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J Biol Chem. 2008 Jun 6;283(23):15965-74. doi: 10.1074/jbc.M801354200. Epub 2008 Apr 8.

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.

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  • 1Section on Eukaryotic Transposable Elements, Laboratory of Gene Regulation and Development, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.

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.

PMID:
18397885
[PubMed - indexed for MEDLINE]
PMCID:
PMC2414268
Free PMC Article
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