NORMAL GENOMIC

• Kusakabe T, Richardson CC
• The role of the zinc motif in sequence recognition by DNA primases.
• J Biol Chem. 1996; 271: 19563-70
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• The DNA primase of bacteriophage T7 has a zinc-binding motif that is essential for the recognition of the sequence 3'-CTG-5'. The T7 primase also catalyzes helicase activity, a reaction coupled to nucleotide hydrolysis. We have replaced the zinc motif of the T7 primase with those found in the gene 61 primase of phage T4 and the DnaG primase of Escherichia coli. The T4 and E. coli primases recognize the sequences 3'-T(C/T)G-5' and 3'-GTC-5', respectively. Both chimeric proteins can partially replace T7 primase in vivo. The two chimeric primases catalyze the synthesis of oligoribonucleotides albeit at a reduced rate and DNA dependent dTTPase activity is reduced by 3-10-fold. Both chimeric proteins recognize 3'-(A/G)CG-5' sites on single-stranded DNA, sites that differ from those recognized by the T7, T4, or E. coli primases, indicating that the zinc motif is only one determinant in site-specific recognition.

• Masai H, Arai K
• Mechanisms of primer RNA synthesis and D-loop/R-loop-dependent DNA replication in Escherichia coli.
• Biochimie. 1996; 78: 1109-17
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• In DNA replication, DNA chains are generally initiated from small pieces of ribonucleotides attached to DNA templates. These 'primers' are synthesized by various enzymatic mechanisms in Escherichia coli. Studies on primer RNA synthesis on single-stranded DNA templates containing specific 'priming signals' revealed the presence of two distinct modes, ie immobile and mobile priming. The former includes primer RNA synthesis by primase encoded by dnaG and by RNA polymerase containing a sigma 70 subunit. Priming is initiated at a specific site in immobile priming. Novel immobile priming signals were identified from various plasmid replicons, some of which function in initiation of the leading strand synthesis. The latter, on the other hands involves a protein complex, primosome, which contains DnaB, the replicative helicase for E coli chromosomal replication. Utilizing the energy fueled by ATP hydrolysis of DnaB protein, primosomes are able to translocate on a template DNA and primase synthesizes primer RNAs at multiple sites. Two distinct primosomes, DnaA-dependent and PriA-dependent, have been identified, which are differentially utilized for E coli chromosomal replication. Whereas DnaA-dependent primosome supports normal chromosomal replication from oriC, the PriA-dependent primosome functions in oriC-independent chromosomal replication observed in DNA-damaged cells or cells lacking RNaseH activity. In oriC-independent replication, PriA protein may recognize the D- or R-loop structure, respectively, to initiate assembly of a primosome which mediates primer RNA synthesis and replication fork progression.

• Tougu K, Marians KJ
• The extreme C terminus of primase is required for interaction with DnaB at the replication fork.
• J Biol Chem. 1996; 271: 21391-7
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• We have shown previously that a protein-protein interaction between DnaG and DnaB is required to attract the primase to the replication fork. This interaction was mediated by the C-terminal 16-kDa domain (p16) of the primase. A screen was developed that allowed the detection of mutant p16 proteins that did not interact with DnaB. Various mutagenesis protocols were used to localize this interaction domain to the extreme C terminus of the primase. A mutant primase missing only the C-terminal 16 amino acids was isolated and its activities examined. This mutant enzyme was fully active as a primase, but was incapable of interacting with DnaB. Thus, the mutant primase could not support DNA synthesis in either the general priming reaction or during phiX174 complementary strand DNA replication. Alanine cluster mutagenesis and deletion analysis in p16 allowed the further localization of the interaction domain to the extreme C-terminal 8 amino acids in primase.

• Griep MA
• Primase structure and function.
• Indian J Biochem Biophys. 1995; 32: 171-8
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• Primase is the ssDNA-dependent RNA polymerase that synthesizes RNA primers during DNA replication. In common with all DNA and RNA polymerases, primase has structural and functional features involved in polymer elongation. As RNA polymerase, it has structural and functional features for initiating chain synthesis. As a primase, it has structural and functional features for initiating chain synthesis on ssDNA. Using amino acid sequence analysis the structure of Escherichia coli primase responsible for binding zinc, at least three magnesium, and DnaB helicase has been identified. One of the magnesium binding motifs resembles the ?active magnesium? motif found in all DNA and RNA polymerases. This motif can be considered to be involved in phosphodiester bond formation. The region with the putatuve zinc binding motif is the most highly conserved portion, including more than 25% of identical residues among bacterial primases. The function of the zinc finger may be to bind ssDNA in a sequence-specific manner. Primase has ?RNAP? motif, a sequence found in all RNA polymerases which may be involved in chain initiation. Many of the observations concerning primer synthesis initiation in vivo have been reproduced by several of the in vitro assay systems. Important among these is that Okazaki fragments are initiated in vivo from d(CTG) most of the time. This trinucleotide initiation specificity has been shown to be an intrinsic property of pure primase in vitro. Using artificial ssDNA templates, primase has been shown to be the slowest and most error-prone polymerase yet studied. The rate-determining step is the first phosphodiester bond formed. Any protein which can influence either the dinucleotide synthesis rate or primase-ssDNA binding affinity will also play a key role in the regulation of primer synthesis initiation.

• Sun W, Tormo J, Steitz TA, Godson GN
• Domains of Escherichia coli primase: functional activity of a 47-kDa N-terminal proteolytic fragment.
• Proc Natl Acad Sci U S A. 1994; 91: 11462-6
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• Endoproteinase Asp-N cleaves the 581-amino acid Escherichia coli primase (65,564 Da) into several major fragments. One of these, a 47-kDa fragment containing the complete N terminus and the first 422 amino acids of primase, is capable of primer RNA (pRNA) synthesis in the G4oric/single-stranded DNA binding protein/primase pRNA synthesis system. A cloned 398-amino acid N-terminal fragment of primase can also synthesize pRNA. The sizes of the pRNA synthesized by these N-terminal fragments, however, are smaller than those synthesized by intact primase, suggesting that the C-terminal region of primase plays a role in processivity or regulation of pRNA synthesis. Primase mutants with the last 10 and 40 C-terminal amino acids deleted synthesize pRNA as wild-type primase, indicating that any regulatory sequences must be internal to the C terminus of primase.

• Metzger R et al.
• Characterization of the macromolecular synthesis (MMS) operon from Listeria monocytogenes.
• Gene. 1994; 151: 161-6
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• The macromolecular synthesis (MMS) operon consists of three genes: rpsU, which encodes the S21 ribosomal protein in Bacillus subtilis (Bs), rpsU is replaced by orfP23 which encodes a protein of unknown function), dnaG, encoding the DNA primase involved in the initiation of chromosome replication, and rpoD, which encodes the principal sigma subunit of RNA polymerase. The operon was cloned in three segments from Listeria monocytogenes (Lm), initially using a probe designed from a highly conserved region of RpoD. Analysis of the nucleotide sequence revealed three genes: orfP17 (whose product, P17, is homologous to Bs P23), dnaG and rpoD. The Lm DnaG resembles the primase from Escherichia coli through the first two-thirds of the sequence. C-terminal similarity was observed between DnaG from Lm and Bs. Lm RpoD is similar to Bs SigA, shares identical DNA-binding domains with SigA, and is a member of the sigma 43 subgroup of the sigma 70 family.

• Mendelman LV, Beauchamp BB, Richardson CC
• Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.
• EMBO J. 1994; 13: 3909-16
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• Gene 4 of bacteriophage T7 encodes two proteins, a 63 kDa and a colinear 56 kDa protein. The coding sequence of the 56 kDa protein begins at the residues encoding an internal methionine located 64 amino acids from the N-terminus of the 63 kDa protein. The 56 kDa gene 4 protein is a helicase and the 63 kDa gene 4 protein is a helicase and a primase. The unique 7 kDa N-terminus of the 63 kDa gene 4 protein is essential for primer synthesis and contains sequences with homology to a Cys4 metal binding motif, Cys-X2-Cys-X17-Cys-X2-Cys. The zinc content of the 63 kDa gene 4 protein is 1.1 g-atom/mol protein, while the zinc content of the 56 kDa gene 4 protein is < 0.01, as determined by atomic absorption spectrometry. A bacteriophage deleted for gene 4, T7 delta 4-1, is incapable of growing on Escherichia coli strains that contain plasmids expressing gene 4 proteins with single amino acid substitutions of Ser at each of the four conserved Cys residues (efficiency of plating, 10(-7)). Primase containing a substitution of the third Cys for Ser has been overexpressed in E. coli and purified to homogeneity. This mutant primase cannot catalyze template-directed synthesis of oligoribonucleotides although it is able to catalyze the synthesis of random diribonucleotides in a template-independent fashion. The mutant primase has reduced helicase activity although it catalyzes single-stranded DNA-dependent hydrolysis of dTTP at rates comparable with wild type primase. The zinc content of the mutant primase is 0.5 g-atom/mol protein.

• Ilyina TV, Gorbalenya AE, Koonin EV
• Organization and evolution of bacterial and bacteriophage primase-helicase systems.
• J Mol Evol. 1992; 34: 351-7
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• Amino acid sequences of primases and associated helicases involved in the DNA replication of eubacteria and bacteriophages T7, T3, T4, P4, and P22 were compared by computer-assisted methods. There are two types of such systems, the first one represented by distinct helicase and primase proteins (e.g., DnaB and DnaG proteins of Escherichia coli), and the second one by single polypeptides comprising both activities (gp4 of bacteriophages T7 and T3, and alpha protein of bacteriophage P4). Pronounced sequence similarity was revealed between approximately 250 amino acid residue N-terminal domains of stand-alone primases and the primase-helicase proteins of T7(T3) and P4. All these domains contain, close to their N-termini, a conserved Zn-finger pattern that may be implicated in template DNA recognition by the primases. In addition, they encompass five other conserved motifs some of which may be involved in substrate (NTP) binding. Significant similarity was also observed between the primase-associated helicases (DnaB, gp12 and P22 and gp41 of T4) and the C-terminal domain of T7(T3) gp4. On the other hand the C-terminal domain of P-alpha of P4 is related to another group of DNA and RNA helicases. Tentative phylogenetic trees generated for the primases and the associated helicases showed no grouping of the phage proteins, with the exception of the primase domains of bacteriophages T4 and P4. This may indicate a common origin for one-component primase-helicase systems. Two scenarios for the evolution of primase-helicase systems are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

• Lanka E, Furste JP, Yakobson E, Guiney DG
• Conserved regions at the DNA primase locus of IncP alpha and IncP beta plasmids.
• Plasmid. 1985; 14: 217-23
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• Genes specifying DNA primases (pri) are common in all IncP plasmids examined so far. These plasmids suppress the thermosensitive character of the Escherichia coli dnaG3 mutation. The mechanism of suppression appears to be identical to that known for RP4 and IncI alpha plasmids. The DNA primases of both these plasmid types can substitute for the dnaG protein in chromosomal DNA replication. The pri genes of the alpha and beta subgroup of IncP plasmids are related to each other as judged from Southern hybridization and immunological data. Extensive DNA and protein sequence homology has been detected although the gene products of the alpha and beta subgroups exhibit substantial differences in size. The arrangement of overlapping genes at the pri locus of IncP alpha plasmids also appears to be present in the IncP beta group.

• Smiley BL, Lupski JR, Svec PS, McMacken R, Godson GN
• Sequences of the Escherichia coli dnaG primase gene and regulation of its expression.
• Proc Natl Acad Sci U S A. 1982; 79: 4550-4
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• 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.