Coupling dTTP Hydrolysis with DNA Unwinding by the DNA Helicase of Bacteriophage T7

Satapathy, Ajit K.; Kulczyk, Arkadiusz W.; Ghosh, Sharmistha; Oijen, Antoine M. van; Richardson, Charles C.

doi:10.1074/jbc.m111.283796

Cited by 21 publications

(23 citation statements)

References 41 publications

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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: 72%

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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: 72%

“…S11). We showed earlier that F523 interacts directly with the extruded DNA leading strand (21). The orientation of gp5/trx lead at the replisome helps to stabilize partially unwound dsDNA at the junction between ssDNA and dsDNA, thus helping to separate dsDNA.…”

Section: And Saxs (5)mentioning

confidence: 99%

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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“…6E) is positioned such that the extension of the 5Ј-end of the ssDNA template would position it in the close proximity to the subunit interface ␤-hairpin that contains residue Phe-523 (orange patch at the front surface of DNA helicase). We demonstrated earlier that the subunit interface ␤-hairpin, specifically Phe-523, interacts directly with the extruded DNA leading strand during the process of DNA unwinding (17). The lagging strand passes through the central channel in the hexameric ring of DNA helicase, making interactions with the residues in the central ␤-hairpins located at the inner surface of the channel.…”

Section: Discussionmentioning

confidence: 99%

“…In the SPR, EM, and small angle x-ray scattering (SAXS) experiments, we use a gp5/trx in which two amino acids (Asp-5 and Glu-7) in the exonuclease domain of gp5 are replaced with Ala; the 3Ј to 5Ј proofreading exonuclease activity is reduced by a factor of 10 6 (6). The biochemical assays have been described in detail earlier: DNA binding and oligomerization assays (16), unwinding assay (17), and minicircle assay (18).…”

Section: Methodsmentioning

confidence: 99%

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An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Kulczyk

Akabayov

Lee

et al. 2012

Journal of Biological Chemistry

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Background:Interactions of DNA polymerase and DNA helicase are crucial in DNA synthesis. Results: Two distinct interactions are involved in formation of the DNA polymerase/DNA helicase complex. Conclusion:The multiple interactions between DNA polymerase and DNA helicase account for the high processivity of leading strand synthesis. Significance: Understanding of the replication process in bacteriophage T7 facilitates studies in more complex systems.

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Replication | DNA Helicases: Hexameric Enzyme Action

Singh¹,

Patel²

2021

Encyclopedia of Biological Chemistry III

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Coupling dTTP Hydrolysis with DNA Unwinding by the DNA Helicase of Bacteriophage T7

Cited by 21 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

An Interaction between DNA Polymerase and Helicase Is Essential for the High Processivity of the Bacteriophage T7 Replisome

Replication | DNA Helicases: Hexameric Enzyme Action

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