Repair of gaps in retroviral DNA integration intermediates

Diverse mobile DNA elements are believed to pirate host cell enzymes to com plete DNA transfer. Prominent examples are provided by retroviral cDNA inte gration and transposon insertion. These reactions initially involve the att achment of each element 3' DNA end to staggered sites in the host DNA by el ement-encoded integrase or transposase enzymes. Unfolding of such intermedi ates yields DNA gaps at each junction. It has been widely assumed that host DNA repair enzymes complete attachment of the remaining DNA ends, but the enzymes involved have not been identified for any system. We have synthesiz ed DNA substrates containing the expected gap and 5' two-base flap structur e present in retroviral integration intermediates and tested candidate enzy mes for the ability to support repair in vitro. We find three required acti vities, two of which can be satisfied by multiple enzymes. These are a poly merase (polymerase beta, polymerase delta and its cofactor PCNA, or reverse transcriptase), a nuclease (flap endonuclease), and a ligase (ligase I, II I, or IV and its cofactor XRCC4). A proposed pathway involving retroviral i ntegrase and reverse transcriptase did not carry out repair under the condi tions tested. In addition, prebinding of integrase protein to gapped DNA in hibited repair reactions, indicating that gap repair in vivo may require ac tive disassembly of the integrase complex.