Rescue of stalled replication forks by RecG: Simultaneous translocation on the leading and lagging strand templates supports an active DNA unwinding model of fork reversal and Holliday junction formation

Modification of damaged replication forks is emerging as a crucial factor for efficient chromosomal duplication and the avoidance of genetic instability. The RecG helicase of Escherichia coli, which is involved in recombination and DNA repair, has been postulated to act on stalled replication forks to promote replication restart via the formation of a four-stranded (Holliday) junction. Here we show that RecG can actively unwind the leading and lagging strand arms of model replication fork structures in vitro. Unwinding is achieved in each case by simultaneous interaction with and translocation along both the leading and lagging strand templates at a fork. Disruption of either of these interactions dramatically inhibits unwinding of the opposing duplex arm. Thus, RecG translocates simultaneously along two DNA strands, one with 5'-3' and the other with 3'-5' polarity. The unwinding of both nascent strands at a damaged fork, and their subsequent annealing to form a Holliday junction, may explain the ability of RecG to promote replication restart. Moreover, the preferential binding of partial forks lacking a leading strand suggests that RecG may have the ability to target stalled replication intermediates in vivo in which lagging strand synthesis has continued beyond the leading strand.     

Topological challenges to DNA replication: Conformations at the fork

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (-) supercoiled DNA that is not being replicated. The replicating DNA can form (+) precatenanes, in which the daughter DNAs are intertwined, and (+) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (+) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (-) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.     

Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.     

Coordinated DNA Replication by the Bacteriophage T4 Replisome

The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.     

Regions of Single-Stranded DNA in the Growing Points of Replicating Bacteriophage T7 Chromosomes

In partially replicated T7 chromosomes, the points where parental strands are separating and new DNA is being synthesized can be seen in the electron microscope to contain regions of single-stranded template DNA. The single-stranded regions are located on only one of the two daughter arms of the replicating chromosome. Inman and Schnös observed such single-stranded regions in 50% of the growing points of replicating lambda DNA, and, as reported in this paper, we find them in about 85% of the growing points of T7 DNA. Both studies support the conclusion that DNA synthesis involves the direct elongation of one daughter strand in the growing point. Evidently, this elongation is accompanied by the unwinding of the parental double helix to expose a region of single-stranded DNA which is then converted to the duplex state by a discontinuous mechanism involving the synthesis of DNA fragments.     

Single-strand interruptions in replicating chromosomes cause double-strand breaks

Replication-dependent chromosomal breakage suggests that replication forks occasionally run into nicks in template DNA and collapse, generating double-strand ends. To model replication fork collapse in vivo, I constructed phage lambda chromosomes carrying the nicking site of M13 bacteriophage and infected with these substrates Escherichia coli cells, producing M13 nicking enzyme. I detected double-strand breaks at the nicking sites in lambda DNA purified from these cells. The double-strand breakage depends on (i) the presence of the nicking site; (ii) the production of the nicking enzyme; and (iii) replication of the nick-containing chromosome. Replication fork collapse at nicks in template DNA explains diverse phenomena, including eukaryotic cell killing by DNA topoisomerase inhibitors and inviability of recombination-deficient vertebrate cell lines.     

A mechanism of duplex DNA replication revealed by enzymatic studies of phage phi X174: catalytic strand separation in advance of replication.

The enzyme system for duplicating the duplex, circular DNA of phage phi X174 (replicative form) in stage II of the replicative life cycle was shown to proceed in two steps: synthesis of the viral (+) strand ]stage II(+)], followed by synthesis of the complementary (-) strand ]stage II(-)] [Eisenberg et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3151-3155]. Novel features of the mechanism of the stage II(+) reaction have now been observed. The product, synthesized in extensive net quantities, is a covalently closed, circular, single-stranded DNA. The supercoiled replicative form I template and three of the four required proteins--the phage-induced cistron A protein (cis A), the host rep protein (rep), and the DNA polymerase III holoenzyme (holoenzyme)--act catalytically; the Escherichia coli DNA unwinding (or binding) protein binds the product stoichiometrically. In a reaction uncoupled from replication, cis A, rep, DNA binding protein, ATP, and Mg2+ separate the supercoiled replicative form I into its component single strands coated with DNA binding protein. In the presence of Mg2+, cis A, nicks the replicative form I; rep, ATP, and Mg2+ achieve strand separation with a concurrent cleavage of ATP and binding of DNA binding protein to the single strands. rep exhibits a single-stranded DNA-dependent ATPase activity. These observations suggest that the rep enzymatically melts the duplex at the replicating fork, using energy provided by ATP; this mechanism may apply to the replication of the E. coli chromosome as well.     

Mutagenic replication in human cell extracts of DNA containing site-specific N-2-acetylaminofluorene adducts.

We have analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of bidirectional replication of double-stranded DNA in a human cell extract. Plasmid vectors were constructed containing the simian virus 40 origin of replication and single AAF adducts at one of three guanines in the Nar I sequence GGCGCC in a lacZ reporter gene. The presence of an AAF adduct diminishes replication efficiency in HeLa cell extracts by 70-80%. Replication product analyses reveal unique termination sites with each damaged vector, suggesting that when the replication fork encounters an AAF adduct, it often stops before incorporation opposite the adduct. We also observed a higher proportion of products representing replication of the undamaged strand compared to the damaged strand. This suggests that the undamaged strand is replicated more readily, either by uncoupling the first fork to encounter the lesion or by replication using the fork arriving from the other direction. Also included among replication products are covalently closed monomer-length molecules resistant to cleavage at the AAF-modified Nar I site. This resistance is characteristic of substrates containing the AAF adduct, suggesting that translesion bypass had occurred. Transformation of Escherichia coli cells with the replicated damaged DNA yielded lacZ alpha revertant frequencies significantly above values obtained with undamaged DNA or with damaged DNA not replicated in vitro. This increase was only seen with the substrate modified at the third guanine position. Analysis of mutant DNA demonstrated the loss of a GC dinucleotide at the Nar I sequence. Generation of this position-dependent AAF-induced frameshift error in a human replication system is consistent with previous observations in E. coli suggesting that, after incorporation of dCMP opposite modified guanine in the third position, realignment of the template-primer occurs to form an intermediate with two unpaired nucleotides in the template strand.     

DNA intermediates at the Escherichia coli replication fork: effect of dUTP.

We have directly tested the hypothesis that elevated levels of dUTP cause the formation of small DNA fragments at the replication fork of Escherichia coli. Addition of increasing levels of dUTP to lysates on cellophane discs results in an increasing number of strand scissions in the newly replicated DNA. Lysates of strains defective in dUTPase produce many more scissions at the same level of dUTP. The size distribution of Okazaki pieces obtained in vivo can be reconstituted in vitro on cellophane discs if appropriate levels of dUTP are present. Although uracil excision leads to the apparent production of Okazaki pieces from both daughter strands, DNA synthesis is actually asymmetric under these conditions. De novo chain initiation events occur on only one strand. It is suggested that asymmetry of synthesis in vivo may be masked by uracil excision and other postreplication processing mechanisms.     

DNA Helicase–Polymerase Coupling in Bacteriophage DNA Replication

Bacteriophages have long been model systems to study the molecular mechanisms of DNA replication. During DNA replication, a DNA helicase and a DNA polymerase cooperatively unwind the parental DNA. By surveying recent data from three bacteriophage replication systems, we summarized the mechanistic basis of DNA replication by helicases and polymerases. Kinetic data have suggested that a polymerase or a helicase alone is a passive motor that is sensitive to the base-pairing energy of the DNA. When coupled together, the helicase–polymerase complex is able to unwind DNA actively. In bacteriophage T7, helicase and polymerase reside right at the replication fork where the parental DNA is separated into two daughter strands. The two motors pull the two daughter strands to opposite directions, while the polymerase provides a separation pin to split the fork. Although independently evolved and containing different replisome components, bacteriophage T4 replisome shares mechanistic features of Hel–Pol coupling that are similar to T7. Interestingly, in bacteriophages with a limited size of genome like Φ29, DNA polymerase itself can form a tunnel-like structure, which encircles the DNA template strand and facilitates strand displacement synthesis in the absence of a helicase. Studies on bacteriophage replication provide implications for the more complicated replication systems in bacteria, archaeal, and eukaryotic systems, as well as the RNA genome replication in RNA viruses.     

In Vivo Repair of the Single-Strand Interruptions Contained in Bacteriophage T5 DNA

Bacteriophage T5 is known to contain several unique single-strand interruptions in only one strand of the duplex DNA. Analysis of labeled parental phage DNA from infected Escherichia coli shows that these nicks are repaired in vivo to yield intact double-stranded molecules. Sealing begins at about 6 min after infection and is independent of DNA replication. Repair may be an ordered process that starts at a unique end of the molecule.     

Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaA-ATP

Initiation of DNA replication in eukaryotes, archea, and eubacteria requires interaction of structurally conserved ATP-binding initiator proteins and origin DNA to mediate assembly of replisomes. However, the specific requirement for ATP in the early steps of initiation remains unclear. This is true even for the well studied Escherichia coli replication origin, oriC, where the ATP form of initiator DnaA is necessary and sufficient for initial DNA strand separation, but the five DnaA-binding sites (R boxes) with consensus sequence 5'TGTGNAT/AAA bind both active ATP-DnaA and inactive ADP-DnaA with equal affinity. By using dimethyl sulfate footprinting, we recently identified two initiator-binding sites, I2 and I3, with sequence 5'TG/TGGATCAG/A. We now show that sites I2 and I3 preferentially bind DnaA-ATP and are required for origin unwinding. Guanine at position 3 determines DnaA-ATP preference, and changing this base to thymine at both I sites allows DnaA-ADP to bind and open oriC, although DNA strand separation is not precisely localized in the AT-rich region. These observations indicate that specific initiator binding sites within a replication origin can be important determinants of an ATP-dependent molecular switch regulating DNA strand separation.     

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.During DNA replication, it is essential to completely unwind and remove all of the intertwining between the two strands of the template DNA double helix. This is achieved by the combined action of replicative helicases and topoisomerases. During elongation, replicative helicases force the strands apart, generating compensatory topological overwinding stress in the unreplicated region ahead of the fork. If overwinding accumulates, it prevents further DNA replication (1, 2). Relaxation of the stress is achieved either by topoisomerase action ahead of the fork, directly on the overwound region, or by coupling helicase action with rotation of the whole fork relative to the unreplicated DNA (Fig. S1). This latter pathway relaxes topological stress ahead of the fork at the expense of generating double-stranded intertwines behind the fork, often referred to as DNA precatenanes (3, 4). These intertwines are subsequently resolved by the action of type II topoisomerases. If type II topoisomerases do not completely resolve either the precatenanes or the full DNA catenanes formed at the completion of replication, the unresolved intertwines will cause chromosome bridging, nondisjunction, and aneuploidy (5). Fork rotation and DNA precatenation appear to be the primary pathway of unlinking when forks come together at the termination of DNA replication (6, 7). In addition, fork rotation appears to be a frequent event during elongation in vitro; it can support ongoing replication, and extensive precatenation is observed behind elongating forks (8–10). Therefore, the prevailing view is that the topological stress caused by DNA unwinding is resolved stochastically during elongation by both topoisomerase action ahead of the fork and fork rotation and DNA decatenation behind the fork (3, 11). At termination, the diminishing distance between converging replisomes is thought to prevent topoisomerase action ahead of the fork, leaving fork rotation as the primary pathway for DNA unwinding in this context. However, unlike viral replisomes and replication complexes established in in vitro systems, the eukaryotic replisome holoenzyme is composed of a far greater number of proteins and activities. These factors are thought to facilitate replication through the highly variable eukaryotic genomic landscape and coordinate DNA synthesis with other chromatid maturation processes such as chromatin assembly and cohesion establishment (5). These latter processes act on the same newly replicated DNA potentially braided by precatenation. How these activities occurring in the wake of the fork may be impeded by precatenation is unexplored (5).Open in a separate windowFig. S1.Model of topological stress generation and relaxation during elongation of DNA replication. (A) During elongation, unwinding of the parental template separates the parental strands but does not resolve the linkages that exist between the two strands. (B) The linkages between the strands are displaced into the region ahead of the fork, leading to this becoming overwound, that is, positively supercoiled. (C and D) This tension is normally resolved by the action of either topoisomerase I (Top1) or topoisomerase II (Top2) (C), which act effectively as “swivelases” ahead of the fork to generate a relaxed replication region (D). (E) However, Champoux and Been (4) proposed a second mode of topological stress unwinding where the helical tension is relaxed by rotation of the fork to generate DNA precatenation behind the fork. Although these linkages should not arrest forward elongation of replication, it is essential that the type II topoisomerase resolve all DNA catenation before the completion of cell division.To study these events in vivo, we have directly examined fork rotation and precatenation in budding yeast. We show that fork rotation and DNA precatenation are not stochastic but rather are actively restricted to distinct contexts by the evolutionarily conserved homologs of Timeless/Tipin, Tof1/Csm3. Failure to regulate fork rotation leads to significantly elevated levels of DNA damage, particularly at known fragile sites. Therefore, the eukaryotic replisome appears to minimize rotation and precatenation, so that they are used to unwind DNA only when absolutely necessary to maintain genome stability.     

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7

Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding. We have measured the single-turnover kinetics of DNA unwinding and globally fit the data to a modified stepping model to obtain the unwinding parameters. The comparison of the unwinding properties of T7 helicase with its translocation properties on single-stranded (ss)DNA has provided insights into the mechanism of strand separation that is likely to be general for ring helicases. T7 helicase unwinds DNA with a rate of 15 bp/s, which is 9-fold slower than the translocation speed along ssDNA. T7 helicase is therefore primarily an ssDNA translocase that does not directly destabilize duplex DNA. We propose that T7 helicase achieves DNA unwinding by its ability to bind ssDNA because it translocates unidirectionally, excluding the complementary strand from its central channel. The results also imply that T7 helicase by itself is not an efficient helicase and most likely becomes proficient at unwinding when it is engaged in a replication complex.     

ϕX174 cistron A protein is a multifunctional enzyme in DNA replication

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication.The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex.The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.     

Remodeling of DNA replication structures by the branch point translocase FANCM

Fanconi anemia (FA) is a genetically heterogeneous chromosome instability syndrome associated with congenital abnormalities, bone marrow failure, and cancer predisposition. Eight FA proteins form a nuclear core complex, which promotes tolerance of DNA lesions in S phase, but the underlying mechanisms are still elusive. We reported recently that the FA core complex protein FANCM can translocate Holliday junctions. Here we show that FANCM promotes reversal of model replication forks via concerted displacement and annealing of the nascent and parental DNA strands. Fork reversal by FANCM also occurs when the lagging strand template is partially single-stranded and bound by RPA. The combined fork reversal and branch migration activities of FANCM lead to extensive regression of model replication forks. These observations provide evidence that FANCM can remodel replication fork structures and suggest a mechanism by which FANCM could promote DNA damage tolerance in S phase.     

Isolation of an intermediate which precedes dnaG RNA polymerase participation in enzymatic replication of bacteriophage phi X174 DNA.

Conversion of phi X174 single-stranded DNA to the duplex replicative form (RF) in vitro requires at least 10 purified proteins. Three stages - strand initiation, elongation, and termination - comprise this conversion. We now identify a separate stage in strand initiation which precedes dnaG RNA polymerase participation. Incubation of five proteins - protein i, protein n, DNA unwinding protein, dnaB protein, and dnaC protein - with ATP and phi X174 DNA forms an intermediate which enables subsequent stages measured by DNA synthesis to proceed 20 times faster. The intermediate can be isolated in quantitative yield by gel filtration or by ultracentrifugation. Protein i and protein n are required in less than stoichiometric amounts and appear to be absent from the isolated intermediate. Whereas formation of the intermediate is sensitive to antibody to protein i and to N-ethylmaleimide (an inhibitor of protein n and dnaC protein), the intermediate itself is resistant to these reagents. DNA unwinding protein complexes the DNA in a ratio of 60 molecules per circle. Synthesis of the intermediate appears to require stoichiometric quantities of dnaB protein and dnaC PROTEin but their presence in the intermediate has not been established as yet.     

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.     

Expression of a DNA strand initiation sequence of ColE1 plasmid in a single-stranded DNA phage.

In order to investigate initiation of H-strand (lagging strand) replication of the plasmid ColE1, the origin region fragment (Hae II-E) of ColE1 was inserted into the intergenic region of filamentous DNA phage M13 and cloned. A site capable of promoting DNA strand initiation on a single-stranded DNA template has been detected on the L-strand (leading strand) of the cloned fragment. The site, named rri-1 rifampicin-resistant initiation), directs conversion of chimeric phage single-stranded DNA to parental replicative form in the presence of rifampicin, which blocks the function of the complementary strand origin of M13. The function of rri-1 is dependent on both the dnaG and dnaB gene products. It is postulated that rri-1 might be an initiation site for synthesis of the lagging DNA strand during unidirectional replication of ColE1 DNA.