Initiation of DNA replication at the primary origin of bacteriophage T7 by purified proteins: requirement for T7 RNA polymerase.

The primary origin of bacteriophage T7 DNA replication is located 15% of the distance from the left end of the T7 DNA molecule. This intergenic segment is A + T-rich, contains a single gene 4 protein recognition site, and is preceded by two tandem promoters for T7 RNA polymerase [RNA nucleotidyltransferase (DNA-directed), EC 2.7.7.6]. Analysis by electron microscopy shows that T7 DNA polymerase [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] and gene 4 protein initiate DNA synthesis at randomly located nicks on duplex DNA to produce branched molecules. However, upon the addition of T7 RNA polymerase and ribonucleoside triphosphates 14% of the product molecules have replication bubbles, all of which are located near the primary origin observed in vivo; no such initiation occurs on T7 deletion mutant LG37 DNA, which lacks the primary origin. We have also studied initiation by using plasmids into which fragments of T7 DNA have been inserted. DNA synthesis on these templates is also dependent on the presence of T7 RNA polymerase and ribonucleoside triphosphates. DNA synthesis is specific for plasmids containing the primary origin, provided they are first converted to linear forms.     

Nucleotide sequence of the region of an origin of replication of the antibiotic resistance plasmid R6K.

A 2.1-kilobase segment of the antibiotic resistance plasmid R6K carries sufficient information to replicate as a plasmid in Escherichia coli. This segment contains a functional origin of replication and a structural gene for a protein, designated pi, that is required for the initiation of R6K replication. The nucleotide sequence of a 520-base-pair portion of this 2.1-kilobase segment that includes the functional origin of replication and the region adjacent to the start of the pi structural gene was determined. A striking feature of the sequence is the presence of seven 22-base-pair direct repeats joined in tandem in the region adjacent to the start of the pi gene. A possible role of the tandem repeats in the regulation of expression of the pi protein and the control of initiation of replication of the plasmid R6K is discussed.     

RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence.

RepA, an initiation protein of R1 plasmid replication, was purified from an Escherichia coli strain overproducing the protein. The purified RepA protein specifically initiated replication in vitro of plasmid DNA bearing the replication origin of R1 plasmid (oriR). The replication, strictly dependent on added RepA protein, was independent of host RNA polymerase but required other host replication functions (DnaB and DnaC proteins, the single-stranded-DNA-binding protein SSB, and DNA gyrase). The replication was also completely dependent on the host DnaA function. In filter binding assays in high salt (0.5 M KCl) conditions, RepA specifically binds to both supercoiled and linear plasmid DNA containing the oriR sequence, whereas it binds to nonspecific DNA in low salt. DNase I-protection studies on a linearized DNA fragment revealed that DnaA protein specifically binds to a 9-base-pair DnaA-recognition sequence ("DnaA box") within oriR only when RepA is bound to the sequence immediately downstream of the DnaA box. These results indicate that initiation of R1 plasmid replication is triggered by interaction of RepA and DnaA proteins with the oriR sequence.     

Replication of lambda dv plasmid in vitro promoted by purified lambda O and P proteins.

An in vitro system for replication of lambda dv plasmid DNA has been constructed. This system consists of an ammonium sulfate fraction from Escherichia coli extract, exogenously added purified lambda O and P proteins, and lambda dv DNA in closed circular form. More than 85% of the added template DNA replicated semiconservatively. In the same system, another plasmid, pBR322, also replicated, but less efficiently than lambda dv. Furthermore, its replication was independent of O and P proteins. Inhibitors of DNA gyrase entirely blocked the replication activity, whereas rifampicin, an inhibitor of RNA polymerase, showed a significant effect only when added prior to initiation of the DNA replication. DNA replication was initiated from a region near to or within the four direct repeats in lambda origin (lambda ori) and proceeded bidirectionally, as examined by DNA chain elongation termination with dideoxy CTP. A cloned DNA carrying a 350-base-pair region including the initiation site also initiated replication, dependent on O and P proteins, and its initiation occurred at the same position as with native lambda dv DNA. An A + T-rich structure neighboring the repeats was found to be essential for lambda DNA replication. Regions corresponding to ice and oop were not required for O,P-dependent initiation.     

Physical mapping of primary and secondary origins of bacteriophage T7 DNA replication.

Deletion mutants of bacteriophage T7 have been used to identify and to map, by electron microscopy, the origins of T7 DNA replication. The primary origin of phage T7 DNA replication lies within a 100-base-pair region located 14.75-15.0% of the distance from the genetic left end of the DNA molecule. T7 phage whose DNA contains a deletion of this region initiate replication at secondary origins, the predominant one of which is located at a distance approximately 4% from the left end of the molecule.     

Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter.

Escherichia coli RNA polymerase (EC 2.7.7.6), bound in a tight complex at an early T7 promoter, protects 41 to 43 base pairs of DNA from digestion by DNase. I. The protected DNA fragment contains both the binding site for RNA polymerase and the mRNA initiation point for the promoter. The sequence of the DNA fragment and the sequence of the mRNA that it codes for are presented here. A seven-base-pair sequence, apparently common to all promoters, is implicated in the formation of a tight binary complex with RNA polymerase.     

Template recognition sequence for RNA primer synthesis by gene 4 protein of bacteriophage T7

The gene 4 protein of bacteriophage T7 recognizes specific sequences on single-stranded DNA and then catalyzes the synthesis of tetraribonucleotide primers complementary to the template. With phi X174 DNA as a template, the gene 4 protein enables T7 DNA polymerase (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) to initiate DNA synthesis at 13 major sites. DNA sequence analysis of the 5' termini of the newly synthesized DNA shows the predominant recognition sequences for the gene 4 protein to be 3'-C-T-G-G-G-5' or 3'-C-T-G-G-T-5'; the products of synthesis at these sites are RNA primers having the sequences pppA-C-C-C or pppA-C-C-A. The gene 4 protein can also synthesize primers at the sequences 3'-C-T-G-G-AC-5' and 3'-C-T-G-T-N-5', although these sites are used less than 10% as frequently as the predominant sites. Comparison of the utilization of primer sites suggests that the gene 4 protein binds randomly to single-stranded DNA and then translocates along the DNA in a unidirectional 5'-to-3' direction with regard to the DNA strand in search of recognition sequences. Models are presented for the role of the gene 4 protein in the initiation of lagging-strand synthesis and in the initiation of DNA replication at the origin.     

Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.     

Mutational analysis of the simian virus 40 replicon: pseudorevertants of mutants with a defective replication origin.

The circular genome of simian virus 40 is a model mammalian replicon, containing a unique origin of replication (ori) and coding for a protein (SV40 T antigen) known to be involved in initiation of viral DNA replication and to bind in vitro to the origin region. Mutations within the ori sequence lead to defective viral DNA replication and the formation of small viral plaques after infection of a cell monolayer. Second-site revertants (pseudorevertants) of ori mutants were isolated by random local mutagenesis of mutant DNA followed by transfection of cultured cells and the selection of large plaques. In each case, reversion of the plaque phenotype was associated with an increased rate of viral DNA replication. The second-site mutations that suppressed the replication defects were localized by in vitro recombination or marker rescue experiments to the gene for T antigen. Their map positions differ from those of previously described T antigen mutants, possibly reflecting a specific ori-binding domain of T antigen. From these results we infer that T antigen interacts with the ori signal during virus development as it does in vitro and that this interaction regulates the rate of viral DNA replication.     

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.