Our long-range goal is to understand virus assembly at the molecular level. DNA packaging is a critical step in the assembly of many double-stranded DNA viruses, including poxvirus, adenovirus, herpesviruses, and many bacteriophages. Terminase enzymes are common to these viruses and function to package viral DNA into the capsid. Common mechanisms for genome packaging have been proposed for these all viruses. The process of genome packaging is unique to viruses, and thus represents an ideal target for antiviral therapy; however, the lack of mechanistic detail precludes a rational approach to drug design. Bacteriophage lambda presents an ideal system to study DNA packaging. Lambda terminase is a central component of an ordered series of packaging intermediates, and is an integral part of the packaging motor. The goal of this project is to define a coherent physical and kinetic model for the assembly of a viral DNA packaging motor. A mechanistic model for any complex biological process requires a description of the physical nature of the macromolecular complexes involved, a kinetic dissection of the catalytic activities required for the process, and a mechanism that clearly describes the linkage between catalysis, structure, and function. Unfortunately, virtually nothing is known about the physical properties of the packaging intermediates in lambda. Moreover, a mechanistic link between catalytic activity and function remains elusive, due in part to the dearth of structural information. This project seeks to (i) characterize the interactions responsible for specific assembly of the packaging motor on viral DNA, (ii) define the assembly state of the functional packaging motor, to (iii) characterize critical packaging intermediates, and (iv) characterize the activity of defined enzyme species to provide a direct link between stucture and function of termiase. A combined structural, biophysical, and kinetic approach will define critical aspects central to our understanding of the mechanism and enzymology of DNA packaging.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM063943-04A1
Application #
6922345
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Basavappa, Ravi
Project Start
2001-09-10
Project End
2006-07-31
Budget Start
2005-05-01
Budget End
2006-07-31
Support Year
4
Fiscal Year
2005
Total Cost
$279,903
Indirect Cost
Name
University of Colorado Denver
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045
Tsay, James M; Sippy, Jean; DelToro, Damian et al. (2010) Mutations altering a structurally conserved loop-helix-loop region of a viral packaging motor change DNA translocation velocity and processivity. J Biol Chem 285:24282-9
Fuller, Derek N; Raymer, Dorian M; Rickgauer, John Peter et al. (2007) Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations. J Mol Biol 373:1113-22
Ortega, Marcos E; Gaussier, Helene; Catalano, Carlos E (2007) The DNA maturation domain of gpA, the DNA packaging motor protein of bacteriophage lambda, contains an ATPase site associated with endonuclease activity. J Mol Biol 373:851-65
Maluf, Nasib Karl; Gaussier, Helene; Bogner, Elke et al. (2006) Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine. Biochemistry 45:15259-68
Ortega, Marcos E; Catalano, Carlos E (2006) Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor. Biochemistry 45:5180-9
Gaussier, Helene; Ortega, Marcos E; Maluf, Nasib K et al. (2005) Nucleotides regulate the conformational state of the small terminase subunit from bacteriophage lambda: implications for the assembly of a viral genome-packaging motor. Biochemistry 44:9645-56
Yang, Qin; Catalano, Carlos Enrique (2004) A minimal kinetic model for a viral DNA packaging machine. Biochemistry 43:289-99