Derek N. Fuller

Single phage T4 DNA packaging motors exhibit large force generation, high velocity, and dynamic variability

Terminase enzyme complexes, which facilitate ATP-driven DNA packaging in phages and in many eukaryotic viruses, constitute a wide and potentially diverse family of molecular motors about which little dynamic or mechanistic information is available. Here we report optical tweezers measurements of single DNA molecule packaging dynamics in phage T4, a large, tailed Escherichia coli virus that is an important model system in molecular biology. We show that a complex is formed between the empty prohead and the large terminase protein (gp17) that can capture and begin packaging a target DNA molecule within a few seconds, thus demonstrating a distinct viral assembly pathway. The motor generates forces >60 pN, similar to those measured with phage φ29, suggesting that high force generation is a common property of viral DNA packaging motors. However, the DNA translocation rate for T4 was strikingly higher than that for φ29, averaging ≈700 bp/s and ranging up to ≈2,000 bp/s, consistent with packaging by phage T4 of an enormous, 171-kb genome in <10 min during viral infection and implying high ATP turnover rates of >300 s−1. The motor velocity decreased with applied load but averaged 320 bp/s at 45 pN, indicating very high power generation. Interestingly, the motor also exhibited large dynamic changes in velocity, suggesting that it can assume multiple active conformational states gearing different translocation rates. This capability, in addition to the reversible pausing and slipping capabilities that were observed, may allow phage T4 to coordinate DNA packaging with other ongoing processes, including viral DNA transcription, recombination, and repair.

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

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 φ29 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 Mg2+ 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 Mg2+ was the dominant ion or with the addition of 1 mM Co3+. Forces resisting DNA confinement were up to ≈80% higher with Na+ as the dominant counterion, and only ≈90% 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 λ.

Initiation of bacteriophage ø29 DNA packaging studied by optical tweezers manipulation of single DNA molecules

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.

A general method for manipulating DNA sequences from any organism with optical tweezers

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.

Studies of viral DNA packaging motors with optical tweezers: a comparison of motor function in bacteriophages φ29, λ, and T4

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.

Dependence of bacteriophage ø29 DNA packaging on ionic conditions studied by optical tweezers manipulation of single DNA molecules

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.