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Journal of Virology, February 2001, p. 1132-1141, Vol. 75, No. 3
Department of Biochemistry, Case Western
Reserve University School of Medicine, Cleveland, Ohio
44106-49351; Washington State
University, Pullman, Washington 991642; and
Department of Microbiology and Immunology, Northwestern
University School of Medicine, Chicago, Illinois
606113
Received 17 August 2000/Accepted 25 October 2000
We have described a reconstituted avian sarcoma virus (ASV)
concerted DNA integration system with specially designed mini-donor DNA
containing a supF transcription unit, a supercoiled plasmid acceptor, purified bacterially expressed ASV integrase (IN), and human
high-mobility-group protein I(Y). Integration in this system is
dependent upon the mini-donor DNA having IN recognition sequences at
both ends and upon both ends of the same donor integrating 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 from bacteria containing drug-resistant markers with amber
mutations. This system 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 conserved 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 integrants from reactions with
mutated U5 but not U3 IN recognition sequences were products that
contain large deletions in the acceptor DNA. Base-pair substitutions at positions 5 and 6 in U3 mostly reduce the efficiency of integration of
the modified donor. Together, these results indicate that sequences directly 5' to the conserved CA dinucleotide are very important for the
process of concerted DNA integration. Furthermore, IN interacts with U3
and U5 termini differently, and aberrant end-processing events leading
to nonconcerted DNA integration are more common in U5 than in U3.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.3.1132-1141.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Base-Pair Substitutions in Avian Sarcoma Virus U5
and U3 Long Terminal Repeat Sequences Alter the Process of DNA
Integration In Vitro
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Northwestern University School of
Medicine, 303 Chicago Ave., Chicago, IL 60611. Phone: (312) 503-1166. Fax: (312) 503-1339. E-mail: j-leis{at}northwestern.edu.
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