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R McMacken

Publications and source records attributed to R McMacken.

At least 37 records · Page 2Linked to original sources
PubMed1988

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.

Bacteriophage lambda↗
PubMed1986

The Escherichia coli dnaB replication protein is a DNA helicase.

Genetic and biochemical analyses indicate that the Escherichia coli dnaB replication protein functions in the propagation of replication forks in the bacterial chromosome. We have found that the dnaB protein is a DNA helicase that is capable of unwinding extensive stretches of double-stranded DNA. We constructed a partially duplex DNA substrate, containing two preformed forks of single-stranded DNA, which was used to characterize this helicase activity. The dnaB helicase depends on the presence of a hydrolyzable ribonucleoside triphosphate, is maximally stimulated by a combination of E. coli single-stranded DNA-binding protein and E. coli primase, is inhibited by antibody directed against dnaB protein, and is inhibited by prior coating of the single-stranded regions of the helicase substrate with the E. coli single-stranded DNA-binding protein. It was determined that the dnaB protein moves 5' to 3' along single-stranded DNA, apparently in a processive fashion. To invade the duplex portion of the helicase substrate, the dnaB protein requires a 3'-terminal extension of single-stranded DNA in the strand to which it is not bound. Under optimal conditions at 30 degrees C, greater than 1 kilobase pair of duplex DNA can be unwound within 30 s. Based on these findings and other available data, we propose that the dnaB protein is the primary replicative helicase of E. coli and that it actively and processively migrates along the lagging strand template, serving both to unwind the DNA duplex in advance of the leading strand and to potentiate synthesis by the bacterial primase of RNA primers for the nascent (Okazaki) fragments of the lagging strand.

Antibodies↗
PubMed1986

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.

Adenosine Triphosphate↗
PubMed1985

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.

Bacterial Proteins↗
PubMed1985

Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: complexes with lambda O protein and with lambda O, lambda P, and Escherichia coli DnaB proteins.

The O protein of bacteriophage lambda is required for initiation of DNA replication at the lambda replicative origin designated ori lambda. The binding sites for O protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage lambda O protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage lambda O and P proteins and host DnaB protein interact at ori lambda to generate a larger structure than that formed by O protein alone; P and DnaB proteins fail to form any observable complex when O protein is excluded from the reaction mixture. We conclude that the specialized nucleoprotein structure formed by phage lambda O protein and ori lambda provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure. Specialized nucleoprotein structures may be a general means to confer exceptional accuracy on DNA transactions requiring extraordinary precision.

Bacterial Proteins↗
PubMed1984

The bacteriophage lambda O and P protein initiators promote the replication of single-stranded DNA.

A soluble enzyme system that specifically initiates lambda dv plasmid DNA replication at a bacteriophage lambda replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176-6180] is also capable of replicating the single-stranded circular chromosomes of phages M13 and phi X174 to a duplex form. This chain initiation on single-stranded templates is novel in that it is absolutely dependent on the lambda O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the lambda chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein-dependent pre-priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single-stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage lambda in vivo.

Bacteriophage lambda↗
PubMed1984

Initiation of DNA synthesis on single-stranded DNA templates in vitro promoted by the bacteriophage lambda O and P replication proteins.

The bacteriophage lambda O and P protein replication initiators, in conjunction with six purified Escherichia coli replication proteins, replicate the single-stranded chromosomes of phages M13 and phi X174 to a duplex form. Several discrete steps are involved in this DNA synthesis reaction. In an ATP-dependent step that precedes priming, the lambda O and P proteins interact with the Escherichia coli dnaJ and dnaK proteins to transfer the bacterial dnaB protein onto DNA coated with single-stranded DNA binding protein. This creates a stable prepriming intermediate, isolable by gel filtration, that is rapidly primed and replicated upon the addition of primase and DNA polymerase III holoenzyme. Each of the eight proteins required for this nonspecific single strand replication reaction also have physiological roles in the replication of the bacteriophage lambda chromosome in vivo. We propose a scheme for the lambda O and P protein-dependent initiation of DNA synthesis that may be relevant to strand initiation events occurring during lambda DNA replication.

Bacteriophage lambda↗
PubMed1983

The bacteriophage lambda O replication protein: isolation and characterization of the amplified initiator.

The bacteriophage lambda O protein participates in the initiation of lambda DNA replication. The lambda O gene was cloned into plasmid pKC30 such that its expression was controlled by the lambda PL promoter. A lambda prophage-coded thermosensitive cI repressor was used to regulate transcription of the cloned O gene. Thermal inactivation of the lambda cI repressor resulted in overproduction of the O protein until it constituted approximately 20% of the total cellular protein of Escherichia coli. A simple three-step purification protocol was developed that yields several milligrams of homogeneous O protein per gram of cell paste. The precise position of the O gene in the known lambda DNA sequence was identified from the amino-terminal sequence of the isolated O protein. Purified O protein stimulated the replication of plasmid lambda dv DNA in vitro and specifically bound to duplex DNA fragments carrying the lambda replication origin.

Amino Acid Sequence↗
PubMed1983

The dnaK protein of Escherichia coli possesses an ATPase and autophosphorylating activity and is essential in an in vitro DNA replication system.

The Escherichia coli dnaK gene product, originally defined by mutations that blocked lambda phage DNA replication, is known to be necessary for E. coli viability. We have purified dnaK protein to homogeneity and have demonstrated that it possesses a weak DNA-independent ATPase activity, which results in the production of ADP and Pi. The proof that this ATPase activity is encoded by the dnaK+ gene relies primarily on the fact that the dnaK756 mutation results in the production of an ATPase activity with altered physical properties. The dnaK protein is phosphorylated in vitro and in vivo, probably as a result of an autophosphorylation reaction. The lambda O and P replication proteins were shown to interact in vitro with the dnaK protein. The ATPase activity of the dnaK protein was inhibited by purified lambda P protein and stimulated by purified lambda O protein. Moreover, the dnaK protein participates in the initiation of DNA synthesis in an in vitro DNA replication system that is dependent on the O and P proteins. Anti-dnaK protein immunoglobulin specifically inhibited DNA synthesis in this system.

Adenosine Triphosphatases↗
PubMed1982

Sequences of the Escherichia coli dnaG primase gene and regulation of its expression.

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the dnaG primase gene [Lupski, J., Smiley, B., Blattner, F. & Godson, G. N. (1982) Mol. Gen, Genet. 185, 120--128] has been determined. The region coding for the dnaG primase has been identified by NH2-terminal and tryptic peptide amino acid analysis of the dnaG protein. The coding region is 1,740 base pairs long (580 amino acids) and is preceded by an unusual ribosome-binding site sequence (G-G-G-G). The dnaG gene is read in the same direction as the adjacent rpoD gene, but no obvious promoter sequences can be found for either gene within several hundred nucleotides upstream. Other unusual features of the dnaG gene that may explain the maintenance of its product at low copy number are the presence of a RNA polymerase terminator 31 nucleotides upstream from the ATG initiator codon and greater use (3--10 times) of certain condons that occur infrequently in other E. coli genes. The nucleotide sequence has also been correlated with data from transposon Tn5 insertional inactivation mapping.

Amino Acid Sequence↗
PubMed1982

Regulation of expression of the Escherichia coli dnaG gene and amplification of the dnaG primase.

We have isolated lambda transducing phages carrying the Escherichia coli primase gene (dnaG) and mapped restriction sites in the cloned bacterial DNA segments. Several different DNA fragments containing the dnaG gene were inserted into multicopy plasmids. An analysis of the primase levels in cells harboring such plasmids indicates that sequences far upstream from the dnaG gene are required for optimal primase expression. Using this knowledge, we constructed a plasmid with a thermoinducible copy-number, pRLM61, which was employed to amplify intracellular primase levels approximately 100-fold. The dnaG gene is transcribed clockwise with respect to the E. coli genetic map, and a HindIII site located 180 base pairs upstream from the dnaG gene separates the gene from its primary promoter. An apparent transcription termination signal is positioned 30-70 base pairs in front of the primase gene. Transcription proceeds past this strong terminator only when RNA polymerase has first transcribed the bacterial DNA segment proximal to the HindIII site. We suggest that primase expression in E. coli is positively regulated by a mechanism of transcription antitermination mediated by a bacterial factor. We propose, furthermore, that the neighboring structural genes for primase and for the sigma subunit of RNA polymerase are coordinately regulated as part of an operon. This arrangement may enable the bacterial cell to readily control the level of initiation of DNA and RNA synthesis and thus to respond quickly and efficiently to changing conditions.

Bacterial Proteins↗
PubMed1982

Initiation of bacteriophage lambda DNA replication in vitro with purified lambda replication proteins.

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.

Bacteriophage lambda↗
PubMed1981

Purification and properties of Escherichia coli protein i, a prepriming protein in phi X174 DNA replication.

Protein i, one of seven Escherichia coli proteins essential for primosome initiation of DNA chains in the in vitro conversion of single-stranded phi X174 DNA to duplex replicative form, has been purified approximately 15,000-fold to more than 98% purity. The protein is an oligomer of 22,000-dalton subunits migrating as a single electrophoretic band on native, as well as on denaturing polyacrylamide gels. Estimates of a Stokes radius of 41 A, a sedimentation coefficient of 3.5 S, a Mr = 61,000, and a frictional coefficient of 1.57 suggest that native protein i is a highly asymmetric oligomer composed of three identical subunits. About 50 such molecules are present/cell. Cross-linking the protein with dimethylsuberimidate or dimethyladipimidate produced three major bands corresponding to the monomer, dimer, and trimer, as well as two minor bands corresponding to the tetramer and pentamer. Incorporation of 3H-labeled "trimeric" protein i into the prepriming replication intermediate (primosome) occurs at a stage requiring participation of dnaB and dnaC proteins, and follows the actions of proteins n, n', and n". After extension of primers by DNA polymerase III holoenzyme, protein i is not retained in the isolated primosome complex. Thus, protein i is essential in the assembly of a functional primosome, but its precise physiologic role and genetic locus are still unknown.

Amino Acids↗
PubMed1978

A multienzyme system for priming the replication of phiX174 viral DNA.

Synthesis of the oligonucleotides that prime replication of phiX174 single-stranded DNA employs complex protein machinery of the host cell which is probably used by the cell to replicate its own chromosome. Primer synthesis depends on at least five proteins (DNA binding protein, dnaB and dnaC proteins, protein i, and protein n) and ATP to form a replication intermediate and another protein, primase (dnaG protein), to assemble the oligonucleotide by template transcription. The data in this paper show that ribo- and deoxyribonucleoside triphosphates can serve as substrates and form hybrid primers when present together. Both RNA and DNA primers were initiated with ATP. At least three of the four base-pairing nucleoside triphosphates were required for the transcription that generates effective primers. Over 90% of the RNA and DNA transcripts were extended into complementary strands by DNA polymerase III holoenzyme. At optimal triphosphate concentrations, the rate and extent of primer formation were greater from ribonucleoside triphosphates than from deoxyribonucleoside triphosphates. Uncoupled from DNA replication, the length of RNA primers was 14 to 50 residues, the DNA primers 4 to 20 residues. The fingerprint pattern of an RNase digest of RNA primers has a complexity suggestive of transcription from many sites on the phiX174 template. The multienzyme priming system is highly specific for phiX174 DNA as template.

Coliphages↗
PubMed1977

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.

Adenosine Triphosphatases↗