Forces and Pressures in DNA Packaging and Release from Viral Capsids

Tzlil, Shelly; Kindt, James T.; Gelbart, William M.; Ben‐Shaul, Avinoam

doi:10.1016/s0006-3495(03)74971-6

Cited by 251 publications

(404 citation statements)

References 20 publications

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“…5A. The rate is constant during the first 20% of genome packaging, consistent with negligible internal force resisting DNA packaging in this low capsid filling regime, in accord with our findings with ϕ29 in the absence of Na + and theoretical predictions 6,19,21,22,23 . The average rate of 580 bp/s (SD 120 bp/s) is approximately equal to that at 5 pN determined in our velocity vs. load measurements (Fig.…”

Section: Internal Force Buildup During Packagingsupporting

confidence: 89%

“…5B), as described in Methods. We find that the force rises steeply with filling during the latter half of genome packaging, as observed with ϕ29 6, 7 and predicted theoretically 19,21,22,23,24,25 , and reaches 25 ± 6 pN with 90% of the genome length packaged. This force is notably two to three-fold lower than that measured for ϕ29 in a similar ionic condition 6 .…”

Section: Internal Force Buildup During Packagingsupporting

confidence: 87%

“…1B, Methods). These measurements were made with <20% of the native λ genome length packaged, where the internal forces resisting DNA confinement are expected to be small 19,20,21,22,23,24,25 . Under these conditions, the entire load on the motor is due to the externally applied force.…”

Section: The λ Dna Packaging Motor Generates High Forcesmentioning

confidence: 99%

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Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Fuller

Raymer

Rickgauer

et al. 2007

Journal of Molecular Biology

160

204

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Molecular motors drive genome packaging into preformed procapsids in many dsDNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage λ. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage ϕ29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged ~600 bp/s, which is 3.5-fold higher than ϕ29, and the motor processivity was also 3-fold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging in many cases, and greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid.

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Section: Internal Force Buildup During Packagingsupporting

confidence: 89%

Section: Internal Force Buildup During Packagingsupporting

confidence: 87%

See 1 more Smart Citation

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Fuller

Raymer

Rickgauer

et al. 2007

Journal of Molecular Biology

160

204

View full text Add to dashboard Cite

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“…This effect was taken into account in subsequent models of DNA packing. [16][17][18][28][29][30] These models are generally consistent with each other, but they focus on different kinds of predictions, such as the structure of the DNA, the forces and pressures involved in DNA confinement, and the effect of . This view is looking into the capsid at the portal (the entry site for DNA) and the green hoops reflect density corresponding to the packed DNA.…”

Section: Dna Virusesmentioning

confidence: 80%

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

et al. 2006

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The mechanical properties of DNA play a critical role in many biological functions. For example, DNA packing in viruses involves confining the viral genome in a volume (the viral capsid) with dimensions that are comparable to the DNA persistence length. Similarly, eukaryotic DNA is packed in DNA-protein complexes (nucleosomes) in which DNA is tightly bent around protein spools. DNA is also tightly bent by many proteins that regulate transcription, resulting in a variation in gene expression that is amenable to quantitative analysis. In these cases, DNA loops are formed with lengths that are comparable to or smaller than the DNA persistence length. The aim of this review is to describe the physical forces associated with tightly bent DNA in all of these settings and to explore the biological consequences of such bending, as increasingly accessible by single-molecule techniques.

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“…Because the size of viral capsids is on the order of the persistence length of the DNA (Ϸ50 nm), significant DNA bending must occur during packaging (2)(3)(4)(5)(6)(7). Moreover, due to the negatively charged phosphate backbone of DNA, a large repulsive electrostatic barrier must be overcome during DNA confinement (2)(3)(4)(5)(6)(7).…”

mentioning

confidence: 99%

Ionic effects on viral DNA packaging and portal motor function in bacteriophage φ29

Fuller

Rickgauer

Jardine³

et al. 2007

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

140

163

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In many viruses, DNA is confined at such high density that its bending rigidity and electrostatic self-repulsion present a strong energy barrier in viral assembly. Therefore, a powerful molecular motor is needed to package the DNA into the viral capsid. Here, we investigate the role of electrostatic repulsion on single DNA packaging dynamics in bacteriophage 29 via optical tweezers measurements. We show that ionic screening strongly affects the packing forces, confirming the importance of electrostatic repulsion. Separately, we find that ions affect the motor function. We separate these effects through constant force measurements and velocity versus load measurements at both low and high capsid filling. Regarding motor function, we find that eliminating free Mg 2؉ blocks initiation of packaging. In contrast, Na ؉ is not required, but it increases the motor velocity by up to 50% at low load. Regarding internal resistance, we find that the internal force was lowest when Mg 2؉ was the dominant ion or with the addition of 1 mM Co 3؉ . Forces resisting DNA confinement were up to Ϸ80% higher with Na ؉ as the dominant counterion, and only Ϸ90% of the genome length could be packaged in this condition. The observed trend of the packing forces is in accord with that predicted by DNA charge-screening theory. However, the forces are up to six times higher than predicted by models that assume coaxial spooling of the DNA and interaction potentials derived from DNA condensation experiments. The forces are also severalfold higher than ejection forces measured with bacteriophage .optical tweezers ͉ single molecule D uring the assembly of many dsDNA viruses, the genome is compacted to near-crystalline density (1). Because the size of viral capsids is on the order of the persistence length of the DNA (Ϸ50 nm), significant DNA bending must occur during packaging (2-7). Moreover, due to the negatively charged phosphate backbone of DNA, a large repulsive electrostatic barrier must be overcome during DNA confinement (2-7). In some cases, more than half of the physically available space inside the capsid is taken up by the viral genome (1, 7).In the case of bacteriophage 29, the 19.3-kbp genome (Ϸ6.5 m in length) is packed inside a prolate icosahedral capsid Ϸ45-nm wide and 54-nm long (8). As with many other dsDNA viruses, DNA is translocated into the preformed precursor capsid (prohead) by an ATP-powered molecular motor (9-11). The 29 motor is situated at a unique vertex of the prohead and consists of a ring of RNA molecules (pRNA) sandwiched between two protein rings: the head-tail connector (gene product 10, gp10) and the packaging ATPase (gp16) (12).Previously, we developed an optical tweezers assay that allowed us to measure the packaging of a single DNA molecule into a single 29 prohead (9). We found that the rate of packaging decreased during capsid filling or when an external force was applied to the DNA substrate. From these measurements, we showed that a large internal force builds during packaging because of DNA confinement ...

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Forces and Pressures in DNA Packaging and Release from Viral Capsids

Cited by 251 publications

References 20 publications

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Measurements of Single DNA Molecule Packaging Dynamics in Bacteriophage λ Reveal High Forces, High Motor Processivity, and Capsid Transformations

Biological consequences of tightly bent DNA: The other life of a macromolecular celebrity

Ionic effects on viral DNA packaging and portal motor function in bacteriophage φ29

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