Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.

Mendelman, Lynn V.; Beauchamp, Benjamin; Richardson, Charles C.

doi:10.1002/j.1460-2075.1994.tb06702.x

Cited by 66 publications

(120 citation statements)

References 50 publications

(11 reference statements)

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“…Four highly conserved cysteine residues within the ZBD coordinate a zinc atom that is undoubtedly important for protein folding (15). These cysteines were not mutated because the bound metal is required for the interaction of the primase with the DNA template (31). We confirmed by CD spectroscopy that the alanine scanning mutants of the ZBD are folded and yield a CD spectrum similar to that of the wild-type ZBD.…”

Section: T7 Primase-polymerase Complexmentioning

confidence: 72%

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Kato

Ito

Wagner

et al. 2004

Journal of Biological Chemistry

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The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 singlestranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.DNA synthesis is initiated on the lagging strand of the replication fork by a site-specific RNA polymerase known as a DNA primase (1). The primase synthesizes a short RNA primer that is extended by DNA polymerase to create an Okazaki fragment (2, 3). During replication, interactions between the primase and other replicative proteins such as DNA polymerase, DNA helicase, and single-stranded DNA (ssDNA) 1 -binding protein are required to initiate DNA synthesis. It has been difficult to identify these essential interactions because they occur transiently and involve weak interactions between multiple partners.We have been studying the physical and functional interactions between proteins of the comparatively simple bacteriophage T7 replication system. Phage T7 replicates DNA using four proteins, T7 gene 4 primase-helicase protein (gp4), T7 gene 2.5 ssDNA-binding protein (gp2.5), and T7 DNA polymerase (gp5) complexed with its processivity factor, Escherichia coli thioredoxin (4). The reactions catalyzed by the individual proteins are coordinated during replication by the assembly of a multiprotein complex that moves with the replication fork (5, 6). During replication, the primase-helicase directly contacts both the DNA polymerase and gp2.5 (7-10). A C-terminal acidic segment of the primase-helicase is required for its interaction with the DNA polymerase (10). An interaction between gp2.5 and T7 DNA polymerase is required for the coordinated synthesis of both leading and lagging strands of the replication fork (5, 6). A C-terminal 21-residue region of gp2.5 is essential not only for the interactions with the DNA polymerase and the primase-helicase but also for dimerization of gp2.5 (9). Although many pairwise interactions between T7 replication proteins have been identified, the overall physical arrangement of subunits and their stoichiometries in the replication complex are unknown.Crystal structures of all the individual T7 replication proteins (11-16) have been determined, and their enzymatic ac...

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Section: T7 Primase-polymerase Complexmentioning

confidence: 72%

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Kato

Ito

Wagner

et al. 2004

Journal of Biological Chemistry

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“…3). By analogy to the phage T7 gene 4 product (gp4), the motif might be involved in Zn 2ϩ binding and play a role in primase activity (6,37,41). In another region of gp␣, an amino acid sequence (positions 501 to 508; GPGGSGKS) matches the type A NBS found in other DNA helicases (20,61).…”

Section: Resultsmentioning

confidence: 99%

“…This could be tested by using an N-terminal gp␣ peptide variant of gp␣⌬2 carrying a mutation in the proposed metal ion-binding cluster. The T7 gene 4 protein (63 kDa, 567 residues) combines two functions, primase and helicase, within one polypeptide chain, which are probably organized as two functional and structural domains (37,38). The primase domain resides in the N-terminal and the helicase in the C-terminal portion, as was suggested by sequence comparison.…”

Section: Discussionmentioning

confidence: 99%

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Domain structure of phage P4 alpha protein deduced by mutational analysis

et al. 1995

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Bacteriophage P4 DNA replication depends on the product of the ␣ gene, which has origin recognition ability, DNA helicase activity, and DNA primase activity. One temperature-sensitive and four amber mutations that eliminate DNA replication in vivo were sequenced and located in the ␣ gene. Sequence analysis of the entire gene predicted a domain structure for the ␣ polypeptide chain (777 amino acid residues, M r 84,900), with the N terminus providing the catalytic activity for the primase and the middle part providing that for the helicase/nucleoside triphosphatase. This model was confirmed experimentally in vivo and in vitro. In addition, the ori DNA recognition ability was found to be associated with the C-terminal third of the ␣ polypeptide chain. The type A nucleotide-binding site is required for P4 replication in vivo, as shown for ␣ mutations at G-506 and K-507. In the absence of an active DnaG protein, the primase function is also essential for P4 replication. Primase-null and helicase-null mutants retain the two remaining activities functionally in vitro and in vivo. The latter was demonstrated by trans complementation studies, indicating the assembly of active P4 replisomes by a primase-null and a helicase-null mutant.Bacteriophage P4 is a temperate satellite phage of Escherichia coli that relies on morphogenetic and lysis functions of the helper phage P2 for its propagation (30). The P4 prophage state is maintained either by integration into the host chromosome or by autonomous replication as a multicopy plasmid. Lysogenization of the host, DNA replication during the lytic cycle, and establishment of the plasmid state all occur independently of helper phage (30). Replication of P4 DNA requires only the P4 ␣ gene product (gp␣) and two cis-acting elements, namely, the origin of replication ori and the sequence-related region crr (cis replication region) (7,26,30,63). Another P4 gene, cnr (copy number regulation), maps directly upstream of ␣; its product has been suggested to control P4 DNA replication, in particular at the plasmid level, maybe by direct interaction of Cnr with gp␣ (59). Thus, protein-protein interaction might modulate at least one specific function of the ␣ protein.The ␣ protein is a multifunctional phage replication protein that recognizes ori and crr by binding to octamer sequences (type I repeats, TGTTCACC [16]) (64). In addition, gp␣ catalyzes the unwinding of DNA via its helicase activity. DNA is unwound with a 3Ј to 5Ј polarity with respect to the strand that gp␣ has bound. The helicase action is fueled by nucleoside triphosphate (NTP) hydrolysis of an interrelated singlestranded DNA-dependent NTPase. gp␣ also functions as a primase, with a template-dependent oligonucleotide-synthesizing activity (56, 64). The primase activity of gp␣ can be replaced by the primase of the host, the DnaG protein (56). However, the P4 burst sizes in the presence of a primase-null ␣ protein and an active DnaG protein were always small, indicating that DnaG can only partially substitute for the primase fu...

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“…The crystal structure of the zinc motif of the primase of Bacillus stearo-thermophilus has shown that it is a member of the zinc ribbon subfamily of zinc binding motifs such as that found in the yeast RNA polymerase II subunit 9 (22). Gene 4 protein contains 1 g atom of zinc per mole of gene 4 protein (26). In vitro mutagenesis of residues in this region as well as studies with chimeric T7 primases have demonstrated a role of the zinc motif in recognition of the trinucleotide sequence 5Ј-GTC-3Ј (27,28).…”

mentioning

confidence: 99%

Essential Lysine Residues in the RNA Polymerase Domain of the Gene 4 Primase-Helicase of Bacteriophage T7

Lee

Richardson

2001

Journal of Biological Chemistry

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At a replication fork DNA primase synthesizes oligoribonucleotides that serve as primers for the lagging strand DNA polymerase. In the bacteriophage T7 replication system, DNA primase is encoded by gene 4 of the phage. The 63-kDa gene 4 protein is composed of two major domains, a helicase domain and a primase domain located in the C-and N-terminal halves of the protein, respectively. T7 DNA primase recognizes the sequence 5-NNGTC-3 via a zinc motif and catalyzes the templatedirected synthesis of tetraribonucleotides pppACNN. T7 DNA primase, like other primases, shares limited homology with DNA-dependent RNA polymerases. To identify the catalytic core of the T7 DNA primase, single-point mutations were introduced into a basic region that shares sequence homology with RNA polymerases. The genetically altered gene 4 proteins were examined for their ability to support phage growth, to synthesize functional primers, and to recognize primase recognition sites. Two lysine residues, Lys-122 and Lys-128, are essential for phage growth. The two residues play a key role in the synthesis of phosphodiester bonds but are not involved in other activities mediated by the protein. The altered primases are unable to either synthesize or extend an oligoribonucleotide. However, the altered primases do recognize the primase recognition sequence, anneal an exogenous primer 5-ACCC-3 at the site, and transfer the primer to T7 DNA polymerase. Other lysines in the vicinity are not essential for the synthesis of primers.

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Requirement for a zinc motif for template recognition by the bacteriophage T7 primase.

Cited by 66 publications

References 50 publications

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

A Molecular Handoff between Bacteriophage T7 DNA Primase and T7 DNA Polymerase Initiates DNA Synthesis

Domain structure of phage P4 alpha protein deduced by mutational analysis

Essential Lysine Residues in the RNA Polymerase Domain of the Gene 4 Primase-Helicase of Bacteriophage T7

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