Kinetic Analysis of the Genome Packaging Reaction in Bacteriophage λ

Bacteriophage λ is a double-stranded DNA virus that infects the <i>Escherichia coli</i> bacterium. λ genomic DNA is replicated via rolling circle replication, resulting in multiple genomes linked head to tail at the <i>cos</i> site. To insert a single λ genome into the viral capsid, the λ terminase enzyme introduces symmetric nicks, 12 bp apart, at the <i>cos</i> site, and then promotes a strand separation reaction, releasing the tail end of the previous genome and leaving a binary complex consisting of λ terminase bound to the head end of the adjacent genome. Next, the genome is translocated into the interior of the capsid particle, in a process that requires ATP hydrolysis by λ terminase. Even though DNA packaging has been studied extensively, currently no bulk assays are available that have been optimized to report directly on DNA translocation. Rather, these assays are sensitive to assembly steps reflecting formation of the active, DNA packaging machine. In this work, we have modified the DNase protection assay commonly used to study DNA packaging in several bacteriophage systems, such that it reports directly on the kinetics of the DNA packaging reaction. We have analyzed our DNA packaging data according to an <i>N</i>-step sequential minimal kinetic model and have estimated an overall packaging rate of 119 ± 8 bp/s, at 4 °C and 1 mM ATP. Furthermore, we have measured an apparent step size for the this reaction (<i>m</i><sub>obs</sub>) of 410 ± 150 bp. The magnitude of this value indicates that our assay is most likely sensitive to both mechanical steps associated with DNA insertion as well as occasional slow steps that are repeated every >410 bp. These slow steps may be reflective of the pausing events observed in recent single-molecule studies of DNA packaging in bacteriophage λ [Fuller, D. N., et al. (2007) <i>J. Mol. Biol. 373</i>, 1113−1122]. Finally, we show that either ATP or ADP is required for terminase cutting at <i>cos</i>, to generate the active, DNA packaging complex.