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Bioengineering Seminar Series: Lindsay Black
Wednesday, October 24, 2007
11:00 a.m.
2108 Chemical and Nuclear Engineering Building
For More Information:
Professor Peter Kofinas
(301) 405-7335
kofinas@umd.edu

Bacteriophage DNA Packaging Nanomotor

Presented by Lindsay Black
Department of Biochemistry and Molecular Biology
University of Maryland School of Medicine

Phages, herpes and other eukaryotic viruses package DNA into an empty preassembled procapsid container by a conserved biological mechanism. The phage ATP driven packaging nanomotor is among the strongest characterized and packs genomic DNA at ~1000 base pairs/sec to near crystalline density. The phage T4 terminase packaging enzyme complexed to the portal dodecamer ring of the procapsid packages linear DNA of any sequence in vitro and duplex DNAs 20-200 bp in length were ~100% efficiently packaged. Dye labeled DNAs were tolerated by the motor, whereas 20 base extensions, hairpin ends, 20 bp DNA-RNA hybrid, and dsRNA substrates were not packaged. A fluorescence correlation spectroscopy (FCS)-based assay revealed the kinetics of packaging of multiple segments into a procapsid and showed by FRET that dye labeled DNA was packed into a GFP containing procapsid interior. Molecules 60 bp long with 10 mismatched bases were efficiently translocated; substrates with 20 mismatched bases, a related D-loop structure or ones with 20 base single-strand regions were not packaged. A single nick in a 100 bp duplex almost completely blocked translocation, but the deleterious effect of a single nick lessened with increasing length of substrate. FCS revealed that small nicked substrates which inhibit translocation do not remain stably bound by the terminase-prohead. Thus, two unbroken DNA strands seem important for packaging, consistent with our proposed torsional-compression translocation model. Our aim is to demonstrate this mechanism employing fluorescence and other technologies applied to efficient T4 single packaging motor complexes.

This Event is For: Graduate • Undergraduate • Faculty • Post-Docs

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