Nucleotide sequences of the separate origins of synthesis of bacteriophage G4 viral and complementary DNA strands.

Bacteriophage G4 has physically separated origins of synthesis of its viral and complementary DNA strands. Chain termination and "plus and minus" DNA sequencing methods have been used to obtain the nucleotide sequence of these two origins. The unique origin at which the complementary DNA strand is initiated has located in the untranslated region between genes F and G. This sequence, which has considerable secondary structure, contains a stretch which is complementary to the RNA primer that is observed during synthesis in vitro of the G4 complementary DNA strand [Bouché, J.P., Rowen, L. & Kornberg, A. (1978) J. Biol. Chem., in press]. This G4 origin shows extensive sequence homology with the bacteriophage lambda origin of DNA replication [Denniston-Thompson, K., Moore, D. D., Kruger, D. E., Furth, M. E. & Blattner, F. R. (1977) Science 198, 1051-1056]. The sequence around the site in gene A at which G4 viral DNA strand synthesis is initiated by the nicking action of the cistron A protein is very similar to that of bacteriophage phiX174. An (A + T)-rich stretch flanked by (G + C)-rich sequences may be involved in the interaction between the DNA and protein.     

Migration of Escherichia coli dnaB protein on the template DNA strand as a mechanism in initiating DNA replication

The first step in conversion of varphiX174 singlestranded DNA to the duplex replicative form in vitro is the synthesis of a nucleoprotein intermediate [Weiner, J. H., McMacken, R. & Kornberg, A. (1976) Proc. Natl. Acad. Sci. USA 73, 752-756]. We now demonstrate that dnaB protein (approximately one molecule per DNA circle) is an essential component of the intermediate and retains its ATPase activity. Synthesis of RNA primers, dependent on dnaG protein (primase), occurred only on DNA that had been converted to the intermediate form. In a coupled RNA priming-DNA replication reaction the first primer synthesized was extended by DNA polymerase III holoenzyme into full-length complementary strand DNA. In RNA priming uncoupled from replication, multiple RNA primers were initiated on a varphiX174 circle. The single dnaB protein molecule present on each DNA circle participated in initiation of each of the RNA primers, which appear to be aligned at regular intervals along the template strand. We propose that dnaB protein, once bound to the template, migrates in a processive fashion along the DNA strand, perhaps utilizing energy released by hydrolysis of ATP for propulsion; in this scheme the actively moving dnaB protein acts as a "mobile promoter" signal for dnaG protein (primase) to produce many RNA primers. Schemes are proposed for participation of dnaB protein both in the initiation of replication at the origin of the Escherichia coli chromosome and in the initiation of primers for nascent (Okazaki) fragments at a replication fork.     

Enzyme-catalyzed DNA unwinding: Studies on Escherichia coli rep protein

Replication in vitro of the replicative form (RF) I DNA of bacteriophage varphiX174 requires the phage-induced cistron A (cisA) protein, the host rep protein, DNA-binding protein, ATP, and DNA polymerase III plus replication factors. The rep protein is a single-stranded DNA-dependent ATPase. In this paper we show that varphiX174 RF I DNA cut by the cisA protein acts as a duplex DNA cofactor for the rep protein ATPase activity, provided that DNA-binding protein is present. In this latter reaction the duplex DNA is unwound by the rep protein with concomitant hydrolysis of ATP. The extents of ATP hydrolysis, DNA unwinding, and, where appropriate, DNA synthesis are proportional to the amounts of DNA-binding protein present. Two ATP molecules are hydrolyzed per base pair unwound. We propose that the obligatory requirement for the cisA protein in the unwinding of varphiX174 RF I DNA is not simply due to its endonuclease activity but rather is due to its provision of a site for the binding of the rep protein. The rep protein in the presence of DNA-binding protein, but in the absence of cisA protein, unwinds duplex DNA when one strand extends to generate a single-stranded leader region preceding the duplex. We show that rep protein translocates along the leader single strand in a 5'-to-3' direction only and then invades the duplex DNA. The rep protein shows a directional specificity for translocation and unwinding. A model is presented to explain the mechanism of DNA unwinding catalyzed by the rep protein.     

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.     

Viable deletions of the M13 complementary strand origin

The single-stranded DNA of bacteriophage M13 is converted to a duplex replicative form by a mechanism involving RNA-primed initiation at a single unique site on the viral DNA. The DNA sequence that specifies the RNA primer is contained largely within one of two adjacent hairpin structures protected from DNase degradation by RNA polymerase. We have used in vitro techniques to construct a series of M13 mutants having deletions in the region of the complementary strand origin. Deletions of the duplex replicative form DNA range in size from 54 to 201 base pairs. The largest deletions remove both of the RNA polymerase-protected hairpins and the entire sequence specifying the primer RNA. Mutants lacking one or both hairpins form faint plaques, give reduced phage yields, and show a lag in phage production of >30 min. The rate of conversion of the single-stranded viral DNA to the parental replicative form is reduced both in vivo and in vitro. These results indicate that both the RNA polymerase-protected hairpins and the RNA primer-coding sequence are important, but not essential, for replication. Other sequences within the origin region, or possibly elsewhere in the genome, may play a role in complementary strand initiation in these mutant phages. The M13 viral strand is initiated by extension of the 3′ terminus generated by site-specific nicking of the viral strand of the replicative form DNA by the M13 gene II protein. This specific nicking site is retained in all of the M13 deletion mutants. Deletion end points do not extend into a 13-nucleotide sequence preceding the viral strand nicking site. We propose that a sequence including these 13 nucleotides is required for gene II protein action at this site.     

Cis-Limited Action of the Gene-A Product of Bacteriophage ϕX174 and the Essential Bacterial Site

Parental replicative-form (RF(*)) DNA of bacteriophage varphiX174 in a replication-deficient host cell (rep(3) (-)) exhibits two characteristic features that correlate the function of viral gene A with the initiation of viral DNA replication: a specific discontinuity in the viral strand of a constant number of RF molecules and elongation of the viral strand to yield replicative-intermediate DNA forms with single-stranded tails. At high multiplicities of infection, these initiation events are limited to an average of four specifically nicked RFII molecules per cell. The limiting factor from the host cell may be related (or identical) to the essential bacterial sites known to limit the participation of parental genomes in RF replication. Double-infection experiments with wild-type phage and phage carrying an amber mutation in gene A show that the formation of gene A-specific RFII and RI is cis-limited to only the wild-type DNA. These results provide a basis at the DNA level for the known asymmetric complementation of gene A.     

Conservation of the primosome in successive stages of phi X174 DNA replication.

Synthesis of a complementary strand to match the single-stranded, circular, viral (+) DNA strand of phage phi X174 creates a parental duplex circle (replicative form, RF). This synthesis is initiated by the assembly and action of a priming system, called the primosome [Arai, K. & Kornberg, A (1981) Proc. Natl. Acad. Sci. USA 78, 69-73; Arai, K., Low, R. L. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 707-711]. Of the seven proteins that participate in the assembly and function of the primosome, most all of the components remain even after the DNA duplex is completed and covalently sealed. Remarkably, the primosome in the isolated RF obviates the need for supercoiling of RF by DNA gyrase, an action previously considered essential for the site-specific cleavage by gene A protein that starts viral strand synthesis in the second stage of phi X174 DNA replication. Finally, priming of the synthesis of complementary strands on the nascent viral strands to produce many copies of progeny RF utilizes the same primosome, requiring the addition only of prepriming protein i. thus a single primosome, which becomes associated with the incoming viral DNA in the initial stage of replication, may function repeatedly in the initiation of complementary strands at the subsequent stage of RF multiplication. These patterns of phi X174 DNA replication suggest that a conserved primosome also functions in the progress of the replicating fork of the Escherichia coli chromosome, particularly in initiating the synthesis of nascent (Okazaki) fragments.     

Conversion of ϕX174 Viral DNA to Double-Stranded Form by Purified Escherichia coli Proteins

The E. coli proteins that catalyze the conversion of varphiX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires varphiX174 viral DNA, dNTPs, Mg(+2), and ATP. The product synthesized is full-length linear varphiX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and varphiX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.     

Replication of the plasmid pBR322 under the control of a cloned replication origin from the single-stranded DNA phage M13.

The replication origins of viral and complementary strands of bacteriophage M13 DNA are contained within a 507-nucleotide intergenic region of the viral genome. Chimeric plasmids have been constructed by inserting restriction endonuclease fragments of the M13 intergenic region into the plasmid pBR322. Replication of these hybrid plasmids, under conditions not permissive for the plasmid replicon, depends on specific segments of the M13 origin region and on the presence of M13 helper virus. Thus M13-infected polA- Escherichia coli can be transformed to ampicillin resistance by hybrid plasmids that have a functional M13 origin. Cells transformed to drug resistance by plasmids bearing M13 origin sequences contain the duplex chimeric DNA at high copy number but do not accumulate significant amounts of single-stranded plasmid DNA. Rare transducing phages carrying single-stranded chimeric DNA are produced and can be detected by their ability to transduce cells to ampicillin resistance. Plasmids containing a 270-nucleotide fragment from the gene II-proximal half of the intergenic region produce transformants at high frequency under nonpermissive conditions. A central Hae III fragment, Hae III-G, containing the nucleotide sequence coding for the RNA primer for the complementary strand and the nicking site for gene II protein, is sufficient for plasmid replication in M13-infected polA- cells but not for high frequency transformation. Additional sequence information on the gene II side of the Hae III-G fragment is necessary for efficient transformation by the plasmid DNA.     

PRE-FORK SYNTHESIS: A MODEL FOR DNA REPLICATION

A model of DNA replication is presented in which DNA synthesis is continuously initiated from parental strand nicks and occurs, with conservation of helix winding number, ahead of the so-called replicating fork. The fork in this model is the locus of unwinding of already replicated, but presumably unstable, DNA. The model, involving Okazaki's notion of multiple initiation, is based upon the properties of Kornberg's DNA polymerase and accounts for the presence of single-stranded nascent DNA fragments in cell lysates. In addition to acting as sites of initiation, the parental strand nicks are implicated as sites of free rotation allowing unwinding of the replicated DNA.     

Initiation of DNA replication on single-stranded DNA templates catalyzed by purified replication proteins of bacteriophage lambda and Escherichia coli.

Initiation of bacteriophage lambda DNA replication at the chromosomal origin depends on the lambda O and P replication proteins. These two viral initiators, together with an Escherichia coli protein fraction, promote the replication in vitro of single-stranded circular DNA chromosomes such as that of bacteriophage M13. This nonspecific strand initiation reaction, which we have termed the "lambda single-strand replication reaction," has now been established with eight purified proteins, each of which is also required for replication of the phage lambda chromosome in vivo. An early rate-limiting step in the overall reaction is the ATP-dependent assembly of an activated nucleoprotein prepriming complex. In this step the lambda O and P initiators cooperate with the E. coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto M13 DNA that is coated with the single-stranded DNA-binding protein. Multiple RNA primers are synthesized on each DNA circle when isolated prepriming complex is incubated with primase and rNTPs. In the complete system, DNA polymerase III holoenzyme extends the first primer synthesized into full-length complementary strands. Because the properties of this system are closely analogous to those found for the replication of phi X174 viral DNA by E. coli proteins, we infer that a mobile prepriming or priming complex (primosome) operates in the lambda single-strand replication reaction.     

DNA sequences required for the in vitro replication of adenovirus DNA.

Initiation of adenovirus (Ad) DNA replication occurs on viral DNA containing a 55-kilodalton (kDa) protein at the 5' terminus of each viral DNA strand and on plasmid DNAs containing the origin of Ad replication but lacking the 55-kDa terminal protein (TP). Initiation of replication proceeds via the synthesis of a covalent complex between an 80-kDa precursor to the TP (pTP) and the 5'-terminal deoxynucleotide, dCMP. Formation of the covalent pTP-dCMP initiation complex with Ad DNA as the template requires the viral-encoded pTP and DNA polymerase and, in the presence of the Ad DNA binding protein, is dependent upon a 47-kDa host protein, nuclear factor I. Initiation of replication with recombinant plasmid templates requires the aforementioned proteins and an additional host protein, factor pL. Deletion mutants of the Ad DNA replication origin contained within the 6.6-kilobase plasmid pLA1 were used to analyze the nucleotide sequences required for the formation and subsequent elongation of the pTP-dCMP initiation complex. The existence of two domains within the first 50 base pairs of the Ad genome, both of which are required for the efficient use of recombinant DNA molecules as templates in an in vitro DNA replication system, was demonstrated. The first domain, consisting of a 10-base-pair "core" sequence located at nucleotide positions 9-18, has been identified tentatively as a binding site for the pTP [ Rijinders , A. W. M., van Bergen, B. G. M., van der Vliet , P. C. & Sussenbach , J. S. (1983) Nucleic Acids Res. 11, 8777-8789]. The second domain, consisting of a 32-base-pair region spanning nucleotides 17-48, was shown to be essential for the binding of nuclear factor I.     

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.     

Origin and direction of synthesis of bacteriophage fl DNA.

The origin and direction of synthesis in vivo of the viral and complementary strands of f1 DNA were studied by measuring the distribution of radioactivity along the genome after a short pulse of [3H]thymidine. The results indicate that the origins of replication of viral and complementary strands are located close to one another, probably both within a restriction fragment (HaeIII-G) which is about 120 bases long. Replication of both viral and complementary strands proceeds in the 5' leads to 3' overall direction. Thus, the viral strand is elongated in the counterclockwise and the complementary strand in the clockwise direction on the standard genetic map. A model is proposed in which only two mechanisms are invoked to generate all f1 DNA species: (1) the conversion of single-stranded viral DNA into double-stranded molecules and (2) the synthesis of viral single strands from double strands.     

Reconstruction of bacteriophage T4 DNA replication apparatus from purified components: rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.

The protein products of T4 bacteriophage genes 41, 43, 45, 44, and 62 have been purified to near homogeneity using an assay which measures their stimulation of DNA synthesis in a crude lysate of Escherichia coli cells in fected by an appropriate mutant phage. When all of these proteins and T4 gene 32 protein are incubated in the presence of deoxyribonucleoside and ribonucleoside triphosphates, extensive DNA synthesis occurs on both single and double-stranded DNA templates. Analysis of this in vitro system reveals most of the features attributed to in vivo DNA replication: (1) De novo DNA chain initiation is found on a single-stranded DNA template only if ribonucleoside triphosphates are present (as expected for RNA priming of Okazaki pieces on the "lagging" strand of a replication fork). (2) With single-stranded circular DNA as template, synthesis continues for many doublings. The products after extensive synthesis resemble a rolling circle as visualized in the electron microscope, with discontinuous "lagging" strand synthesis generating a long, unbranched double-stranded tail. The fact that all six mutationally identified T4 replication gene products are required for these syntheses suggests the existence of a large multienzyme complex, constituting the T4 replication apparatus.     

In vitro assembly of relaxosomes at the transfer origin of plasmid RP4.

During initiation of conjugative transfer of DNA containing the transfer origin (oriT) of the promiscuous plasmid RP4, the proteins TraI, TraJ, and TraH interact and assemble a specialized nucleoprotein complex (the relaxosome) at oriT. The structure can be visualized on electron micrographs. Site- and strand-specific nicking at the transfer origin in vitro is dependent on the proteins TraI and TraJ and on Mg2+ ions. Substrate specificity is directed exclusively towards the cognate transfer origin: the RP4-specified TraJ protein cannot recognize the closely related oriT of plasmid R751. After nicking, TraI protein remains attached to the 5'-terminal 2'-deoxycytidyl residue at the nic site [Pansegrau, W., Ziegelin, G. & Lanka, E. (1990) J. Biol. Chem. 265, 10637-10644]. Nicking and relaxosome formation require supercoiled DNA. Thus, a complicated structure involving multiple plasmid-specified proteins and a defined region of DNA must be formed at the transfer origin to prepare the plasmid for generating the single strand to be transferred.     

Replication of single-stranded plasmid pT181 DNA in vitro.

Plasmid pT181 is a 4437-base-pair, multicopy plasmid of Staphylococcus aureus that encodes tetracycline resistance. The replication of the leading strand of pT181 DNA initiates by covalent extension of a site-specific nick generated by the initiator protein at the origin of replication and proceeds by an asymmetric rolling circle mechanism. The origin of the leading strand synthesis also serves as the site for termination of replication. Replication of pT181 DNA in vivo and in vitro has been shown to generate a single-stranded intermediate that corresponds to the leading strand of the DNA. In vivo results have suggested that a palindromic sequence, palA, located near the leading strand termination site acts as the lagging strand origin. In this paper we report the development and characterization of an in vitro system for the replication of single-stranded pT181 DNA. Synthesis of the lagging strand of pT181 proceeded in the absence of the leading strand synthesis and did not require the pT181-encoded initiator protein, RepC. The replication of the lagging strand required RNA polymerase-dependent synthesis of an RNA primer. Replication of single-stranded pT181 DNA was found to be greatly stimulated in the presence of the palA sequence. We also show that palA acts as the lagging strand origin and that DNA synthesis initiates within this region.     

Identification of a primosome assembly site in the region of the ori 2 replication origin of the Escherichia coli mini-F plasmid.

A primosome assembly site for F plasmid DNA replication has been identified. This site, which we term rriA (F), is localized to one strand of a 385-base-pair Sau3A restriction fragment very close to ori 2 and within the 2.25-kilobase DNA sequence required for replication and incompatibility of the entire F plasmid. rriA (F) was isolated by cloning into the deletion phage vector M13 delta Elac. This phage forms very faint plaques due to a deletion of the M13 complementary strand origin but forms large wild-type plaques when DNA single-strand initiation determinants are inserted. The single-stranded viral DNA of the Sau3A F-M13 delta Elac recombinant provides an effector site of dATP hydrolysis by the primosomal protein n'. It also provides an assembly site for the Escherichia coli primosome protein complex that directs the in vitro conversion of the single-stranded DNA to a double-stranded form by the same mechanism as that used by phi X174. Homologies of the nucleotide sequence between this F DNA sequence and the previously identified primosome assembly sites in phi X174 phage DNA and in ColE1 plasmid DNA (rriA and rriB) have been found. The sequences 5' G-T-G-A-G-C-G 3' and 5' G-N-G-G-A-A-G-C 3' or variations of these sequences occur from two to five times within each assembly locus. In addition, two distinct 15-base-pair sequences in rriA (F) are perfectly homologous to corresponding sequences in rriA (ColE1).     

On the molecular mechanisms of transposition

We present a model for transposition that allows a choice between cointegrate formation (replicon fusion) and direct transposition. We propose that initiation of the process occurs by invasion of the target DNA by a single-stranded end of the transposable element. This leads to nicking of one of the DNA strands of the target molecule and ligation of this strand to that of the invading transposon. Transposition then occurs in a processive way by replication of the element from the invading end into the target site in a looped rolling-circle mode similar to replication of phage phi X174 replicative form to viral strand. The choice between cointegrate formation and direct transposition occurs at the nick-ligation step, which terminates the process. We suggest that the choice is determined by the topology of the transposition enzymes and could be related to whether the element generates five- or nine-base-pair repeats in the target DNA on insertion.     

A DNA fragment from the origin of single-strand to double-strand DNA replication of bacteriophage fd.

A specific complex is formed between fd DNA, Escherichia coli DNA unwinding protein, and RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) during the first steps in the conversion of the single-stranded viral DNA to the double-stranded replicative form. In this complex a unique DNA fragment of about 120 nucleotides is protected against nuclease digestion. Both the requirements for its isolation and its position on the map of the phage genome indicate that the fragment contains the origin of single-strand to double-strand DNA replication. The isolated DNA fragment possesses double-strand-like characteristics, which protect it from being covered by the DNA unwinding protein and thus indirectly positions the RNA polymerase to the origin of replication.