Abstract
We have shown previously that the active form of avian sarcoma virus integrase (ASV IN) is a multimer. In this report we investigate IN multimerization properties by a variety of methods that include size exclusion chromatography, chemical cross-linking, and protein overlay assays. We show that removal of the nonconserved C-terminal region of IN results in a reduced capacity for multimerization, whereas deletion of the first 38 amino acids has little effect on the oligomeric state. Binding of a full-length IN fusion protein to various IN fragments indicates that sequences in both the catalytic core (residues 50-207) and a C-terminal region (residues 201-240) contribute to IN self-association. We also observe that the isolated C-terminal fragment (residues 201-286) is capable of self-association. Finally, a single amino acid substitution in the core domain (S85G) produces a severe defect in multimerization. We conclude from these analyses that both the catalytic core and a region in the nonconserved C terminus are involved in ASV integrase multimerization. These results enhance our understanding of intergrase self-association determinants and define a major role of the C-terminal region of ASV integrase in this process.
Highlights
§ To whom correspondence should be addressed: Fox Chase Cancer Center, Institute for Cancer Research, 7701 Burholme Ave., Philadelphia, PA 19111
Catalytic functions have been localized to the central core domain, which is resistant to limited proteolysis [10], and contains a conserved triad of acidic residues [D,D(35)E] that is presumed to bind the divalent cations that are required for catalytic activity [5, 11]
We have used a variety of chemical cross-linking reagents to identify the multimeric forms of avian sarcoma virus integrase (ASV IN) that exist in solution
Summary
The isolated catalytic domain is competent to perform a concerted cleavage-ligation activity [12,13,14] and contributes to the recognition of conserved CA residues present at the 3Ј ends of retroviral long terminal repeats and other transposable elements [15]. It cannot perform the viral DNA end processing and joining reactions required for insertion of viral DNA into target DNA sequences. In addition to demonstrating a role for the conserved catalytic core domain in ASV IN dimerization, our results un-
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