title: The GP(Y/F) domain of TF1 integrase multimerizes when present in a fragment, and substitutions in this domain reduce enzymatic activity of the full-length protein.
abstract: Integrases (INs) of retroviruses and long terminal repeat retrotransposons possess a C-terminal domain with DNA binding activity. Other than this binding activity, little is known about how the C-terminal domain contributes to integration. A stretch of conserved amino acids called the GP(Y/F) domain has been identified within the C-terminal IN domains of two distantly related families, the gamma-retroviruses and the metavirus retrotransposons. To enhance understanding of the C-terminal domain, we examined the function of the GP(Y/F) domain in the IN of Tf1, a long terminal repeat retrotransposon of Schizosaccharomyces pombe. The activities of recombinant IN were measured with an assay that modeled the reverse of integration called disintegration. Although deletion of the entire C-terminal domain disrupted disintegration activity, an alanine substitution (P365A) in a conserved amino acid of the GP(Y/F) domain did not significantly reduce disintegration. When assayed for the ability to join two molecules of DNA in a reaction that modeled forward integration, the P365A substitution disrupted activity. UV cross-linking experiments detected DNA binding activity in the C-terminal domain and found that this activity was not reduced by substitutions in two conserved amino acids of the GP(Y/F) domain, G364A and P365A. Gel filtration and cross-linking of a 71-amino acid fragment containing the GP(Y/F) domain revealed a surprising ability to form dimers, trimers, and tetramers that was disrupted by the G364A and P365A substitutions. These results suggest that the GP(Y/F) residues may play roles in promoting multimerization and intermolecular strand joining.
authors.affiliation: Section on Eukaryotic Transposable Elements, Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Ebina
authors.forename: Atreyi Ghatak
authors.initials: AG
authors.lastname: Chatterjee
authors.forename: Robert L
authors.initials: RL
authors.lastname: Judson
authors.forename: Henry L
authors.initials: HL
authors.lastname: Levin
mesh_terms: D019943:Amino Acid Substitution; D019426:Integrases / Q000737:chemistry* / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018626:Retroelements / Q000502:physiology; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry* / Q000378:metabolism; D020079:Terminal Repeat Sequences / Q000502:physiology
chemical_list: D011994:Recombinant Proteins; D018626:Retroelements; D029702:Schizosaccharomyces pombe Proteins; D019426:Integrases