Bacteriophage T4 DNA primase-helicase. Characterization of the DNA synthesis primed by T4 61 protein in the absence of T4 41 protein

The bacteriophage T4 61/41 protein primase-helicase is part of a seven T4 protein system needed for DNA synthesis in vitro. Although both 41 and 61 proteins are required for the synthesis and utilization of the normal pppApC(pN)3 pentanucleotide primer, we show in the accompanying paper (Hinton, D. M., and Nossal, N. G. (1987) J. Biol. Chem. 262, 10873-10878) that high concentrations of 61 protein alone carry out a limited, template-dependent oligonucleotide synthesis with the dimers pppApC and pppGpC as the major products labeled with [alpha-32P]CTP. At these high concentrations, 61 protein alone primes DNA synthesis by T4 DNA polymerase and the T4 genes 44/62 and 45 polymerase accessory proteins, or by Escherichia coli DNA polymerase I. The addition of T4 replication proteins other than 41 protein does not change the size distribution of oligonucleotides made by 61 protein. However, the primers used for DNA synthesis in the absence of 41 protein are not dimers, but rather trace quantities of longer oligonucleotides (5 to about 45 bases) which begin predominantly with pppGpC. These results show that 41 protein is required to prime with oligonucleotides beginning with pppApC and suggest that 41 protein, either alone or in conjunction with 61 protein, helps to stabilize the usual short pentamer primers on the template until they are elongated by the DNA polymerase. Moreover, since 61 protein by itself can only initiate DNA synthesis with primers beginning with pppGpC, but cannot make oligonucleotides starting with pppGpC on T4 DNA in which all the C is glucosylated and hydroxymethylated, both the T4 41 and 61 proteins are essential to prime DNA synthesis on their normal template. In our analysis of RNA-primed DNA, we demonstrate that although RNA primers at the 5' ends of DNA chains are relatively resistant to the 3' to 5' exonuclease of T4 DNA polymerase (Kurosawa, Y., and Okazaki, T. (1979) J. Mol. Biol. 135, 841-861), pppNpNpNpNpN oligomers are digested to a greater extent than the dephosphorylated pentamers NpNpNpNpN.     

Site-specific recognition of bacteriophage T4 DNA by T4 type II DNA topoisomerase and Escherichia coli DNA gyrase

The site specificity of bacteriophage T4-induced type II DNA topoisomerase action on double-stranded DNA has been explored by studying the sites where DNA cleavages are induced by the enzyme. Oxolinic acid addition increases the frequency at which phi X174 duplex DNA is cut by the enzyme by about 100-fold, to the point where nearly every topoisomerase molecule causes a double-stranded DNA cleavage event. The effect of oxolinic acid on the enzyme is very similar to its effect on another type II DNA topoisomerase, the Escherichia coli DNA gyrase. A filter-binding method was developed that allows efficient purification of topoisomerase-cleaved DNA fragments by selecting for the covalent attachment of this DNA to the enzyme. Using this method, T4 topoisomerase recognition of mutant cytosine-containing T4 DNA was found to be relatively nonspecific, whereas quite specific recognition sites were observed on native T4 DNA, which contains glucosylated hydroxymethylcytosine residues. The increased specificity of native T4 DNA recognition seems to be due entirely to the glucose modification. In contrast, E. coli DNA gyrase shows a high level of specificity for both the mutant cytosine-containing DNA and native T4 DNA, recognizing about five strong cleavage sites on both substrates. An unexpected feature of DNA recognition by the T4 topoisomerase is that the addition of the cofactor ATP strongly stimulates the topoisomerase-induced cleavage of native T4 DNA, but has only a slight effect on cleavage of cytosine-containing T4 DNA.     

Structure of the bacteriophage T4 DNA adenine methyltransferase

DNA-adenine methylation at certain GATC sites plays a pivotal role in bacterial and phage gene expression as well as bacterial virulence. We report here the crystal structures of the bacteriophage T4Dam DNA adenine methyltransferase (MTase) in a binary complex with the methyl-donor product S-adenosyl-L-homocysteine (AdoHcy) and in a ternary complex with a synthetic 12-bp DNA duplex and AdoHcy. T4Dam contains two domains: a seven-stranded catalytic domain that harbors the binding site for AdoHcy and a DNA binding domain consisting of a five-helix bundle and a beta-hairpin that is conserved in the family of GATC-related MTase orthologs. Unexpectedly, the sequence-specific T4Dam bound to DNA in a nonspecific mode that contained two Dam monomers per synthetic duplex, even though the DNA contains a single GATC site. The ternary structure provides a rare snapshot of an enzyme poised for linear diffusion along the DNA.     

Replicative Hybrid of T4 Bacteriophage DNA

Hybrid density replicative T4 DNA was isolated from CsCl, sheared, and reanalyzed in CsCl. The results rule out a branched model for T4 DNA replication and confirm that T4 DNA replicates to a conventional, semiconservative, colinear hybrid.     

Recombination-dependent DNA replication in phage T4

Studies in the 1960s implied that bacteriophage T4 tightly couples DNA replication to genetic recombination. This contradicted the prevailing wisdom of the time, which staunchly supported recombination as a simple cut-and-paste process. More-recent investigations have shown how recombination triggers DNA synthesis and why the coupling of these two processes is important. Results from T4 were instrumental in our understanding of many important replication and recombination proteins, including the newly recognized replication/recombination mediator proteins. Recombination-dependent DNA replication is crucial to the T4 life cycle as it is the major mode of DNA replication and is also central to the repair of DNA breaks and other damage.     

The bacteriophage T4 DNA injection machine

The tail of bacteriophage T4 consists of a contractile sheath surrounding a rigid tube and terminating in a multiprotein baseplate, to which the long and short tail fibers of the phage are attached. Upon binding of the fibers to their cell receptors, the baseplate undergoes a large conformational switch, which initiates sheath contraction and culminates in transfer of the phage DNA from the capsid into the host cell through the tail tube. The baseplate has a dome-shaped sixfold-symmetric structure, which is stabilized by a garland of six short tail fibers, running around the periphery of the dome. In the center of the dome, there is a membrane-puncturing device, containing three lysozyme domains, which disrupts the intermembrane peptidoglycan layer during infection.     

Template-independent ligation of single-stranded DNA by T4 DNA ligase

T4 DNA ligase is one of the workhorses of molecular biology and used in various biotechnological applications. Here we report that this ligase, unlike Escherichia coli DNA ligase, Taq DNA ligase and Ampligase, is able to join the ends of single-stranded DNA in the absence of any duplex DNA structure at the ligation site. Such nontemplated ligation of DNA oligomers catalyzed by T4 DNA ligase occurs with a very low yield, as assessed by quantitative competitive PCR, between 10(-6) and 10(-4) at oligonucleotide concentrations in the range 0.1-10 nm, and thus is insignificant in many molecular biological applications of T4 DNA ligase. However, this side reaction may be of paramount importance for diagnostic detection methods that rely on template-dependent or target-dependent DNA probe ligation in combination with amplification techniques, such as PCR or rolling-circle amplification, because it can lead to nonspecific background signals or false positives. Comparison of ligation yields obtained with substrates differing in their strandedness at the terminal segments involved in ligation shows that an acceptor duplex DNA segment bearing a 3'-hydroxy end, but lacking a 5'-phosphate end, is sufficient to play a role as a cofactor in blunt-end ligation.     

Making AppDNA using T4 DNA ligase

5('),5(')-Adenylyl pyrophosphoryl DNA (AppDNA) contains a high-energy pyrophosphate linkage and can be exploited as an activated DNA substrate to derive new DNA enzymes for carrying out various DNA modification reactions. For this reason, enzymatic synthesis of AppDNA is highly desirable. AppDNA is a known intermediate in DNA ligase mediated DNA ligation reactions, but rarely accumulates under normal reaction conditions. Here we report that T4 DNA ligase can quantitatively convert 5(')-phosphoryl DNA donor into AppDNA in the absence of acceptor DNA but in the presence of a template DNA that contains at least one unpaired nucleotide opposite the 5(')-phosphoryl DNA donor site. This adenylylation behavior of T4 DNA ligase is not observed with Thermus aquaticus (Taq) and Escherichia coli DNA ligases. We further found that a donor-template duplex of 11-bp in length is required by T4 DNA ligase for the formation of AppDNA.     

Function and assembly of the bacteriophage T4 DNA replication complex: interactions of the T4 polymerase with various model DNA constructs

Complexes formed between DNA polymerase and genomic DNA at the replication fork are key elements of the replication machinery. We used sedimentation velocity, fluorescence anisotropy, and surface plasmon resonance to measure the binding interactions between bacteriophage T4 DNA polymerase (gp43) and various model DNA constructs. These results provide quantitative insight into how this replication polymerase performs template-directed 5' --> 3' DNA synthesis and how this function is coordinated with the activities of the other proteins of the replication complex. We find that short (single- and double-stranded) DNA molecules bind a single gp43 polymerase in a nonspecific (overlap) binding mode with moderate affinity (Kd approximately 150 nm) and a binding site size of approximately 10 nucleotides for single-stranded DNA and approximately 13 bp for double-stranded DNA. In contrast, gp43 binds in a site-specific (nonoverlap) mode and significantly more tightly (Kd approximately 5 nm) to DNA constructs carrying a primer-template junction, with the polymerase covering approximately 5 nucleotides downstream and approximately 6-7 bp upstream of the 3'-primer terminus. The rate of this specific binding interaction is close to diffusion-controlled. The affinity of gp43 for the primer-template junction is modulated specifically by dNTP substrates, with the next "correct" dNTP strengthening the interaction and an incorrect dNTP weakening the observed binding. These results are discussed in terms of the individual steps of the polymerase-catalyzed single nucleotide addition cycle and the replication complex assembly process. We suggest that changes in the kinetics and thermodynamics of these steps by auxiliary replication proteins constitute a basic mechanism for protein coupling within the replication complex.     

Hydration pattern of A4T4 and T4A4 DNA: a molecular dynamics study

Hydration pattern and energetics of 'A-tract' containing duplexes have been studied using molecular dynamics on 12-mer self-complementary sequences 5'-d(GCA4T4GC)-3' and 5'-d(CGT4A4CG)-3'. The structural features for the simulated duplexes showed correlation with the corresponding experimental structures. Analysis of the hydration pattern confirmed that water network around the simulated duplexes is more conformation specific rather than sequence specific. The calculated heat capacity change upon duplex formation showed that the process is entropically driven for both the sequences. Furthermore, the theoretical free energy estimates calculated using MMPBSA approach showed a higher net electrostatic contribution for A4T4 duplex formation than for T4A4, however, energetically both the duplexes are observed to be equally stable.     

Characterization of the bacteriophage T4 gene 41 DNA helicase

The T4 gene 41 protein and the gene 61 protein function together as a primase-helicase within the seven protein bacteriophage T4 multienzyme complex that replicates duplex DNA in vitro. We have previously shown that the 41 protein is a 5' to 3' helicase that requires a single-stranded region on the 5' side of the duplex to be unwound and is stimulated by the 61 protein (Venkatesan, M., Silver L. L., and Nossal, N. G. (1982) J. biol. Chem. 257, 12426-12434). The 41 protein, in turn, is required for pentamer primer synthesis by the 61 protein. We now show that the 41 protein helicase unwinds a partially duplex DNA molecule containing a performed fork more efficiently than a DNA molecule without a fork. Optimal helicase activity requires greater than 29 nucleotides of single-stranded DNA on the 3' side of the duplex (analogous to the leading strand template). This result suggests the 41 protein helicase interacts with the leading strand template as well as the lagging strand template as it unwinds the duplex region at the replication fork. As the single-stranded DNA on the 3' side of a short duplex (51 base pairs) is lengthened, the stimulation of the 41 protein helicase by the 61 protein is diminished. However, both the 61 protein and a preformed fork are essential for efficient unwinding of longer duplex regions (650 base pairs). These findings suggest that the 61 protein promotes both the initial unwinding of the duplex to form a fork and subsequent unwinding of longer duplexes by the 41 protein. A stable protein-DNA complex, detected by a gel mobility shift of phi X174 single-stranded DNA, requires both the 41 and 61 proteins and a rNTP (preferably rATP or rGTP, the nucleotides with the greatest effect on the helicase activity). In the accompanying paper, we report the altered properties of a proteolytic fragment of the 41 protein helicase and its effect on in vitro DNA synthesis in the T4 multienzyme replication system.     

Identification of the breakage-reunion subunit of T4 DNA topoisomerase

The antitumor drug 4'-(9-acridinylamino)methanesulfon-m-anisidide which stimulates the cleavable complex formation between mammalian DNA topoisomerase II and DNA also stimulates the cleavable complex formation between bacteriophage T4-induced DNA topoisomerase and DNA. In the presence of 4'-(9-acridinylamino)methanesulfon-m-anisidide, T4 DNA topoisomerase and DNA form a "cleavable complex" which is characterized by its sensitivity to protein-denaturant treatment. Upon protein-denaturant treatment, the phosphodiester bond of DNA is cleaved, and the gene 52 protein subunit of the topoisomerase becomes covalently linked to the 5'-end of the broken DNA. The covalent protein-DNA linkage has been determined by both paper electrophoresis and thin layer chromatography to be tyrosyl phosphate.     

Control of bacteriophage T4 DNA polymerase synthesis

Analysis of sodium dodecyl sulphate/acrylamide gels of ¹⁴C-labelled proteins from phage-infected bacteria suggests the existence of a self-regulatory control mechanism in bacteriophage T4.Infection of Escherichia coli with phage T4 carrying a mutation in gene 43 (which codes for the phage DNA polymerase) results in a greatly increased rate of synthesis of the gene 43 protein. Such overproduction of defective polymerase occurs in restrictive infections with all gene 43 amber and most gene 43 temperature-sensitive mutants tested. Gene 43 protein synthesis in gene 43⁺ infections or increased synthesis in gene 43^? infections appears to require no additional function of other phage proteins essential for DNA synthesis. Functional gene 43 protein is needed continuously to keep its own levels down to normal.     

T4 DNA replication and viral gene expression

The normal dependence of “late” T4 gene expression on concurrent viral DNA replication is circumvented in cells infected with a triple mutant in which viral DNA polymerase, DNA ligase, and the exonuclease functions of genes 46 or 47 are defective. Acrylamide gel electrophoresis of labeled proteins from infected cells has made possible an extension of the analysis of replication-uncoupled T4 protein synthesis. We find a number of late T4 proteins synthesized: the products of genes 34, 37, 18, 23 and 24. Processing of the gene 23 product, normally headassembly dependent, occurs, but with considerably diminished efficiency compared to wild-type infection. Late T4 protein synthesis in replication-uncoupled infection retains a requirement for T4 gene 33 and gene 55 function. Finally, a number of “early” T4 gene products, normally shut off late in wildtype infection, continue to be synthesized late in replication-uncoupled infection, concurrently with the late proteins.