Testing a proposed paradigm shift in analysis of phage DNA packaging

Serwer, Philip; Wright, Elena T.

doi:10.1080/21597081.2016.1268664

Cited by 7 publications

(20 citation statements)

References 31 publications

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“…This NLD–NHD separation was caused by a Nycodenz impermeability-generated high hydration of the NLD ipDNA-capsids, as previously shown for DNA-free capsids [14,21,22]. Vertical arrows under NLD in Figure 4b indicate sub-peaks within the NLD region; D indicates the position of protein-free DNA, identified by agarose gel electrophoresis; φ indicates the position of infective phage.…”

Section: Resultssupporting

confidence: 61%

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ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

Serwer

Wright

2017

Viruses

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Adenosine triphosphate (ATP) cleavage powers packaging of a double-stranded DNA (dsDNA) molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA) and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM) confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration) causes contraction of hyper-expanded, low-permeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supra-physiological concentration) or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

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Section: Resultssupporting

confidence: 61%

“…For NLD ipDNA-capsids that had the lowest Nycodenz density in both density gradients of Figure 4 (e.g., 1.086 g/mL fraction indicated by leftmost arrow under NLD in Figure 4b), EM revealed characteristics previously observed [14] for DNA-free, hyper-expanded NLD capsid II. The specimens were prepared by negative staining.…”

Section: Resultssupporting

confidence: 59%

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

Serwer

Wright

2017

Viruses

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“…9; Serwer and Wright 2016). In Serwer and Wright (2016), (1) the protein composition is found to be that of T3 capsid II, (2) analysis of the EM images confirms the hyper-expansion and low permeability, and (3) the EM also reveals some contracted capsid II, as also seen in Fig. 9.…”

Section: Bdu: Fractionation By Capsid Permeabilitymentioning

confidence: 63%

“…The unusually low density is caused by unusually high hydration, caused, in turn, by impermeability of this capsid II to N ycodenz. Impermeability-derived hydration causes the density to progressively decrease as the gp10 shell radius increases (Serwer and Wright 2016). We will review details of this technique, given its development via the investigation of capsid shell flexibility.…”

Section: Dna Packaging: Additional Shell Dynamicsmentioning

confidence: 99%

“…Isolation/characterization studies reveal flexibility of the shell of T3 capsid II during in vivo DNA packaging. Some flexibility is found via the isolation of hyper-expanded capsid II (Serwer and Wright 2016), as reviewed below. Additional flexibility, accompanied by dynamism, is observed when some ipDNA-containing, hyper-expanded capsid II is contracted in vitro by incubation with magnesium ATP at physiological ATP concentration (Serwer and Wright 2017).…”

Section: Dna Packaging-associated Shell Flexibility and Dynamismmentioning

confidence: 99%

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States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM

et al. 2017

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Mature double-stranded DNA bacteriophages have capsids with symmetrical shells that typically resist disruption, as they must to survive in the wild. However, flexibility and associated dynamism assist function. We describe biochemistry-oriented procedures used to find previously obscure flexibility for capsids of the related phages, T3 and T7. The primary procedures are hydration-based buoyant density ultracentrifugation and purified particle-based cryo-electron microscopy (cryo-EM). We review the buoyant density centrifugation in detail. The mature, stable T3/T7 capsid is a shell flexibility-derived conversion product of an initially assembled procapsid (capsid I). During DNA packaging, capsid I expands and loses a scaffolding protein to form capsid II. The following are observations made with capsid II. (1) The in vivo DNA packaging of wild type T3 generates capsid II that has a slight (1.4%), cryo-EM-detected hyper-expansion relative to the mature phage capsid. (2) DNA packaging in some altered conditions generates more extensive hyper-expansion of capsid II, initially detected by hydration-based preparative buoyant density centrifugation in Nycodenz density gradients. (3) Capsid contraction sometimes occurs, e.g., during quantized leakage of DNA from mature T3 capsids without a tail.

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Hypothesis for the cause and therapy of neurodegenerative diseases

Serwer

2018

Medical Hypotheses

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Testing a proposed paradigm shift in analysis of phage DNA packaging

Cited by 7 publications

References 31 publications

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

States of phage T3/T7 capsids: buoyant density centrifugation and cryo-EM

Hypothesis for the cause and therapy of neurodegenerative diseases

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