase: this would act preferentially upon a covalently linked single-stranded nucleic acid ring from which, by displacement, serial repetitions of the complementary sequence could be produced. A likely predecessor for such a duplicase would be a less complex molecule capable of copying the parental sequence but not of displacing it so as to allow continued polynucleotide synthesis. A possible analogous mechanism still operating may be found in the-replication of FMD virus (Brown & Martin. 1965). It seems likely. however. that this staee has no functional contemporary equivalent. (c) A later duplic& now acting preferent~lly upon the more stable duplex DNA circles. The displaced DNA shown here is duplex. However, in the analogous contemporary process of transcription, only one strand is displaced. (d) A contemporary duplicase acting on duplex circular DNA as found in the E. coli genome. Elongation of the template no longer occurs.
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the combination would have (a) been selected for and (b) been potentially self-replicating. A fraction of such “multi-cistronic” polynucleotides might themselves have condensed into circular contigurations, a percentage of which would again be capable of self-replication and elongation while containing potentially novel information. It is characteristic of the bacterial genome that functionally related genes tend to occur close together. Cistrons related to one compound are often contiguous and closely mimic the reaction sequence they determine (e.g. the tryptophan operon). Others are contiguous but do not mimic the reaction sequence they determine (the histidine operon). Others are merely clustered in a small region of the chromosome while a few map in widely separated areas (the arginine operon). This type of distribution is exactly what one would expect on the basis of the statistical circularity concept presented above. It also allows for selective pressure to concentrate related genes so as to permit co-ordinate regulation of enzyme synthesis (Jacob & Monod, 1961). The selective pressure to maintain concentrated genes would have resulted in many cases from the original circumstances of their genesis. Thus the above concept has the flexibility to account for the evolutionary origin of a great number of diverse proteins together with the inbuilt constraints necessary to facilitate grouping of certain of these proteins into biosynthetic pathways. For example, the enzymes of the GAL operon all recognize galactose in diferent biochemical reactions. On the basis of the circularity hypothesis, these enzymes were generated by a process in which information was replicated, modified in varying degrees, to give a series of enzymes of related function. Whether the base sequences in the coding nucleic acid remained similar or diverged would depend upon the relative selective advantages of the proteins they specified. The presence of multiple copies of information in DNA resulting from transcription on a circular template may have infIuenced the selection of circular templates. Multiple copies of genes permit mutation to occur in one copy without loss of all function. It is likely that this would have been advantageous to the early evolutionary process, permitting a “trial and error” selection in which loss of gene function did not immediately eliminate the primitive organism. For the proposed mechanism of DNA elongation to operate plausibly, breakage and recyclization of the DNA product would be necessary (Fig. 1). The possibility of linear DNA molecules cyclizing spontaneously at high dilution has been discussed (see section 2). Under the putative primitive earth conditions selection would have favoured cyclic molecules since circular DNA molecules (e.g. circular lambda DNA) are relatively stable while long linear DNA’s are fragile and easily sheared (Smith, 1967). Thus
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cyclization would confer enhanced survival value on replicated DNA’s It would in effect “protect” information gathered during elongation. An additional advantage would arise if exposure of the earth to U.V. radiation, or other circumstances caused greatly increased numbers of breaks in DNA. Circular DNA molecules would not lose information when broken (c.f. linear DNA’s), provided cyclization rapidly occurred to “repair” the break. The above factors may have favoured the early evolution of a DNA-ligase enzyme, to catalyse (a) a rapid recyclization of elongated DNA’s and (b) immediate repair of broken DNA circles. This latter process would prevent loss of information in the event of a second break. 5. The Relevance of Circularity
in Contemporary Genomes It is probably futile to look among contemporary replicative processes for any mechanism formally analogous to that postulated to occur under conditions which may have vanished from the earth over 1.5 billion years ago. The following discussion is not meant to imply that modern circular genomes have preserved ancient mechanisms of nucleic acid replication or that such genomes have evolved specifically to meet the needs of novel situations which are evolutionary cul-de-sacs. Rather it is intended to highlight features, unique to circular nucleic acids, which may have played and may still play an important role in the selection and maintenance of genetic circularity in contemporary genomes. For primitive cells replication and translation were both statistical mechanisms (see above and Woese, 1965). As the translation apparatus became more accurate, so in proportion, would the fidelity with which the genetic information was replicated, increase. Whereas the problem of the primitive cell was to decrease variability, subsequent generations with precise modes of protein synthesis were also faced with the task of enhancing genetic variation in order to facilitate further evolution. Numerous means of promoting and storing variation have been exploited during evolution, the most effective “innovation” being the union of homologous DNA molecules within one cell and the concomitant mechanism of recombination. E. coli, normally haploid, possesses a primitive form of sexuality. Insertion of the circular F factor into the continuous bacterial chromosome enables a polarized transfer of information to take place between donor and recipient cells (Jacob & Wollman, 1961). The probability that any given marker will be transferred is a function of its distance from the F insertion site (the breakage point or origin). In assessing the mechanism of this phenomenon one basic advantage of a continuous chromosome becomes apparent. Only a circular structure can break at any point and still yield molecules of identical size. Random breakage of a linear molecule would yield fragments of varying
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size and thereby exclude many markers from transfer. For a linear molecule which did not break prior to transfer there would be an invariant gradient of transfer probability as measured from either free end. However, for a circular chromosome every marker has an equal probability of transfer provided the probability of breakage is evenly distributed around the length of the chromosome. The genomes of the different hfr strains analysed to date appear to break at different points prior to transfer (Hayes, 1964). Moreover, the fact that the polarity of transfer can be either clockwise or anticlockwise (Hayes, 1964) confers an additional plasticity. Taking the markers from azi-r to ade in the E. coli genome and assuming a break just prior to ade; if polarization were clockwise ade would have a higher probability of transfer than azi-r; assuming an identical breakage point counterclockwise polarization reverses the probability of transfer for these two markers. Thus a circular chromosome is a far more $exible instrument for promoting genetic variation while retaining the total information in the popula tion than a linear molecule of equivalent size. Circularity in the E. coli chromosome may have influenced or been influenced by selection of a circular form of the F factor which mediates
sex transfer. Circularity in the episome is economical since it ensures that the regions of homology between the F and host DNA’s are concentrated in a small area. If the episome were linear the chromosomal region of homology would presumably require separating and after breakage the broken ends of the host chromosome would have to separate appreciably to permit insertion to occur. Similar arguments apply to the reassociation of disconnected chromomeres during pachytene as envisaged by Whitehouse (1967), if in fact the cycloid model of the chromosome is correct. The modern circular genomes which seem most rigorously analogous in their function to those postulated in section 4 of this paper are those which occur in phage infected cells together with long reiterative copies of the genetic information. Brown & Martin (1965), for example, claimed to have found an RNA species apparently longer than the viral genome in baby hampster kidney cells infected with foot-and-mouth disease virus. On the basis of this finding they proposed a model for the production and extension of the viral RNA involving cyclization of the minus strand. Smith (1967) has estimated that the molecular weight of the replicating form of lambda DNA in infected cells is greater than 250 million daltons; this implies a length of many viral DNA subunits. The existence of covalently linked circular DNA in the previral nucleic acid pool is interesting and might seem to support a mechanism whereby long linear precursors of lambda DNA were generated by repeating passage of duplicase round a circular template. Covalently linked circular DNA found in infected cells could thus represent
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an obligatory but nonmaturing intermediate, prevented by virtue of its circular structure, from being broken down to mature phage DNA equivalents as occurs with the linear repetitive copies produced from it. This would account for the small quantities of cyclic material found in infected cells (Weissbach & Salzman, 1967). However, the kinetics of the appearance of the covalently closed circles and the concatenated DNA’s at present do not appear entirely consistent with a precursor relationship of the former for the latter. The recent discovery of a DNA joining enzyme or DNA ligase (Gellert, 1967; Olivera & Lehman, 1967; Goulian, Kornberg & Sinsheimer, 1967) suggests that in uiuo this enzyme may link phage DNA’s by their cohesive ends to form linear concatenated intermediates. On this basis cyclic DNA’s might result from fortuitous ring closure by DNA ligase and be by-products of no fundamental importance. Thus DNA ligase could in theory account for all the multiple length DNA’s found in infected systems, e.g. cells infected with T4 phage (Frankel, 1966) and 4X174 (Rush, KleinSchmidt, Hellmann & Warner, 1967). If any contemporary process retains analogy with the model proposed here it would not be formal replication but transcription. Unlike replication, transcription involves the displacement of a newly synthesized polynucleotide from its templating strand. This raises the interesting possibility that “transcriptase” (DNA dependent RNA polymerase) may be more closely related to the original “duplicase” than DNA polymerase itself. Transcriptase may be a close evolutionary relative of duplicase, modified to fulfil a specialized function. The third “polymerase” (DNA ligase) may also be of extreme antiquity in view of the necessity for continued cyclization of elongating DNA’s (section 4). All three enzymes have in common the ability to catalyse the formation of phosphodiester bonds. The circularity hypothesis provides an ideal mechanism for generating the three enzymes by statistical modification of a common progenitor nucleotide sequence (cf. the genesis of the GAL enzymes: section 4). Thus it will be of considerable interest to determine (a) whether the three corresponding genes are closely linked and (b) whether these enzymes retain significant homologies in primary structure. REFERENCES BERNAL, J. (1951). “The Physical Basis of Life”. London: Routledge and Kegan BUX)M, B. (1967). Perspect. Biol. Med. 10,269. BROWN, F. & MARTIN, S. (1965). Nature, Loti. 208,861. CAIRNS, J. (1963). Cold Spring Harb. Symp. quant. Biol. 28, 43. CALLAN, H. G. (1967). .I. cell Sci. 2, 1. CANTOR, C. & JUKES, T. H. (1966). Proc. narn. Acad. Sci. U.S. A. 56, 177. ~AWFORD, L. V. (1965). J. molec. Biol. 13, 362. DULBECCO, R. & VOGT, M. (1963). Proc. nutn. Acud. Sci. U.S.A. 50, 236. ECK, R. & DAYHOFF, M. (1966). Science, iV.Y. 152,363. FIERS, W. & SINSHEIMER, R. L. (1962). J. molec. Biol. 5, 424.
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FOX, S. W. (1965). In “Evolving Genes and Proteins” (V. Bryson & H. Vogel, 4~). New York: Academic Press. FRANKEL, F. R. (1966). .Z.molec. Biol. 18, 127. GELLERT, M. (1967). Proc. nutn. Acad. Sci. U.S.A. 57, 148. GOULIAN, M., KORNBERG, A. & SINSHEIMER, R. L. (1967). Proc. natn. Acad. Sci. U.S.A. 58, 2321. HARADA, K. & Fox, S. W. (1965). In “Origin of Pre-biological Systems” (S. W. FOX, ed.). New York: Academic Press. HARuNA, I. & SPIEGELMAN, S. (1965). Proc. natn. Acad. Sci. U.S.A. 54, 1189. HAYES, W. (1964). In ‘The Genetics of Bacteria and their Viruses”. Oxford: Blackwell Scientific Publications. HOTTA, Y. & BASSEL, A. (1965). Proc. natn. Acad. Sci. U.S. A. 53, 356. JACOB, F. & MONOD, J. (1961). J. molec. Biol. 3, 318. JACOB, F. & WOLLMAN, E. L. (1958). In “The Biological Replication of Macromolecules”. Sytnp. Sot. Expl. Biol. 12,15. JACOB, F. & WOLLMAN, E. L. (1961). In “Sexuality and the Genetics of Bacteria”. New York: Academic Press. JOSSE, J. & EIGN~R, J. (1966). Ann. Rev. Biochem. 35, 789. JUKES, T. H. (1965). Biochem. biophys. Res. Commun. 19, 391. KLEINSCHMIDT, A. K., BURTON, A. & SINSHEIMER, R. L. (1963). Science, N.Y. 142, 961. MCCARTHY, B. J., HOLLAND, J. J. & BUCK, C. A. (1966). Cold Spring Harb. Symp. quant. Biol. 31,683. MCLAUGHLIN, G. S., DONDON, J., GRUNBERG-MANAGO, M., MICHELSON, A. & SAUNDERS, G. (1966). Cold Spring Harb. Symp. quant. Biol. 31, 601. NAYUIR, R. & GIWAM, P. (1966). Biochemistry, N. Y. 5, 2722. NIRENBERO, M., CASKEY, T. & LEVIN, J. (1967). Abstracts. 7th Int. Con@. Biochem. Supplement I, 1068. OLIVERA, B. M. &LEHMAN, I. R. (1967). Proc. natn. Acad. Sci. U.S.A. 57, 1426. OPARIN, A. I. (1965). Adv. Enzymol. 27,347. PONNAMPERUMA, C. & PETERSON, E. (1965). Science, N. Y. 147, 1572. POUWELS, P. H. & JANSZ, H. S. (1964). Biochim. biophys. Acta, 91,177. RALPH, R. K., CONNORS, W. J., SCHALLER, H. & KHORANA, H. G. (1963). J. Am. them. Sot. 85,1983. RALPH, R. K. & KHORANA, H. G. (1961). J. Am. them. Sot. 83,2926. REANNEY, D. C. & RALPH, R. K. (1967). J. Theoret. Biol. 15,41. RITOSSA, F. M., ATWOOD, K. C. & SPIEGELMAN, S. (1966). Genetics, Princeton, 54,663. RUSH, M. G., KCEINSCHMIDT, A. K., HELLMANN, W. & WARNER, R. C. (1967). Proc. natn. Acad. Sci. U.S.A. 58,1676. SINCLAIR, J. H. & STEVENS,B. J. (1966). Proc. natn. Acad. Sci. U.S.A. 56, 508. SMITH, A. E., BELLWARE, F. T. & SILVER, J. J. (1967). Nature, Lond. 214, 1038. SMUH, M. G. (1967). IX Intemat. Congr. Microbial., Moscow, p. 483. Oxford: Pergamon Press. STEBBINS, G. L. (1966). Science, N. Y. 152, 1463. STEINMAN, G., LEMMON, R. M. & CALVIN, M. (1965). Science, N. Y. 147, 1574. STRAZIELLE, C., BENOIT, H. & HIRTH, L. (1965). J. molec. Biol. 13, 735. TENER, G. M., KHORANA, H. G., MARKHAM, R. G. & POL, E. H. (1958). J. Am. them. Sot. 80, 6223. TITOMAS, C. A. (1967). J. cell Physioi. 70, 13. THOMAS, C. A. & MACHATI’IE, L. A. (1967). Ann. Rev. Biochem. 36,485. VINOGRAD, J. & LEBOWITZ, J. (1966). J. gen. Physiol. 49, 103. WEIL, R. & VINOGRAD, J. (1963). Proc. natn. Acad. Sci. U.S.A. 50, 730. WEISSBACH, A. & SALZMAN, A. L. (1967). Proc. natn. Acad. Sci. U.S.A. 58, 1096. W~HOUSE, H. L. K. (1967). J. cell Sci. 2, 9. WOES, C. (1965). Proc. natn. Acad. Sci. U.S.A. 54, 1546. YOUNG, E. T. & SINSHEIMER, R. L. (1964). J. molec. Biol. 10, 562.