Integrase catalyzes insertion of a retroviral genome into the host chromosome.
Following reverse transcription, integrase binds specifically to the ends of the duplex retroviral
DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity),
and inserts these ends into the host DNA (the joining activity) in a concerted manner.
Additionally, it has been observed that integrase can catalyze the removal of inserted viral ends
(the disintegration activity) in vitro. Presteady-state experiments were performed using synapsed
substrates to probe the processing reaction and a disintegration substrate to determine the
number of protomers in a functional multimeric complex. In single-turnover studies, a novel
"splicing" reaction was observed that revealed complications with accurate quantification of
enzymatic activity using the synapsed substrates. The splicing reaction was further used to gain
insight into the selection of nucleophiles and electrophiles at the binding site. To reduce the
complexity introduced by the integrase-catalyzed splicing reaction, 5'-5' reverse-polarity
synapsed substrates were designed that were not susceptible to the splicing reaction and that
allowed direct comparison of LTR ends simultaneously bound at the active site. Analysis of the
presteady-state assays using these reverse-polarity substrates revealed that the concurrent binding
of the biologically relevant U3/U5 combination of viral ends facilitates maximal activity of the
processing reaction. A disintegration substrate was used in presteady-state active site titrations to
determine a reaction stoichiometry of four integrase protomers per one substrate molecule for
the disintegration reaction. A tetrameric active complex was then confirmed using atomic force
microscopy to image integrase-DNA complexes during the first catalytic turnover. The
observed increase of the tetramer population in the presence of substrate DNA demonstrates that
the binding of the disintegration substrate induces assembly of the active tetramer and suggests
that tetramer assembly may be an integral and dynamic component of the catalytic pathway. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/30239 |
| Date | 19 September 2002 |
| Creators | Bao, Kogan K. |
| Contributors | Wong, Isaac |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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