We focus on the assembly of double-stranded DNA bacteriophages. These bacteriophages undergo assembly in the following two stages: First, a DNA-free container of protein is assembled. This particle is called a procapsid. The procapsid subsequently both binds and packages a double-stranded DNA genome. DNA packaging is an example of complex biochemistry because DNA packaging requires co-ordination of several biochemical activities. These activities include (a) binding of the procapsid to a genome, sometimes at a specific site called a Pac site, (b) unknown activities involved in finding the Pac site, (c) in some cases, the cleavage of the genome from a longer, end-to-end polymer (called a concatemer) of genomes, (d) the aligning of one DNA end (the cleaved end) to enter a channel formed by a dodecameric ring, called a connector, (e) ATP-dependent motor-driven entry of DNA through the connector's channel into a cavity of the procapsid, (f) termination of packaging that is accompanied by a second cleavage in the case of the packaging of a concatemer. The figure below illustrates our best current model for bacteriophage T7 DNA packaging.¹ Changes in the structure of the procapsid accompany these events. The final capsid of the mature bacteriophage is typically more angular, larger, less protease sensitive, more stable and less electrically charged at its surface than the procapsid.

       Bacteriophage DNA packaging involves a complex process of initiation and also a biological motor. Complex initiation is a means for leaving the world of pure physical chemistry and entering the world of natural selection, i.e., the world of biology. Complex initiation can optimize the probability that the genome packaged is undamaged. Complex initiation can also reduce the chance that too much packaging activity occurs and, therefore, that ATP is exhausted before any packaging is completed.² Therefore, one focus of our research is the initiation of bacteriophage DNA packaging.

       Bacteriophage DNA packaging motors are examples of ATP-dependent motors that are essential to all biological systems. These systems include the sliding filaments of muscle and the transport vesicles/microtubules of all cells. We adopt the working hypothesis that, thus far, in vitro DNA packaging systems are much more complete than in vitro systems used to analyze the motors of higher organisms. An incomplete system can be missing a motor component essential to grasping even the rudiments of how the motor works. For example, the evidence supports the presence of a feedback loop that is essential even to the most primitive function of bacteriophage DNA packaging motors.³ Feedback loops of this type have not even surfaced in the data for other motors, even though their existence is reasonably expected. So, a second focus of our research is the mechanism of bacteriophage DNA packaging motors.

       Our analysis of DNA packaging uses fluorescence microscopy of single biochemical events. To give an example of this "single-particle" analysis, one of our previous studies used single-particle (single concatemer, in this case) fluorescence microscopy to observe stained DNA during the packaging of concatemer-associated genomes of bacteriophage T7. The result was surprising at first. Packaging was clustered in time for the multiple genomes of a single concatemer. The resultant hypothesis is that packaging is co-operative among the several capsids that package the genomes associated with a single concatemer.3 Before our observations were made, nobody had thought that DNA packaging might be co-operative. Nonetheless, co-operative packaging was a reasonable hypothesis given the conditions inside of a bacteriophage-infected cell. These conditions require control of packaging much tighter than the control required in a purified in vitro system.

       To analyze bacteriophage DNA packaging in more detail, I consider bacteriophage DNA packaging from the perspective of (A) the DNA, (B) the procapsid and (C) the complete system used for in vitro bacteriophage DNA packaging.

[1] Sun, M. Louie, D. and Serwer, P. (1999) Single-event analysis of the packaging of bacteriophage T7 DNA concatemers in vitro. Biophys. J. 77, 1627-1637.

[2] Serwer, P. and Cerritelli, M. (2003) T3/T7 DNA packaging, in preparation.

[3] Serwer, P. (2003) Models of bacteriophage DNA packaging motors. J. Struct Biol., in press.