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

Kato, Masato; Ito, Takuhiro; Wagner, Gerhard; Richardson, Charles C.; Ellenberger, Tom

doi:10.1016/s1097-2765(03)00195-3

Cited by 103 publications

(245 citation statements)

References 70 publications

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“…The linker region has the potential for flexibility in that it is exposed to the solvent and susceptible to digestion with proteases (6,17,38). Attempts to crystallize a truncated polypeptide containing the primase domain and the linker region (residues 1-271) have failed whereas a smaller primase fragment (residues 1-255) lacking a portion of the linker region was successfully crystallized (11). The crystal structure of this latter primase fragment reveals the C-terminal region of the polypeptide to be packed tightly within the catalytic core (11).…”

Section: Fig 5 Oligoribonucleotide Synthesismentioning

confidence: 99%

“…Attempts to crystallize a truncated polypeptide containing the primase domain and the linker region (residues 1-271) have failed whereas a smaller primase fragment (residues 1-255) lacking a portion of the linker region was successfully crystallized (11). The crystal structure of this latter primase fragment reveals the C-terminal region of the polypeptide to be packed tightly within the catalytic core (11). The inability to crystallize the primase fragment with the linker region may arise from the flexible linker region hindering the packing of the C-terminal region into a stable crystal array.…”

Section: Fig 5 Oligoribonucleotide Synthesismentioning

confidence: 99%

“…However, each domain, particularly the primase domain, is impaired in specific reactions when it is not coupled to the other domain. The crystal structures of both the helicase (9, 10) and primase (11) domains have been solved.…”

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confidence: 99%

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The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

Lee

Richardson

2004

Journal of Biological Chemistry

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The gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain is responsible for DNA-dependent dTTP hydrolysis, translocation, and DNA unwinding whereas the N-terminal primase domain is responsible for templatedirected oligoribonucleotide synthesis. A 26 amino acid linker region (residues 246 -271) connects the two domains and is essential for the formation of functional hexamers. In order to further dissect the role of the linker region, three residues (Ala 257 , Pro 259 , and Asp 263 ) that was disordered in the crystal structure of the hexameric helicase fragment were substituted with all amino acids, and the altered proteins were analyzed for their ability to support growth of T7 phage lacking gene 4. The in vivo screening revealed Ala 257 and Asp 263 to be essential whereas Pro 259 could be replaced with any amino acid without loss of function. Selected gene 4 proteins with substitution for Ala 257 or Asp 263 were purified and examined for their ability to unwind DNA, hydrolyze dTTP, translocate on ssDNA, and oligomerize. In the presence of Mg 2؉ , all of the altered proteins oligomerize. However, in the absence of divalent ion, alterations at position 257 increase the extent of oligomerization whereas those at position 263 reduce oligomer formation. Although dTTP hydrolysis activity is reduced only 2-3-fold, none of the altered gene 4 proteins can translocate effectively on single-strand DNA, and they cannot mediate the unwinding of duplex DNA. Primer synthesis catalyzed by the altered proteins is relatively normal on a short DNA template but it is severely impaired on longer templates where translocation is required. The results suggest that the linker region not only connects the two domains of the gene 4 protein and participates in oligomerization, but also contributes to helicase activity by mediating conformations within the functional hexamer.

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Section: Fig 5 Oligoribonucleotide Synthesismentioning

confidence: 99%

Section: Fig 5 Oligoribonucleotide Synthesismentioning

confidence: 99%

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The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

Lee

Richardson

2004

Journal of Biological Chemistry

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“…Crystal structures of all the individual T7 replication proteins (11)(12)(13)(14)(15)(16) have been determined, and their enzymatic activities are well characterized (3,4,(17)(18)(19). The primase and helicase activities of phage T7 are fused in a single bifunctional protein, the gp4 primase-helicase (20,21), which consists of three domains separated by proteolytically sensitive linkers (15,22,23).…”

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confidence: 99%

“…The primase and helicase activities of phage T7 are fused in a single bifunctional protein, the gp4 primase-helicase (20,21), which consists of three domains separated by proteolytically sensitive linkers (15,22,23). The C-terminal helicase domain (residues 262-566) assembles into the ring-shaped hexamers that unwind a DNA duplex by moving along one strand that passes through the center of the ring (24,25) and displacing the opposite strand in a reaction requiring the hydrolysis of nucleotide cofactors (26,27).…”

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confidence: 99%

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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Dual‐Acting Small‐Molecule Inhibitors Targeting Mycobacterial DNA Replication

Singh

Ilić

Tam

et al. 2020

Chemistry A European J

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Mycobacterium tuberculosis (Mtb) is a pathogenic bacterium and a causative agent of tuberculosis (TB), a disease that kills more than 1.5 million people worldwide annually. One of the main reasons for this high mortality rate is the evolution of new Mtb strains that are resistant to available antibiotics. Therefore, new therapeutics for TB are in constant demand. Here, we report the development of small‐molecule inhibitors that target two DNA replication enzymes of Mtb, namely DnaG primase and DNA gyrase (Gyr), which share a conserved TOPRIM fold near the inhibitors’ binding site. The molecules were developed on the basis of previously reported inhibitors for T7 DNA primase that bind near the TOPRIM fold. To improve the physicochemical properties of the molecules as well as their inhibitory effect on primase and gyrase, 49 novel compounds have been synthesized as potential drug candidates in three stages of optimization. The last stage of chemical optimization yielded two novel inhibitors for both Mtb DnaG and Gyr that also showed inhibitory activity toward the fast‐growing non‐pathogenic model Mycobacterium smegmatis (Msmg).

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

Cited by 103 publications

References 70 publications

The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

The Linker Region between the Helicase and Primase Domains of the Gene 4 Protein of Bacteriophage T7

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

Dual‐Acting Small‐Molecule Inhibitors Targeting Mycobacterial DNA Replication

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