Structure of nascent replicative form DNA of coliphage M13.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' leads to 3' exonuclease associated uith DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand of the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.     

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.     

Replication of Bacteriophage M13: Specificity of the Escherichia coli dnaB Function for Replication of Double-Stranded M13 DNA

Infection of the temperature-sensitive E. coli mutant HfrH 165/70 (dnaB) with the filamentous single-stranded DNA phage M13 is abortive at the restrictive temperature. Upon infection at 41 degrees , single-stranded phage DNA penetrates the cell and is converted in a rifampicin-sensitive step to the double-stranded replicative form (RF). The parental RF attaches to the cell membrane, but subsequent replication of the RF is blocked. It is concluded that in M13 infection semiconservative RF replication of a double strand to a double strand, in contrast to single-stranded DNA synthesis, depends specifically on the dnaB function.     

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.     

Replication of duplex DNA of phage phi X174 reconstituted with purified enzymes.

Replication of the covalently closed duplex replicative form (RF) of phage phi X174 DNA has been achieved by coupling two known enzyme systems: (i) synthesis of viral strand circles (SS) from RF, and (ii) conversion of SS to nearly complete RF (RF II). In this coupled system, activated RF (gene A . RF II complex) was a more efficient template and generated as many as 10 RF II molecules per RF input, at a rate commensurate with SS synthesis. The 11 proteins required for the two component systems were all needed in the coupled RF duplication system; no new factors were required. Single-stranded DNA binding protein was needed for RF duplication at only 4% the level needed in its stoichiometric participation in SS synthesis. In addition to RF II, more complex replicative forms appeared late in the reaction, and their possible origin is discussed.     

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.     

An enzyme system for replication of duplex circular DNA: the replicative form of phage phi X174.

Viral single strands (SS) are converted to the duplex from (RF) by a soluble enzyme fraction uninfected Escherichia coli [Schekman et al. (1975) J. Biol. Chem. 250, 5859-5865]. When reactions were supplemented with a soluble enzyme fraction from phi X174-infected cells, replication of phi X174 superhelical RF I DNA was observed. The activity supplied by infected cells was absent in cells treated with chloramphenicol or in cells infected with a phi X174 phage mutant in cistron A (cis A). A host function coded by the rep gene, essential in vivo for RF replication (but not for SS leads to RF), was supplied by enzyme fractions from either infected or uninfected cells. Based on complementation assays, the cisA-dependent and the rep-dependent proteins have each been purified about 1000-fold. The synthetic products of the enzymatic reaction were identified as RF I and RF II in which viral (+) and complementary (-) strands were newly synthesized.     

THE PROCESS OF INFECTION WITH BACTERIOPHAGE ϕX174, XXX. REPLICATION OF DOUBLE-STRANDED ϕX DNA

Intermediates involved in the replication of double-stranded varphiX174 RF DNA have been identified and partially characterized. Analysis of pulselabeled RF DNA suggests that the synthesis of progeny RF molecules involves, in part, the addition of nucleotides to linear complementary strands on a circular parental strand as template, so as to produce intermediate DNA strands of greater than viral length. Electron microscopy reveals DNA rings with "tails" and "double rings," which could be the intermediate structures. A model is postulated for the replication process.     

Efficient correction of a mutation by use of chemically synthesized DNA.

The mutated base in the am3 lysis-defective mutant of the bacteriophage phiX174 has been corrected by a combined in vitro enzymatic DNA synthesis and in vivo replication of the heteroduplex product. Chemically synthesized oligodeoxyribonucleotides carrying the wild-type sequence have been used to prime DNA synthesis with am3 phiX174 DNA serving as a template. The resultant semisynthetic heteroduplex composed of an am3(+) strand and a wild-type (-) strand, with one mismatched base pair at position 587 on the phiX174 DNA sequence, was used to infect spheroplasts. The progeny phage were analyzed by a parallel plaque assay on wild-type host, Escherichia coli C, to screen for wild-type phenotype, and on E. coli HF4714, an amber suppressor strain, to determine the total progeny phage. When a 23-base-long synthetic primer was used, about one-third of total progeny were found to be wild type. Shorter primers yielded lower percentages of wild type; they also had poorer priming activity.     

In vitro synthesis of bacteriophage phi X174 by purified components.

An in vitro system capable of synthesizing infectious phi X174 phage particles was reconstituted from purified components. The synthesis required phi X174 supercoiled replicative form DNA, phi X174-encoded proteins A, C, J, and prohead, Escherichia coli DNA polymerase III holoenzyme, rep protein, and deoxyuridinetriphosphatase (dUTPase, dUTP nucleotidohydrolase, EC 3.6.1.23) as well as MgCl2, four deoxyribonucleoside triphosphates, and ATP. Phage production was coupled to the synthesis of viral single-stranded DNA. More than 70% of the synthesized particles sedimented at the position of mature phage in a sucrose gradient and associated with the infectivity. The simple requirement of the host proteins suggests that the mechanism of viral strand synthesis in the phage-synthesizing reaction resembles that of viral strand synthesis during the replication of replicative form DNA.     

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).     

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.     

Replication deficiencies in priA mutants of Escherichia coli lacking the primosomal replication n'' protein.

The priA gene of Escherichia coli encodes the protein that initiates assembly of the promosome, the entity essential for the replication of phage phi X174 and ColE1-like plasmids in vitro. We have prepared a null priA mutant to assess its role in vivo in replication of phages, plasmids, and the host chromosome. Extracts of this mutant are inert in the initial conversion of the phi X174 viral strand to the duplex form, confirming the absence of the PriA activity. In vivo, the priA mutant fails to produce phi X174 phage and, remarkably, is unable to maintain plasmids that depend on the E. coli chromosome origin as well as those of ColE1. Deficiencies in cell growth and cell division are also manifest.     

Integration host factor interacts with the DNA replication enhancer of filamentous phage f1.

We present data which show that the Escherichia coli integration host factor (IHF) is an activator of phage f1 DNA replication. Phage f1 poorly infects bacterial strains lacking IHF because IHF is required for efficient expression of F-pili, the receptor for f1 phage. However, when F- strains are transfected with f1 DNA the phage replicates in IHF mutants (himA, himD, or himA himD) at a rate of only 3% of that in wild-type bacteria. A plasmid dependent on the f1 replicon fails to transform IHF mutants. By gel retardation analysis, we show that IHF specifically binds to the origin of replication. DNase I "footprinting" experiments demonstrate that IHF binds to multiple sites within the replication enhancer sequence, a cis-acting, A + T-rich sequence that potentiates f1 DNA replication. Moreover, the effect of IHF mutation on f1 growth is suppressed by initiator protein (f1 gene II) mutations that restore efficient replication from origins that lack a functional replication enhancer sequence. This genetic evidence supports the conclusion that the replication enhancer sequence is the site of action of IHF.     

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.     

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.     

Enzymatic techniques for the isolation of random single-base substitutions in vitro at high frequency.

A general and efficient method has been developed to generate large numbers of single-base substitution mutations simply and rapidly. A unique f1 phage recombinant DNA cloning vector is described, which contains the phi X174 origin of viral strand DNA synthesis and allows one to direct mutagenesis to any specific segment of DNA. Gapped circular DNA is constructed by annealing viral single-stranded circular DNA [ss(c) DNA] with a mixture of linear duplex DNAs that have had their 3'-OH termini processively digested with Escherichia coli exonuclease III under conditions in which the resulting, newly generated 3'-OH termini present in the various hybrid molecules span the region of interest. Base changes are induced by misincorporation of an alpha-thiodeoxynucleoside triphosphate analog onto this primer-template, followed by DNA repair synthesis. The asymmetric segregation of mutants from wild-type sequences is accomplished by double-stranded replicative form DNA----ss(c) DNA synthesis in vitro, initiated from the phi X174 viral strand origin sequence present on the vector DNA. Mutated ss(c) DNA is screened by the dideoxy chain termination method. In one mutagenesis experiment, 21 independent single-base substitutions were isolated in a 72-nucleotide-long target region. DNA sequence analysis showed that all possible base transversions and transitions were represented.