Base-pair substitutions in avian sarcoma virus U5 and U3 long terminal repeat sequences alter the process of DNA integration in vitro

We have described a reconstituted avian sarcoma virus (ASV) concerted DNA i ntegration system with specially designed mini-donor DNA containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially ex pressed ASV integrase (IN), and human high-mobility-group protein I(Y). Int egration in this system is dependent upon the mini-donor DNA having IN reco gnition sequences at both ends and upon both ends of the same donor integra ting into the acceptor DNA. The integrated DNA product exhibits all of the features associated with integration of viral DNA in vivo (P. Hindmarsh et al., J. Virol., 73:2994-3003, 1999). Individual integrants are isolated fro m bacteria containing drug-resistant markers with amber mutations. This sys tem was used to evaluate the importance of sequences in the terminal U5 and U3 long terminal repeats at positions 5 and/or 6, adjacent to the conserve d CA dinucleotide. Base-pair substitutions introduced at these positions in U5 result in significant reductions in recovered integrants from bacteria, due to increases in one-ended insertion events. Among the recovered integr ants from reactions with mutated U5 but not U3 IN recognition sequences wer e products that contain large deletions in the acceptor DNA. Base-pair subs titutions at positions 5 and 6 in U3 mostly reduce the efficiency of integr ation of the modified donor. Together, these results indicate that sequence s directly 5' to the conserved CA dinucleotide are very important for the p rocess of concerted DNA integration. Furthermore, IN interacts with U3 and U5 termini differently, and aberrant end-processing events leading to nonco ncerted DNA integration are more common in U5 than in U3.