CPB 697 RESEARCH SEMINAR

DEPARTMENT OF COMPARATIVE PATHOBIOLOGY

Tae Jin Lee, MS

Graduate Student in Molecular Virology

Department of Comparative Pathobiology

Purdue University

“Kinetic And Single Molecule Study On

DNA-Dependent ATP Hydrolysis Of gp16 Of Bacterial Virus Phi29”

Thursday, April 19, 2007

VPTH 112

3:30 p.m.

Abstract:

All linear dsDNA viruses translocate their genome into a pre-assembled procapsid to near-crystalline density. The packaging enzyme in the viral DNA-packaging motors generates the driving force to overcome this entropically unfavorable packaging reaction. The putative packaging enzyme gp16 of Bacillus subtilis phage virus phi29 contains a conserved nucleotide triphosphate binding motif.

It was found that gp16 bound to DNA nonspecifically and that gp16 bound to the phi29 packaging RNA (pRNA) -containing procapsid much more strongly than to the pRNA-free procapsid. Gp16 binds to the 5’/3’ paired helical region of pRNA in the pRNA/procapsid complex. The C₁₈C₁₉A₂₀ bulge on pRNA that is essential for DNA packaging was found to be dispensable for gp16 binding.

We also performed the steady-state analysis on the ATPase activity of gp16 using fluorescence-involved system. It shows that gp16 itself is a low-affinity ATPase. However, DNA binding to gp16 stimulates the ATP hydrolysis activity of gp16. In addition, we investigated the interaction of gp16 with nucleic acids using recently developed single molecule system. The technique employs TIRF enable to observe the binding between molecules. The single molecule studies showed that gp16 binds DNA depending on their structures, which agrees with the ATPase stimulation effect of DNA binding.