title: Directed DNA shuffling of retrovirus and retrotransposon integrase protein domains.
abstract: Chimeric proteins are used to study protein domain functions and to recombine protein domains for novel or optimal functions. We used a library of chimeric integrase proteins to study DNA integration specificity. The library was constructed using a directed shuffling method that we adapted from fusion PCR. This method easily and accurately shuffles multiple DNA gene sequences simultaneously at specific base-pair positions, such as protein domain boundaries. It produced all 27 properly-ordered combinations of the amino-terminal, catalytic core, and carboxyl-terminal domains of the integrase gene from human immunodeficiency virus, prototype foamy virus, and Saccharomyces cerevisiae retrotransposon Ty3. Retrotransposons can display dramatic position-specific integration specificity compared to retroviruses. The yeast retrotransposon Ty3 integrase interacts with RNA polymerase III transcription factors to target integration at the transcription initiation site. In vitro assays of the native and chimeric proteins showed that human immunodeficiency virus integrase was active with heterologous substrates, whereas prototype foamy virus and Ty3 integrases were not. This observation was consistent with a lower substrate specificity for human immunodeficiency virus integrase than for other retrovirus integrases. All eight chimeras containing the Ty3 integrase carboxyl-terminal domain, a candidate targeting domain, failed to target strand transfer in the presence of the targeting protein, suggesting that multiple domains of the Ty3 integrase cooperate in this function.
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, California, United States of America.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Qi
authors.forename: Edwin
authors.initials: E
authors.lastname: Vargas
authors.forename: Liza
authors.initials: L
authors.lastname: Larsen
authors.forename: Whitney
authors.initials: W
authors.lastname: Knapp
authors.forename: G Wesley
authors.initials: GW
authors.lastname: Hatfield
authors.forename: Richard
authors.initials: R
authors.lastname: Lathrop
authors.forename: Suzanne
authors.initials: S
authors.lastname: Sandmeyer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D019020:Directed Molecular Evolution / Q000379:methods*; D015723:Gene Library; D015497:HIV-1 / Q000201:enzymology; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D016415:Sequence Alignment; D016092:Spumavirus / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019426:Integrases; D012194:RNA-Directed DNA Polymerase; C496084:reverse transcriptase Ty3, S cerevisiae