Modular Architecture of the Bacteriophage T7 Primase Couples RNA Primer Synthesis to DNA Synthesis

Harvard University, Cambridge, Massachusetts, United States
Molecular Cell (Impact Factor: 14.02). 06/2003; 11(5):1349-60. DOI: 10.1016/S1097-2765(03)00195-3
Source: PubMed


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.

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    • "Purification for the RPD was carried out following the same protocol as described for the full-length primase except that the unbound flow-through fractions from the DEAE column chromatography was collected and used for subsequent steps. The ZBDs were purified using DEAE anion exchange chromatography followed by G-50 gel filtration chromatography (6). All proteins were further cleaned on Mono Q anion exchange column to ensure that proteins were free from any nucleases. "
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    ABSTRACT: DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5′-TGGTC-3′) than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.
    Nucleic Acids Research 03/2010; 38(13):4372-83. DOI:10.1093/nar/gkq205 · 9.11 Impact Factor
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    • "Motifs IV–VI constitute the TOPRIM fold in T7 gp4 in which five residues, E157, D161, D207, D209 and D237 form an acidic patch and bind two Mg 2+ ions for catalysis. Three of these, E157, D207, and D209, are perfectly conserved in proteins that contain a TOPRIM fold [9] [10]. "
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    ABSTRACT: The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase–helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.
    Biochimica et Biophysica Acta 05/2009; 1787(5-1787):290-295. DOI:10.1016/j.bbabio.2008.11.005 · 4.66 Impact Factor
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    • "The N-terminal domain (the primase-related domain) of human TWINKLE lacks several critical motifs required for primer synthesis in the T7 gp4 protein. A zinc-binding domain (ZBD) important for DNA binding is missing [17] [18] and essential amino acids required for nucleotide polymerization are also absent [19] [20]. In accordance with this "
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    ABSTRACT: TWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis.
    Biochimica et Biophysica Acta 12/2008; 1792(2):132-9. DOI:10.1016/j.bbadis.2008.11.009 · 4.66 Impact Factor
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