Carlos Catalano, Principal Investigator
Our laboratory is interested in the molecular mechanisms of genome packaging in the double-stranded DNA viruses. Similar mechanisms for genome packaging have been proposed for all of the dsDNA bacteriophages, and likely apply to mammalian viruses such as adenovirus and the herpesvirus groups. Terminase enzymes are common to all of these viruses and are responsible for the packaging of a single genome within the confines of a pre-formed, empty viral capsid. We study the biochemical and biophysical properties of the bacteriophage lambda terminase, and the nature of the multiple nucleoprotein complexes involved in the packaging process. Phage lambda terminase possesses a site-specific endonuclease activity, a DNA strand-separation activity, and a DNA-stimulated ATPase activity, all of which work in concert to effect genome packaging. This enzyme is an integral part of several nucleoprotein intermediates that are required for successful packaging of the genome within the viral capsid. The experiments performed in our laboratory systematically probe the proteinoprotein and proteinoDNA interactions required to assemble a stable nucleoprotein complex that site-specifically nicks a concatemeric DNA precursor, and the subsequent interactions required to disengage this complex from the assembly site so that packaging may ensue. We couple detailed enzyme kinetic analyses (steady-state and pre-steady-state) with biophysical characterization of proteinoprotein and proteinoDNA interactions (CD & fluorescence spectroscopy, analytical ultracentrifugation, quantitative gel shift and DNase footprinting, surface plasmon resonance, etc.) to characterize the structure and function of these nucleoprotein packaging complexes. NMR studies in our lab have yielded the three dimensional high-resolution structure of the small terminase subunit, and we are currently expanding these studies to define the structure of the holoenzyme complex (NMR, crystallography). While mechanistic details may differ, the data derived from our studies may be used to model DNA packaging by other double- stranded DNA viruses, including assembly in the eucaryotic herpesvirus groups. A major effort is currently under way in our lab to characterize DNA packaging in herpes simplex 1.