Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis

DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.     

Characterization of strand displacement synthesis catalyzed by bacteriophage T7 DNA polymerase

The DNA polymerase induced after infection of Escherichia coli by bacteriophage T7 can exist in two forms. One distinguishing property of Form I, the elimination of nicks in double-stranded DNA templates, strongly suggests that this form of the polymerase catalyzes limited DNA synthesis at nicks, resulting in displacement of the downstream strand. In this paper, we document this reaction by a detailed characterization of the DNA product. DNA synthesis on circular, duplex DNA templates containing a single site-specific nick results in circular molecules bearing duplex branches. Analysis of newly synthesized DNA excised from the product shows that the majority of the branches are less than 500 base pairs in length and that they arise from a limited number of sites. The branches have fully base-paired termini but are attached by two noncomplementary DNA strands that have a combined length of less than 30 nucleotides. The product molecules are topologically constrained as a result of the duplex branch. DNA sequence analysis has provided an unequivocal structure of one such product molecule. We conclude that strand displacement synthesis catalyzed by Form I of T7 DNA polymerase is terminated by a template-switching reaction. We propose two distinct models for template-switching that we call primer relocation and rotational strand exchange. Strand displacement synthesis catalyzed by Form I of T7 DNA polymerase effectively converts T7 DNA circles that are held together by hydrogen bonds in their 160-nucleotide-long terminal redundancy to T7-length linear molecules. We suggest that strand displacement synthesis catalyzed by T7 DNA polymerase is essential in vivo to the processing of a T7 DNA concatemer to mature T7 genomes.     

Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB)

Single-stranded DNA binding protein is a key component in growth of bacteriophage T7. In addition, DNA synthesis by the purified in vitro replication system is markedly stimulated when the DNA template is coated with Escherichia coli single-stranded DNA binding protein (SSB). In an attempt to understand the mechanism for this stimulation, we have studied the effect of E. coli SSB on DNA synthesis by the T7 DNA polymerase using a primed single-stranded M13 DNA template which serves as a model for T7 lagging strand DNA synthesis. Polyacrylamide gel analysis of the DNA product synthesized on this template in the absence of SSB indicated that the T7 DNA polymerase pauses at many specific sites, some stronger than others. By comparing the position of pausing with the DNA sequence of this region and by using a DNA template that contains an extremely stable hairpin structure, it was found that many, but not all, of these pause positions correspond to regions of potential secondary structure. The presence of SSB during synthesis resulted in a large reduction in the frequency of hesitations at many sites that correspond to these secondary structures. However, the facts that a large percentage of the pause sites remain unaffected even at saturating levels of SSB and that SSB stimulates synthesis on a singly primed poly(dA) template suggested that other mechanisms also contribute to the stimulation of DNA synthesis caused by SSB. Using a sucrose gradient analysis, we found that SSB increases the affinity of the polymerase for single-stranded DNA that this increased binding is only noticed when the polymerase concentration is limiting. The effect of this difference in polymerase affinity was clearly observed by a polyacrylamide gel analysis of the product DNA synthesized during a limited DNA synthesis reaction using conditions where only two nucleotides are added to the primer. Under these circumstances, where the presence of hairpin structures should not contribute to the stimulatory effect of SSB, we found that the extension of the primer is stimulated 4-fold if the DNA template is coated with SSB. Furthermore, SSB had no effect on this synthesis at large polymerase to template ratios.     

Murine DNA polymerase alpha fills gaps to completion in a direct assay. Altered kinetics of de novo DNA synthesis at single nucleotide gaps

DNA polymerase alpha was studied in a direct gap-filling assay. Using a defined template, DNA synthesis was primed from the M13 17-mer universal primer and blocked by an oligonucleotide hybridized 56 nucleotides downstream of the primer. DNA polymerase alpha filled this gap to completion. A time course of the reaction showed that in 50% of the substrate molecules, gaps were filled to completion within 10 min. In another 35% of the molecules the final nucleotide was lacking after 10 min. This nucleotide was added at a reduced rate, and was not incorporated into all of the molecules even after 6 h. The reduced rate of incorporation of the final nucleotide is reflected in an increased Km for de novo incorporation of one nucleotide at a single nucleotide gap (0.7 microM), as opposed to the Km for de novo incorporation of one nucleotide into singly primed M13 DNA (0.18 microM). DNA polymerase alpha purified from murine cells infected with the parvovirus minute virus of mice, and HeLa cell DNA polymerase alpha 2, exhibited the same kinetics of gap filling as did DNA polymerase alpha purified from uninfected Ehrlich ascites murine tumor cells. T4 DNA polymerase filled gaps to completion in this assay. Escherichia coli DNA polymerase I Klenow fragment quantitatively displaced the downstream oligonucleotide, and extended nascent DNA chains for an additional 100 nucleotides. Nicks and single-nucleotide gaps produced in gap-filling reactions by murine DNA polymerase alpha and T4 DNA polymerase were sealed by T4 DNA ligase.     

DNA primase-DNA polymerase alpha assembly from mouse FM3A cells. Purification of constituting enzymes, reconstitution, and analysis of RNA priming as coupled to DNA synthesis

The mouse DNA primase-DNA polymerase alpha complex can be resolved with buffer containing 50% ethylene glycol (Suzuki, M., Enomoto, T., Hanaoka, F., and Yamada, M. (1985) J. Biochem. (Tokyo) 98, 581-584). The dissociated primase and DNA polymerase alpha have been purified sufficiently that there was no cross-contamination with each other. By the use of thus isolated DNA primase and DNA polymerase alpha in addition to DNA primase-DNA polymerase alpha complex, we have studied primer RNA synthesis and DNA elongation separately as well as the coupled reaction of the initiation and elongation of DNA chains. In the absence of deoxyribonucleoside triphosphates, the isolated primase synthesized oligoribonucleotides of an apparent length of 7-11 nucleotides (monomeric oligomer) and multiples of a modal length of 9-10 nucleotides (multimeric oligomer) and fd phage single-stranded circular DNA. Monomeric and dimeric oligomers were synthesized processively, and trimeric and larger oligomers were produced by repeated cycles of processive synthesis. The primase complexed with DNA polymerase alpha mainly synthesized monomeric and a small amount of dimeric oligomers. In the presence of deoxyribonucleoside triphosphates at concentrations above 10 microM, the DNA primase-DNA polymerase alpha complex exclusively synthesized monomeric oligomers only, which were utilized as primers for DNA synthesis. On the other hand, the products synthesized by the isolated primase were qualitatively unchanged as compared with those synthesized in the absence of DNA precursors. When the synthesis of oligomers by the isolated primase was coupled with DNA elongation by the addition of the primase-free DNA polymerase alpha, the synthesis of dimeric oligomers was inhibited as a result of efficient DNA elongation from monomeric oligomers.     

Simple methods for the 3′ biotinylation of RNA

Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3′ ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3′-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3′ ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of ϕ29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme''s proofreading activity, can cope with more extended 3′ heterogeneities.     

A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization

The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.     

Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase.

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression. Moreover, after the time taken by an unimpeded enzyme to go from promoter to terminator, the rate of RNA synthesis remained elevated and the number of complete RNA molecules (7000 nucleotides) continued to increase for some time. These results suggest that, if the E. coli RNA polymerase is stopped by an AP site, most often, after a pause, the enzyme resumes elongation of the RNA chain which is continuous over the AP site. Sometimes however, RNA synthesis is definitively interrupted during the pause; the probability of interruption has been estimated to be 0.3 in our experimental conditions. When a nick is placed 5' to the AP site by an AP endonuclease, the results are similar: most often, the RNA chain is synthesized without interruption past the nick in the template strand. The pause of the E. coli RNA polymerase at this combined lesion appears to be shorter than when the AP site is intact. To investigate whether a nucleotide is placed in the RNA chain in front of the AP site in the template strand by E. coli RNA polymerase, RNA synthesis was taken to completion before using this RNA for protein synthesis and measuring the activity of gene-1 product, T7 RNA polymerase. The result suggests that, after pausing, the E. coli RNA polymerase places a nucleotide in the RNA chain when passing over an AP site. The mechanism of the delayed lethality of T7 coliphages treated with monofunctional alkylating agents, which is due to the appearance of AP sites, is discussed.