Presteady-State Analysis of ASV integrase: II. Reverse-Polarity Substrates Identify Preferential Processing of the U3-U5 Pair

doi:10.1074/jbc.M111314200

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Papers In Press, published online ahead of print January 30, 2002
J. Biol. Chem, 10.1074/jbc.M111314200
Submitted on November 28, 2001
Revised on January 29, 2002
Accepted on January 29, 2002

Presteady-State Analysis of ASV integrase: II. Reverse-Polarity Substrates Identify Preferential Processing of the U3-U5 Pair

Kogan K. Bao, Anna Marie Skalka, and Isaac Wong

Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331

Corresponding Author: wongis{at}onid.orst.edu

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactions in vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka [Kukolj, G. and Skalka, A. M. (1995) Genes Dev 9, 2556-67] have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5’-5’ phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral LTR-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction [Bao et al., (2002) in press] that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2s-1 for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair.

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