A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an
empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to
measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We
can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the
capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods
to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems
have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging
motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA
processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by
the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the
confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also
striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and
dynamic disorder, responses to load, and pausing and slipping dynamics.
The bacteriophage φ29 portal motor is capable of packaging the φ29, 19.3 Kbp, genome to high density into its preformed capsid. The packaging process must overcome the forces due to confining the highly negative charge of the DNA to a small volume, as well as the forces due to bending the DNA on length scales smaller than one persistence length. Both of these energetic considerations can be modulated by the ionic nature of the buffer DNA packaging occurs in. To measure the effects of DNA charge shielding on the packaging process, we studied the dynamics of DNA packaging by optical tweezers in a variety of different ionic conditions. We looked at the effects monovalent, divalent, and trivalent cations have on the motor function and its dependence on external force and, we observed the rate of DNA packaging at nominal force as a function of capsid filling. Specifically, we varied the concentrations of Na+, Mg+2, and cobalt hexamine in the solution bathing the bacteriophage during packaging to see what effects, if any, these cations have. From these measurements, we present an inferred internal force as a function of percent filling of the bacteriophage capsid in a variety of ionic environments. Preliminary analysis suggests the ionic environment can modulate internal pressure, with the presence of higher valence cations better shielding the packaged DNA resulting in lower internal pressures.
A key step in the life cycle of many viruses, including bacteriophages, adenoviruses, and herpesviruses, is the packaging of replicated viral genomes into pre-assembled proheads by the action of ATP-dependent portal motor complexes. Here we present a method that allows the initiation of packaging by single complexes to be studied using optical tweezers. A procedure is developed for assembling phage Φ29 prohead-motor complexes, which are demonstrated to bind and begin translocation of a target DNA molecule within only a few seconds. We show that the Φ29 DNA terminal protein (gene product 3), which functions to prime DNA replication, also has a dramatic effect on packaging. The DNA tether length measured immediately after binding varied from ~30-100% of the full length, yet shortened monotonically, indicating that packaging does not strictly begin at the terminal end of the DNA. Removal of the terminal protein eliminated this variability, causing packaging to initiate at or very near the end of the DNA. These findings, taken together with electron microscopy data, suggest that rather than simply threading into the portal, the motor captures and dynamically tensions a DNA loop, and that the function of the terminal protein is to load DNA segments on both sides of the loop junction onto separate DNA translocating units.
Optical tweezers have broad applications in studies of structures and processes in molecular and cellular biophysics. Use of optical tweezers for quantitative molecular-scale measurement requires careful calibration in physical units. Here we show that DNA molecules may be used as metrology standards for force and length measurements. Analysis of DNA molecules of two specific lengths allows simultaneous determination of all essential measurement parameters. We validate this "biological calibration" method experimentally and with simulated data, and show that precisions in determining length scale factor (~0.2%), length offset (~0.03%), force scale factor (~2%), and compliance of the traps (~3%) are limited only by current measurement variation, much of which arises from polydispersity of the microspheres (~2%). We find this procedure to be simpler and more convenient than previous methods, and suggest that it provides an easily replicated standard that can insure uniformity of measurements made in different laboratories.
Here we describe and characterize a method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial, and viral sequences ranging from ~10 to 40 kbp can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength, and pH. Attachment strength is characterized by measuring the unbinding time distribution as a function of applied force.
Mechanical manipulation of single DNA molecules can provide novel information about protein-DNA interactions. Here we review two examples studied by our group. First, we have studied the forced unraveling of nucleosomes assembled on heterogeneous DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). We measure abrupt events releasing ~55 to 95 base pairs of DNA, which are attributable to non-equilibrium unraveling of individual nucleosomes. Wide variations observed in the unraveling force and sudden DNA re-wrapping events may have an important regulatory influence on DNA directed biochemical processes. Second, we have studied the mechanics and dynamics of single DNA looping and cleavage by "two-site" restriction enzymes. Cleavage is measured as a function of DNA tension, incubation time, and enzyme concentration, distinguishing enzymes that require DNA looping from ones that do not. Forced disruption of fixed DNA loops formed in the absence of Mg2+ is observed, allowing the distribution of number of loops, loop length, and disruption force to be measured as a function of time, DNA tension, and ionic conditions.
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