Abstract
Retroviral DNA made by reverse transcription is blunt-ended, and the viral integrase protein must remove two nucleotides from each 3′ end prior to integration into chromosomal DNA. Under most reaction conditions for integration in vitro, the majority of the reaction products are “half-site” products that result from integration of only one viral DNA end into one strand of the target DNA. Preprocessed DNA substrates are more efficient substrates for half-site reactions than are blunt-ended substrates, which require the removal of two nucleotides prior to integration. In contrast, we find that blunt-ended DNA is a better substrate for the biologically relevant reaction of concerted integration of pairs of viral DNA ends. The reaction pathway is channeled to concerted integration, and half-site integration products are reduced with blunt-ended DNA substrate that must first be processed by integrase. In addition, the terminal nucleotide requirements for concerted integration are more stringent than for the half-site reaction. Longer DNA is more efficient for the concerted reaction than is shorter DNA that is capable of efficient half-site integration. This suggests that nonspecific interactions of integrase with viral DNA distant from the termini contribute to the assembly of a complex that is competent for concerted integration. Finally, differential effects of mutation of a residue in the C-terminal domain of integrase on concerted versus half-site integration implicate protein-protein interactions involving this domain as important for concerted integration.
Highlights
Introduction□S The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure and a supplemental table
□S The on-line version of this article contains a supplemental figure and a supplemental table
In contrast to the typical reaction products with purified integrase, the products mostly result from concerted integration of a pair of viral DNA ends into target DNA
Summary
□S The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure and a supplemental table. Under most reaction conditions the products of DNA strand transfer result from the integration of a single viral DNA end into one strand of the target DNA, a reaction that has been termed “half-site” as opposed to concerted integration. Were this to occur in the cell, the viral DNA would fail to integrate, and the viral replication cycle would be aborted. Because PICs are not readily amenable to detailed biochemical analysis at the molecular level, it is necessary to reconstitute complexes with all the features of those isolated from cells to fully understand their functioning To this end, we have investigated the factors that promote concerted integration by HIV-1 integrase. These results suggest that the C-terminal domain is involved in a multimerization interface that is required for concerted integration but is dispensable for the half-site reaction
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