Abstract
A number of studies have demonstrated that the retroviral protein integrase (IN) alone is sufficient to carry out two discrete steps required for retroviral integration: the endonucleolytic processing of viral DNA ends and the cleavage and joining of host DNA to the processed viral DNA termini. Little is known about the biochemical and biophysical mechanisms involved in these reactions. Here, we employ in vitro assays of Rous sarcoma virus IN to demonstrate for the first time that IN is capable of multiple turnover in both the processing and joining reactions. The turnover number calculated for the processing reaction is 0.26 cleavages/min/mol of IN. Our steady state kinetic studies indicate that both the processing and joining activities require a multimeric form of IN. Ultracentrifugation analyses reveal a substrate-independent reversible equilibrium among the monomeric, dimeric, and tetrameric forms of this protein. From these results we conclude that the minimal functional unit for both the processing and joining of each viral DNA end is an IN dimer.
Highlights
A number of studies have demonstrated that the retroviral protein integrase(IN) alone is sufficientto carry out two discrete steps required for retroviral integration: the endonucleolytic processing of viral DNA ends and the cleavage and joining of host DNA to the processed viral DNA termini
The turnover In this paper, we use in vitro assays to demonstrate that number calculated for the processing reaction is 0.26 the IN from Rous sarcoma virus (RSV) cuanndergo multiple cleavages/min/mol of IN
We investigate its activities using steady state kinetic analysis and its self-association potential by equilibrium ultracentrifugation analyses
Summary
Tionare located at the ends of the long terminalrepeats (LTRs). In vitro, IN does not require that the entire viral DNAbeused as a substrate; short duplex oligonucleotides homologous to the viralDNA termini can serve as substrates (Received for publication, May 20, 1992). We employ in vitro function more than once; a singlerecombination eventshould assays of Rous sarcoma virus IN to demonstrate for be sufficient to integrate a provirus, and there are an estithe firsttime that IN is capable of multiple turnover in mated 50-100 mol of IN/virion [9]. From these results we conclude that the minimal functional unit for both the processing and joining of each viral DNA end is an IN dimer
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