I was trained as a biochemist-molecular biologist as a graduate student in the laboratory of David Hogness, then working on lambda phage, in the biochemistry department of Arthur Kornberg in the Stanford Medical School; and then as a Jane Coffin Childs Fund Postdoctoral Fellow in the Institute of Molecular Biology, University of Geneva, Geneva, Switzerland in the laboratory of Eduard Kellenberger, a pioneering electron microscopist and structural biologist. I have maintained my interest in the prokaryotic problems I was introduced to in both of these eminent training centers. My research program has been supported for over 30 years by NIH. During this period I have received an NIH Merit Award.
My primary interest is in viral nucleic acid packaging which is highly conserved. A powerful packaging motor is required to translocate DNA into an empty procapsid. The ATP powered phage motor consists of a packaging enzyme docked to a unique procapsid vertex containing a dodecameric portal. It packages DNA through the portal channel to ~500 mg/ml. We have shown that a long favored rotary portal motor mechanism does not apply (see reference #1 for recent review). Instead packaging in vitro of short dye labeled model DNA substrates provides evidence for a transient linear torsional B form to A form DNA "crunching" or spring-like motor mechanism to translocate DNA by the large terminase subunit (1, 3, 6, 9, 11, 12, 17) . A four strand "synapsis" of two homologous pac sequences in the DNA concatemer by a twin ring form of the small terminase subunit gauges DNA maturation and regulates cutting of the DNA by the large terminase to initiate packaging in vivo (1).
We have developed a bipartite phage display system to study the complex integration of DNA packaging with other viral development steps in vivo (18) and to use for cell docking. Most recently we have pioneered use of fluorescence correlation spectroscopy and smFRET to study viral DNA packaging in real time and to understand packaging structure and dynamics in collaboration with colleagues at the Center for Fluorescence Spectroscopy at UMB (15, 14, 11, 10).
Packaging DNA and protein together into a phage T4 procapsid in vitro with specific proteins and DNAs has useful applications. Virtually any active protein (100s of copies) can be transferred along with any DNA or multiple DNAs (170 kb total) by such a viral nanocontainer into cells. Capsid decoration proteins allow specific cell targeting and uptake of the nanocargo (2, 5, 8).
We have recently studied the giant PhiKZ bacteriophage. Its capsid contains a remarkable protein rod-like structure within the densely packaged DNA that is displaced from the portal apex axis (4). This >10 MD structure is assembled from more than six structural proteins processed (~50 % protein removal) by a morphogenetic protease to make the structure found in the virion (5, 7). The function of this structure is unknown but this bacteriophage and its relatives inject these structural proteins and large enzymatically active proteins such as multi-subunit RNA polymerases into the infected host together with the DNA.
The Tquatrovirinae can contain highly modified hmC (hydroxymethylcytosine) DNA that prevents DNA breakdown by host restriction modification enzymes. A glucose modified restriction gmrS/gmrD enzyme that targets diverse glycosyl-hmC modified phage DNAs has been isolated from pathogenic E. coli CT596 (16). In response T-even phages have evolved capsid-targeted internal protein enzyme inhibitors injected into the host with the DNA to shield it. Analysis of the structures of these inhibitors reveals an evolutionary pathway that has elaborated a surprisingly diverse and specifically fitted set of coevolving attack and defense proteins and DNA modifications (13).
Lab Techniques and Equipment:
We employ standard molecular and biochemical techniques together with electron microscopy for structure-function studies of phage packaging and assembly. We have developed bipartite phage display as well as a phage internal protein packaging system to study structure and assembly as well as to develop new phage derived technology. Most recently we have pioneered use of fluorescence correlation spectroscopy and smFRET to study viral DNA packaging in real time and to understand packaging structure and dynamics in collaboration with colleagues at the Center for Fluorescence Spectroscopy at UMB.