Paired DNA three-way junctions as scaffolds for assembling integrase complexes

Early steps of retroviral replication involve reverse transcription of the viral RNA genome and integration of the resulting cDNA copy into a chromosome of the host cell. The initial DNA breaking and joining steps of integration are carried out by the virus-encoded integrase enzyme. Integrases bind specifically to the ends of the unintegrated viral cDNA but nonspecifically to target DNA. Conventional assays in vitro reveal primarily the nonspecific DNA binding mode, complicating studies of integrase--DNA complexes. Here, we report an investigation of unconventional DNA structures useful for positioning integrase at predetermined sites. We find that paired DNA three-way junctions can be used to mimic branched DNAs normally formed as reaction intermediates. The three-way junctions differ from authentic intermediates in the connectivity of the DNAs, which, in contrast to the authentic intermediate, allow formation of stable DNA structures under physiological conditions. Assays in vitro showed that integrase can direct hydrolysis at sequences resembling the viral cDNA ends within the three-way junction, but not on junctions with mutant sequences. Changing the spacing between the paired three-way junctions disrupted the cleavage pattern, emphasizing the importance of the correct DNA scaffold. DNase I footprinting studies revealed protection of specific bases at the terminus of the LTR in the three-way junction complex, but not on control linear DNA, specifying the locations of tight interactions between integrase and DNA. Paired DNA three-way junctions are attractive reagents for structural studies of integrase-DNA complexes.