Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase

Crampton, Donald J.; Ohi, Melanie D.; Qimron, Udi; Walz, Thomas; Richardson, Charles C.

doi:10.1016/j.jmb.2006.05.037

Cited by 58 publications

(91 citation statements)

References 41 publications

(76 reference statements)

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“…When DNA is not present, no binding between gp4-E343Q and gp5/trx is detected, indicating that DNA is required for assembly of the functional gp4-E343Q-gp5/trx complex. In the absence of DNA, ∼70% helicase rings are heptameric, and ∼20% are hexameric, consistent with previous negative-stain EM analysis (5-8), native gel electrophoresis (5,6,21), and gel-filtration data (9).…”

Section: Significancesupporting

confidence: 89%

“…It binds to ssDNA, hydrolyzes nucleotides, translocates on ssDNA, and unwinds dsDNA (7)(8)(9). In contrast, the heptamer does not bind to ssDNA (6).…”

mentioning

confidence: 90%

“…The negative stain EM revealed that hexamers and heptamers are the most abundant (∼22% and ∼78%, respectively). Upon addition of ssDNA, the equilibrium between the two oligomeric forms changes (∼77% protein rings are now hexameric, and ∼18% are heptameric), indicative of DNA binding (6). The hexamer is an enzymatically active form of gp4.…”

mentioning

confidence: 99%

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Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Kulczyk

Moeller

Meyer

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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We present a structure of the ∼650-kDa functional replisome of bacteriophage T7 assembled on DNA resembling a replication fork. A structure of the complex consisting of six domains of DNA helicase, five domains of RNA primase, two DNA polymerases, and two thioredoxin (processivity factor) molecules was determined by single-particle cryo-electron microscopy. The two molecules of DNA polymerase adopt a different spatial arrangement at the replication fork, reflecting their roles in leading-and lagging-strand synthesis. The structure, in combination with biochemical data, reveals molecular mechanisms for coordination of leading-and laggingstrand synthesis. Because mechanisms of DNA replication are highly conserved, the observations are relevant to other replication systems.cryo-EM structure | replisome | coordination of leading-and lagging-strands synthesis | DNA replication | DNA polymerase T he reverse polarity and antiparallel nature of the two strands in duplex DNA pose a topological problem for their simultaneous synthesis. The "trombone" model of DNA replication postulates that the lagging strand forms a loop such that the leading-and lagging-strand replication proteins contact one another (1). This replication loop contains a nascent Okazaki fragment and allows for coordination of leading-and laggingstrand synthesis. The bacteriophage T7 replisome has been studied extensively (2). Thus, the macromolecular complex of T7 replication proteins provides an excellent model for structural analysis of a replisome. Notably, the human mitochondrial and the T7 replication systems are similar (3).The phage T7 replisome is relatively simple. Four proteins are sufficient for reconstitution of the functional replisome, yet the assembled replisome recapitulates all of the key features of more complex prokaryotic and eukaryotic systems (2, 4). These proteins are the following: DNA polymerase (gp5) and its processivity factor, Escherichia coli thioredoxin (trx), single-stranded DNA (ssDNA)-binding protein (gp2.5), and the bifunctional DNA primase-helicase (gp4).Gp4 forms multiple oligomeric forms (5). The negative stain EM revealed that hexamers and heptamers are the most abundant (∼22% and ∼78%, respectively). Upon addition of ssDNA, the equilibrium between the two oligomeric forms changes (∼77% protein rings are now hexameric, and ∼18% are heptameric), indicative of DNA binding (6). The hexamer is an enzymatically active form of gp4. It binds to ssDNA, hydrolyzes nucleotides, translocates on ssDNA, and unwinds dsDNA (7-9). In contrast, the heptamer does not bind to ssDNA (6).Crystal structures of all of the individual T7 replication proteins have been determined (10-15). However, to date there is no structural information on the T7 replisome or its subassemblies. Consequently, intermolecular interactions involved in coordination of DNA synthesis have not been identified. We have therefore used cryo-electron microscopy (cryo-EM) and single-particle reconstruction methods to determine a structure of the T7 replisome. ResultsR...

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Section: Significancesupporting

confidence: 89%

“…It binds to ssDNA, hydrolyzes nucleotides, translocates on ssDNA, and unwinds dsDNA (7)(8)(9). In contrast, the heptamer does not bind to ssDNA (6).…”

mentioning

confidence: 90%

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Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Kulczyk

Moeller

Meyer

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, based on the locations of these two residues within the heptamer, they could not interact to form the configuration necessary for DNA binding. Indeed, heptamers do not bind ssDNA (25).…”

Section: (5)mentioning

confidence: 99%

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

Lee

Qimron

Richardson

2008

Proc. Natl. Acad. Sci. U.S.A.

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The DNA helicase encoded by bacteriophage T7 consists of six identical subunits that form a ring through which the DNA passes. Binding of ssDNA is a prior step to translocation and unwinding of DNA by the helicase. Arg-493 is located at a conserved structural motif within the interior cavity of the helicase and plays an important role in DNA binding. Replacement of Arg-493 with lysine or histidine reduces the ability of the helicase to bind DNA, hydrolyze dTTP, and unwind dsDNA. In contrast, replacement of Arg-493 with glutamine abolishes these activities, suggesting that positive charge at the position is essential. Based on the crystallographic structure of the helicase, Asp-468 is in the range to form a hydrogen bonding with Arg-493 on the adjacent subunit. In vivo complementation results indicate that an interaction between Asp-468 and Arg-493 is critical for a functional helicase and those residues can be swapped without losing the helicase activity. This study suggests that hydrogen bonding between Arg-493 and Asp-468 from adjacent subunits is critical for DNA binding ability of the T7 hexameric helicase.DNA replication ͉ hydrogen bonding ͉ subunit interaction ͉ residue swappping T he replication, recombination, and repair of DNA all require the unwinding of duplex DNA. Such an important transformation of dsDNA into ssDNA is carried out by a group of proteins designated as DNA helicases. Underlying the overall process of unwinding dsDNA is the ability of the protein to bind to ssDNA and use the hydrolysis of a nucleoside triphosphate to translocate unidirectionally on the DNA (1, 2). Not surprisingly, DNA helicases differ in the mechanism by which they bind to DNA, translocate on ssDNA, and unwind the duplex. On the basis of their amino acid sequences and 3D structures, DNA helicases can be divided into several families or superfamilies (2, 3).The helicase encoded by gene 4 of bacteriophage T7 is one of the most extensively characterized helicases. As a member of the DnaB-like family (family 4), the gene 4 protein forms a hexameric toroid. The oligomeric structure not only gives rise to the nucleotide bindings at the subunit interfaces but also provides a central cavity through which the ssDNA can pass. Binding sites for DNA and nucleotide span between subunits, thus allowing cooperative binding of DNA and efficient coupling of nucleotide hydrolysis to movement of the DNA strand.Electron microscopy and x-ray crystallography illustrated that the protein forms a closed ring-shaped oligomeric structure composed of six or seven subunits (4-7). Biochemical studies have identified regions critical to oligomerization of the protein, including a linker region that connects the C-terminal helicase domain of the protein to the N-terminal primase domain (8, 9). Four motifs were defined based on conserved amino acid sequences among family 4 helicases. Strictly conserved residues in motifs 1 and 2 contact the nucleotide at the nucleotide binding site located at the interface of subunits. These residues participate in h...

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“…On the other hand, studies of several other AAA+ ATPases have revealed surprisingly elastic substrate accommodation. Hexamer to heptamer transitions resulting in larger pore sizes have been described for bacteriophage T7 primase (Crampton et al 2006). Moreover, recent experiments analyzing protein translocation into the AAA+ ClpXP proteosome revealed that this caged protease with an annular entry port can translocate and degrade branched substrates (Bolon et al 2004).…”

mentioning

confidence: 92%

SpoIIIE strips proteins off the DNA during chromosome translocation

Marquis¹,

Nollmann²,

Ptacin³

et al. 2008

Genes Dev.

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The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore.[Keywords: Chromosome segregation; chromosome translocation; sporulation; FtsK; AAA+ ATPase] Supplemental material is available at http://www.genesdev.org.

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Oligomeric States of Bacteriophage T7 Gene 4 Primase/Helicase

Cited by 58 publications

References 41 publications

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7

SpoIIIE strips proteins off the DNA during chromosome translocation

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