Preparation and Characterization of a Set of Linear DNA Molecules for Polymer Physics and Rheology Studies

Laib, Stephan; Robertson, Rae M.; Smith, Douglas E.

doi:10.1021/ma0601464

Cited by 66 publications

(87 citation statements)

References 33 publications

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“…A 45-kbp fosmid DNA construct (pCCFOS1-45) was prepared as described (6). It was treated with topoisomerase I to prepare the relaxed circular form and with ApaI to prepare the linear form (5,6).…”

Section: Resultsmentioning

confidence: 99%

“…It was treated with topoisomerase I to prepare the relaxed circular form and with ApaI to prepare the linear form (5,6). The DNA was concentrated by isopropanol precipitation and resuspended in TE buffer (Tris⅐HCl/EDTA, pH 8) with 10 mM NaCl.…”

Section: Resultsmentioning

confidence: 99%

“…This sample was digested with BamHI to prepare the linear form (6). With this shorter molecule there were only minor effects of topology, even at 1 mg/ml ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Strong effects of molecular topology on diffusion of entangled DNA molecules

Robertson

Smith

2007

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

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101

118

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When long polymers such as DNA are in a highly concentrated state they may become entangled, leading to restricted self-diffusion. Here, we investigate the effect of molecular topology on diffusion in concentrated DNA solutions and find surprisingly large effects, even with molecules of modest length and concentration. We measured the diffusion coefficients of linear and relaxed circular molecules by tracking the Brownian motion of single molecules with fluorescence microscopy. Four possible cases were compared: linear molecules surrounded by linear molecules, circular molecules surrounded by linear molecules, linear molecules surrounded by circles, and circles surrounded by circles. In measurements with 45-kbp DNA at 1 mg/ml, we found that circles diffused Ϸ100 times slower when surrounded by linear molecules than when surrounded by circles. In contrast, linear and circular molecules diffused at nearly the same rate when surrounded by circles, and circles diffused Ϸ10 times slower than linears when surrounded by linears. Thus, diffusion in entangled DNA solutions strongly depends on topology of both the diffusing molecule and the surrounding molecules. This effect also strongly depends on DNA concentration and length. The differences largely disappeared when the concentration was lowered to 0.1 mg/ml or when the DNA length was lowered to 6 kb. Present theories cannot fully explain these effects.polymers ͉ reptation A mong polymers DNA is rather unique in that it is naturally found in a number of different topological forms, including linear, supercoiled circular, relaxed circular, knotted circular, and branched. DNA solutions handled in vitro in molecular biology research are often relatively concentrated (Ϸ1-10 mg/ ml, for example, after lysis of bacterial cells during DNA isolation, or when DNA is redissolved after ethanol precipitation). According to classical theories and experiments in polymer physics, long flexible molecules form random coils that overlap and become entangled as the concentration of solutions is increased (1, 2). In the field of polymer physics and rheology there is considerable fundamental interest in understanding the effect of molecular topology on entangled polymer dynamics (3, 4). In gel electrophoresis it is well known that molecular topology strongly affects the mobility of DNA. However, with few exceptions, most theories and experiments on diffusion in concentrated polymer solutions have examined only linear molecules. Here, we investigate the effect of molecular topology on diffusion of entangled DNA. Although relaxed circular molecules differ from linear molecules only by the presence of one additional pair of phosphodiester bonds (linking head to tail) and diffuse at nearly the same rate in dilute solution (5), we observe large differences in their diffusion rates with molecules of modest size and concentration. ResultsA 45-kbp fosmid DNA construct (pCCFOS1-45) was prepared as described (6). It was treated with topoisomerase I to prepare the relaxed circular form and with ApaI ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Strong effects of molecular topology on diffusion of entangled DNA molecules

Robertson

Smith

2007

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

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101

118

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“…The 115 kbp (38 µm) molecules were prepared by cloning in E. coli. 39 These molecules were treated with topoisomerase I to produce torsionally relaxed, non-interlinked circles. 34 Displacements were made by moving the sample chamber with a piezoelectric nanopositioning stage, and the induced force along the axis of displacement was measured by recording the deflection of the trapping laser, as described previously.…”

Section: Methodsmentioning

confidence: 99%

Direct Measurement of the Confining Forces Imposed on a Single Molecule in a Concentrated Solution of Circular Polymers

Robertson

Smith

2007

Macromolecules

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We measure the forces confining the displacement of a single DNA molecule embedded within a concentrated solution of long relaxed circular DNA molecules (115 kbp at 1 mg/mL) using optical tweezers. We compare these measurements with our previous measurements of forces imposed by entangled linear DNA molecules of the same length and concentration. A tube-like confining field and three relaxation modes were observed, but the tube radius was 25% smaller (=0.6 µm) and the longest relaxation time ∼3 times shorter (=11 s) than with linear molecules, consistent with recent theoretical predictions by Iyer, Lele, and Juvekar. For displacements greater than the tube radius, the confining force imposed by circular polymers was substantially lower and shorter range than that measured with linear polymers.

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“…When this construct is treated with the terminase proteins, they cleave the DNA resulting in a 34.7 kbp DNA-terminase complex (complex I), and a 13.8 kbp cleavage fragment. The 52 kbp and the 75 kbp DNA constructs were prepared from the BAC clone CTD-2342K16 and purified as described previously 46 . The 75 kbp construct was produced by digesting the BAC clone with restriction endonuclease BspEI producing a 5' overhang 74,587 bp upstream of a left end lambda cos site (present in the pBeloBAC11 BAC cloning vector).…”

Section: Dna Constructsmentioning

confidence: 99%

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

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158

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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Preparation and Characterization of a Set of Linear DNA Molecules for Polymer Physics and Rheology Studies

Cited by 66 publications

References 33 publications

Strong effects of molecular topology on diffusion of entangled DNA molecules

Strong effects of molecular topology on diffusion of entangled DNA molecules

Direct Measurement of the Confining Forces Imposed on a Single Molecule in a Concentrated Solution of Circular Polymers

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

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