DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure Function Study of the Zinc-Binding Domain (original) (raw)
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Characterization of a Novel DNA Primase from the Salmonella typhimurium Bacteriophage SP6 †
Biochemistry, 2000
The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5′-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5′-GCA-3′. This trinucleotide sequence, containing a cryptic dA at the 3′-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. coli bacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli-or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys 3 His zinc finger motif that may be involved in DNA binding.
Primer initiation and extension by T7 DNA primase
The EMBO Journal, 2006
T7 DNA primase is composed of a catalytic RNA polymerase domain (RPD) and a zinc-binding domain (ZBD) connected by an unstructured linker. The two domains are required to initiate the synthesis of the diribonucleotide pppAC and its extension into a functional primer pppACCC (de novo synthesis), as well as for the extension of exogenous AC diribonucleotides into an ACCC primer (extension synthesis). To explore the mechanism underlying the RPD and ZBD interactions, we have changed the length of the linker between them. Wild-type T7 DNA primase is 10-fold superior in de novo synthesis compared to T7 DNA primase having a shorter linker. However, the primase having the shorter linker exhibits a twofold enhancement in its extension synthesis. T7 DNA primase does not catalyze extension synthesis by a ZBD of one subunit acting on a RPD of an adjacent subunit (trans mode), whereas de novo synthesis is feasible in this mode. We propose a mechanism for primer initiation and extension based on these findings.
Interaction of Bacteriophage T7 Gene 4 Primase with Its Template Recognition Site
Journal of Biological Chemistry, 1999
The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5-GGGTC-3, 5-TGGTC-3, 5-GTGTC-3, or 5-TTGTC-3). Although it is not copied into the oligoribonucleotides, the cytosine at the 3-position is essential for synthesis and template binding. Two nucleotides flanking the 3end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 M. The tight DNA binding (<1 M) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.
Biochemistry, 2002
Primase is an essential DNA replication enzyme in Escherichia coli and responsible for primer synthesis during lagging strand DNA replication. Although the interaction of primase with single-stranded DNA plays an important role in primer RNA and Okazaki fragment synthesis, the mechanism of DNA binding and site selection for primer synthesis remains unknown. We have analyzed the energetics of DNA binding and the mechanism of site selection for the initiation of primer RNA synthesis on the lagging strand of the replication fork. Quantitative analysis of DNA binding by primase was carried out using a number of oligonucleotide sequences: oligo(dT) 25 and a 30 bp oligonucleotide derived from bacteriophage G4 origin (G4ori-wt). Primase bound both sequences with moderate affinity (K d) 1.2-1.4 × 10-7 M); however, binding was stronger for G4ori-wt. G4ori-wt contained a CTG trinucleotide, which is a preferred site for initiation of primer synthesis. Analysis of DNA binding isotherms derived from primase binding to the oligonucleotide sequences by fluorescence anisotropy indicated that primase bound to DNA as a dimer, and this finding was further substantiated by electrophoretic mobility shift assays (EMSAs) and UV cross-linking of the primase-DNA complex. Dissection of the energetics involved in the primase-DNA interaction revealed a higher affinity of primase for DNA sequences containing the CTG triplet. This sequence preference of primase may likely be responsible for the initiation of primer synthesis in the CTG triplet sites in the E. coli lagging strand as well as in the origin of replication of bacteriophage G4.
Biochemistry, 2002
Primase is an essential DNA replication enzyme in Escherichia coli and responsible for primer synthesis during lagging strand DNA replication. Although the interaction of primase with single-stranded DNA plays an important role in primer RNA and Okazaki fragment synthesis, the mechanism of DNA binding and site selection for primer synthesis remains unknown. We have analyzed the energetics of DNA binding and the mechanism of site selection for the initiation of primer RNA synthesis on the lagging strand of the replication fork. Quantitative analysis of DNA binding by primase was carried out using a number of oligonucleotide sequences: oligo(dT) 25 and a 30 bp oligonucleotide derived from bacteriophage G4 origin (G4ori-wt). Primase bound both sequences with moderate affinity (K d) 1.2-1.4 × 10-7 M); however, binding was stronger for G4ori-wt. G4ori-wt contained a CTG trinucleotide, which is a preferred site for initiation of primer synthesis. Analysis of DNA binding isotherms derived from primase binding to the oligonucleotide sequences by fluorescence anisotropy indicated that primase bound to DNA as a dimer, and this finding was further substantiated by electrophoretic mobility shift assays (EMSAs) and UV cross-linking of the primase-DNA complex. Dissection of the energetics involved in the primase-DNA interaction revealed a higher affinity of primase for DNA sequences containing the CTG triplet. This sequence preference of primase may likely be responsible for the initiation of primer synthesis in the CTG triplet sites in the E. coli lagging strand as well as in the origin of replication of bacteriophage G4.
Structural Insight into the Specific DNA Template Binding to DnaG primase in Bacteria
Scientific reports, 2017
Bacterial primase initiates the repeated synthesis of short RNA primers that are extended by DNA polymerase to synthesize Okazaki fragments on the lagging strand at replication forks. It remains unclear how the enzyme recognizes specific initiation sites. In this study, the DnaG primase from Bacillus subtilis (BsuDnaG) was characterized and the crystal structure of the RNA polymerase domain (RPD) was determined. Structural comparisons revealed that the tethered zinc binding domain plays an important role in the interactions between primase and specific template sequence. Structural and biochemical data defined the ssDNA template binding surface as an L shape, and a model for the template ssDNA binding to primase is proposed. The flexibility of the DnaG primases from B. subtilis and G. stearothermophilus were compared, and the results implied that the intrinsic flexibility of the primase may facilitate the interactions between primase and various partners in the replisome. These resu...
Class-specific restrictions define primase interactions with DNA template and replicative helicase
Nucleic Acids Research, 2010
Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the b4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase-helicase interaction has co-evolved within each species.
Proceedings of the National Academy of Sciences, 1998
Primase and helicase activities of bacteriophage T7 are present in a single polypeptide coded by gene 4. Because the amino terminal region of the gene 4 protein contributes to primase activity, we constructed a truncated gene 4 encoding the N-terminal 271-aa residues. The truncated protein, purified from cells overexpressing the protein, is a dimer in solution; the full-length protein is a hexamer. Although the fragment is devoid of dTTPase and helicase activities, it catalyzes template-directed synthesis of di-, tri-, and tetranucleotides. The rates for tetraribonucleotide synthesis and for dinucleotide extension on a 20-nucleotide template are similar for the full-length and truncated proteins. However, the activity of the primase fragment is unaffected by dTTP whereas the primase activity of the full-length protein is stimulated >14-fold. The primase fragment is defective in the interaction with T7 DNA polymerase in that primer synthesis cannot be coupled to DNA synthesis. MATERIALS AND METHODS Reagents and Strains. Oligonucleotides were obtained from Integrated DNA Technologies (Corralville, IA), and M13 ssDNA was from New England Biolabs. The 56-kDa and 63-kDa gene 4 G64 (13) proteins and the T7 gene 2.5 protein (20) have been described. The 63-kDa gene 4 G64 protein has a glycine substituted for methionine to eliminate the start site of the 56-kDa gene 4 protein but is indistinguishable from the wild-type protein (21). T7 DNA polymerase was provided by S. Tabor (Harvard Medical School). Escherichia coli DH5␣ was obtained from GIBCO͞ BRL, and strain BL21(DE3) was obtained from Novagen. Plasmid pGP4-G64 S10 has been described (13). Mutagenesis of T7 Gene 4. Oligonucleotide primers g4R272Bam (5Ј GCG CGC GGA TCC TCA TCA TAA CGA AAG AGC CGA TAC-3Ј) and gp4Nde 5Ј-AGA TAT ACA TAT GGA CAA TTC GC-3Ј were used in the PCR to attach NdeI and BamHI restriction sites and to amplify T7 gene 4 from the plasmid pGP4-G64 S10 (13). The 950-bp product was purified from an agarose gel, was digested with NdeI and BamHI, was purified, and was ligated into the NdeI and BamHI sites of pET24a plasmid (Novagen). In the resulting plasmid, pETg4P, the truncated gene 4 was under control of a T7(lac) promoter. The plasmid pETg4P was used to transform strain DH5␣ for sequencing and strain Bl21(DE3), for protein expression. Purification of the Primase Fragment. Colonies of strain Bl21(DE3) containing pETg4P were inoculated into Luria-Bertani medium containing 60 g͞ml Kanamycin. At OD 600 of 0.5, isopropyl -D-thiogalactopyranoside was added to a final concentration of 1 mM, and the cells were grown at 37°C for 3 hr. The induced cells were harvested, washed, and collected by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase
Journal of Molecular Biology, 2006
Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or β,γ-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di-and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.