Fluorescence measurement of the kinetics of DNA injection by bacteriophage .lambda. into liposomes

Novick, Steven L.; Baldeschwieler, John D.

doi:10.1021/bi00420a050

Cited by 35 publications

(35 citation statements)

References 31 publications

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“…In this paper, we have shown that the ejection of DNA from bacteriophage can reach speeds of up to 60 kbp/s, comparable to the lower bound of 75 kbp/s found for the translocation speed in T5 (18) and clarifying an earlier bulk experiment (13). The speed may also be compared with the slips of 10 kbp/s or greater observed during 29 DNA packaging under force (24).…”

Section: Discussionsupporting

confidence: 77%

“…A force of tens of piconewtons (pN) is produced by the highly bent and compressed DNA within the capsid (9-11), but not much is known about how fast the DNA transfer occurs, except that ejection reaches completion in vivo in Ͻ2 min (12). One study used lipid vesicles incorporating LamB and filled with ethidium bromide: the DNA was ejected into the vesicles, causing an increase in fluorescence over Ϸ30 s (13). However, the Ϸ1,000 molecules of ethidium bromide in each vesicle were enough for only the first 1 kbp of DNA (14).…”

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confidence: 99%

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Real-time observations of single bacteriophage λ DNA ejections in vitro

Grayson¹,

Lin

Winther

et al. 2007

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

119

191

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fluorescence ͉ microscopy ͉ virus ͉ genome T he transfer of bacteriophage DNA from a capsid into the host cell is an event of great importance to biology and physics. In biology, DNA ejection was a key piece of evidence demonstrating that the genetic material was DNA and not protein (1), phages have long been used to insert foreign genes into bacteria (2), and phage-mediated DNA transfer between species is a challenge to theories of evolution (3). In physics, the translocation of DNA through a pore has been studied from the theoretical and experimental points of view (4-8). Because phage DNA ejection is such a well known example of this process, it is important to understand it from a quantitative point of view. This paper addresses a longstanding, quantitative puzzle about phage DNA ejection: How fast is the ejection process? We use bacteriophage , a typical tailed phage, to answer this question. In a infection, first the phage tail binds to the Escherichia coli outer membrane protein LamB, triggering ejection. Then the genome, 48.5 kbp of double-stranded DNA, moves out of the phage head, through the tail, and into the cytoplasmic space, which requires force on the DNA directed into the cell. A force of tens of piconewtons (pN) is produced by the highly bent and compressed DNA within the capsid (9-11), but not much is known about how fast the DNA transfer occurs, except that ejection reaches completion in vivo in Ͻ2 min (12). One study used lipid vesicles incorporating LamB and filled with ethidium bromide: the DNA was ejected into the vesicles, causing an increase in fluorescence over Ϸ30 s (13). However, the Ϸ1,000 molecules of ethidium bromide in each vesicle were enough for only the first 1 kbp of DNA (14). Also, because the ejections could have started at different times, that experiment says very little about the DNA translocation process. This paper aims to resolve these challenges in describing the ejection process.An important insight from theory is that frictional forces limit the speed of ejection, due to DNA rearrangement in the phage head or sliding forces in the tail (15,16). Because the DNA is in a liquid state (17), we expect friction to behave at least somewhat like macroscopic hydrodynamic drag: stronger at higher speed or at smaller spacings between the moving parts. The DNA-tail interaction does not change during the ejection process, so we expect friction in the tail to remain constant. In contrast, friction in the head should be stronger when the spacing between the loops of DNA is small, i.e., at the beginning of ejection.To quantify the rate of ejection, a single-phage technique is necessary. Single-phage ejections were first observed with fluorescence microscopy on phage T5, revealing an effect of the unique structure of the T5 genome: nicks in the DNA resulted in predefined stopping points and a stepwise translocation process, with speeds that were too high to be quantified, so that further analysis of the speed and source of friction was not possible (18). As we will show here, ejects...

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Section: Discussionsupporting

confidence: 77%

mentioning

confidence: 99%

Real-time observations of single bacteriophage λ DNA ejections in vitro

Grayson¹,

Lin

Winther

et al. 2007

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

119

191

View full text Add to dashboard Cite

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“…The samples were first incubated at 25°C for 15 min to allow phage binding to LamB, followed by DNA ejection (which should be complete within 1 min; ref. 6), and then for 1 h at 37°C to allow full digestion of the ejected DNA by DNase I. Note that the nuclease is present throughout the ejection process to avoid precipitation of the ejected DNA.…”

Section: Methodsmentioning

confidence: 99%

“…Previous in vitro experiments have demonstrated that DNA ejection is fast (occurring on a time scale of seconds) and complete when phage capsids are opened by their receptors in aqueous solution (6). The capsids are permeable to water and salt ions, so there is no difference in hydrostatic pressure between the inside and outside of the capsid, and osmotic equilibrium is also maintained (7).…”

mentioning

confidence: 99%

Osmotic pressure inhibition of DNA ejection from phage

Evilevitch

Lavelle

Knobler

et al. 2003

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

303

381

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Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage , the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.V iral capsids are often highly pressurized. In the case of double-stranded DNA bacteriophages, for example, there are large forces acting to push the genome out through the tail of the viral particle. Recent experiments (1) and theory (2, 3) have established that the forces inside these capsids reach very high values (of order 50 pN) when the genomes are fully packaged. These forces are estimated to correspond to pressures of the order of 50 atm (1 atm ϭ 101.3 kPa; refs. 1-4). Such high values of stress result from the DNA being strongly bent and confined. Specifically, the inner diameter of the capsid is comparable to the DNA persistence length (50 nm), and the typical interaxial spacings are small enough (2.5 nm; ref. 5) that neighboring chains experience strong repulsions. It is precisely these forces that drive the ejection of the genome into the host cell after the tail of the capsid has been bound to its receptor protein in the outer cell membrane; equivalently, it is these forces that must be exerted by the motor protein responsible for packaging of the viral genome (1).Previous in vitro experiments have demonstrated that DNA ejection is fast (occurring on a time scale of seconds) and complete when phage capsids are opened by their receptors in aqueous solution (6). The capsids are permeable to water and salt ions, so there is no difference in hydrostatic pressure between the inside and outside of the capsid, and osmotic equilibrium is also maintained (7). The pressure difference between the capsid and the solution is therefore associated only with the confinement of the DNA. When the capsid is opened, ejection proceeds until this pressure difference falls to zero. Clearly, at this point the force driving the ejection has also dropped to zero. For the in vivo situation, however, ejection does not occur into a simple salt solution. The DNA is ejected into a highly concentrated colloidal suspension, the cell cytoplasm, which is characterized by high concentrations of proteins and other macromolecular structures. In general, then, one expects this colloidal solution to give rise to a force that resists the entry of DNA (8). More explicitly, because the ejection force associated with stress inside the capsid drops monotonically as the ejection proceeds, there will be a...

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“…49 When the DNA was ejected from l particles into the vesicles the ethidium bromide binds to the entering DNA, causing an increase in fluorescence. The timescale of ejection as determined by this experiment was $30 s. However, only $1000 molecules of ethidium bromide were present in each of the vesicles, so that the experiment was only capable of measuring the first few kbp of DNA entry.…”

Section: Measurements Of the Packing And Ejection Processesmentioning

confidence: 99%

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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Fluorescence measurement of the kinetics of DNA injection by bacteriophage .lambda. into liposomes

Cited by 35 publications

References 31 publications

Real-time observations of single bacteriophage λ DNA ejections in vitro

Real-time observations of single bacteriophage λ DNA ejections in vitro

Osmotic pressure inhibition of DNA ejection from phage

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

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