Human Immunodeficiency Virus Nucleocapsid Protein: Analysis of the mechanism of strand exchange and the role of the zinc fingers in nucleic acid chaperone activity.

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dc.contributor.advisorDeStefano, Jeffrey Jen_US
dc.contributor.authorHeath, Megan Joyen_US
dc.contributor.departmentMolecular and Cell Biologyen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2004-07-16T05:19:50Z
dc.date.available2004-07-16T05:19:50Z
dc.date.issued2004-06-15en_US
dc.description.abstractThe human immunodeficiency virus genome is coated by the nucleocapsid protein (NC). NC is a 55 amino acid highly basic protein. It has two zinc fingers that differ by five amino acids. NC contains nucleic acid chaperone activity that aids in the formation of highly stable nucleic acid structures by destabilizing and preventing the formation of weaker structures. This activity is important for genome dimerization and maturation, tRNA:primer binding site annealing, and many steps in reverse transcription. Annealing experiments were performed with four different RNA structures and complementary DNAs. NC enhanced annealing of all structures showing that NC enhances both unwinding of nucleic acid structure and hybridization of unstructured sequences. NC mutant proteins were used in annealing assays. 1.1 NC had two copies of the first zinc finger, 2.2 NC had two copies of the second zinc finger, and 2.1 NC had both zinc fingers with their positions switched. Experiments showed that all mutants could enhance the annealing of weakly structured nucleic acids but only 1.1 NC and 2.1 NC enhanced annealing of strongly structured nucleic acids. Results suggest that finger one is important for nucleic acid unwinding while finger two plays an accessory role in annealing. The mechanism of strand exchange, another important aspect of NC chaperone activity, was also investigated. Experiments were performed using RNA:DNA hybrids with either the DNA or RNA radioactively labeled. Hybrids were incubated with different types of RNA acceptor molecules to which the DNA could transfer. The transfer of the DNA or the displacement of the original donor RNA was monitored. Experiments showed that optimal enhancement of strand exchange by NC occurred with acceptors that had more than 22 nucleotides that could anneal to the single stranded region of the DNA. Also, experiments with acceptors that had point mutations showed that the region of the acceptor that binds to the single stranded region of the DNA should be completely complementary for optimal NC stimulation. These results indicate the annealing of the acceptor and DNA outside the donor:DNA hybrid region can be an important initiation step for NC enhanced strand exchange.en_US
dc.format.extent2949089 bytes
dc.format.mimetypeapplication/pdf
dc.identifier.urihttp://hdl.handle.net/1903/1690
dc.language.isoen_US
dc.subject.pqcontrolledBiology, Microbiologyen_US
dc.subject.pqcontrolledBiology, Generalen_US
dc.subject.pquncontrollednucleic acid annealingen_US
dc.subject.pquncontrolledRNA chaperoneen_US
dc.subject.pquncontrollednucleocapsid proteinen_US
dc.subject.pquncontrolledrecombination and strand exchangeen_US
dc.subject.pquncontrolledzinc fingersen_US
dc.subject.pquncontrolledHIVen_US
dc.titleHuman Immunodeficiency Virus Nucleocapsid Protein: Analysis of the mechanism of strand exchange and the role of the zinc fingers in nucleic acid chaperone activity.en_US
dc.typeDissertationen_US

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