Journal Article

A tripartite DNA-binding element, comprised of the nuclear localization signal and two AT-hook motifs, mediates the association of LEDGF/p75 with chromatin <i>in vivo</i>

Fanny Turlure, Goedele Maertens, Shaila Rahman, Peter Cherepanov and Alan Engelman

in Nucleic Acids Research

Volume 34, issue 5, pages 1653-1665
Published in print March 2006 | ISSN: 0305-1048
Published online March 2006 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/gkl052

More Like This

Show all results sharing these subjects:

  • Chemistry
  • Biochemistry
  • Bioinformatics and Computational Biology
  • Genetics and Genomics
  • Molecular and Cell Biology

GO

Show Summary Details

Preview

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a DNA-binding, transcriptional co-activator that participates in HIV-1 integration site targeting. Using complementary approaches, we determined the mechanisms of LEDGF/p75 DNA-binding in vitro and chromatin-association in living cells. The binding of highly-purified, recombinant protein was assayed by surface plasmon resonance (SPR) and electrophoretic mobility gel shift. Neither assay revealed evidence for sequence-specific DNA-binding. Residues 146–197 spanning the nuclear localization signal (NLS) and two AT-hook motifs mediated non-specific DNA-binding, and DNA-binding deficient mutants retained the ability to efficiently stimulate HIV-1 integrase activity in vitro. Chromatin-association was assessed by visualizing the localization of EGFP fusion proteins in interphase and mitotic cells. Although a conserved N-terminal PWWP domain was not required for binding to condensed mitotic chromosomes, its deletion subtly affected the nucleoplasmic distribution of the protein during interphase. A dual AT-hook mutant associated normally with chromatin, yet when the mutations were combined with NLS changes or deletion of the PWWP domain, chromatin-binding function was lost. As the PWWP domain did not readily bind free DNA in vitro, our results indicate that chromatin-association is primarily affected through DNA-binding, with the PWWP domain likely contributing a protein interaction to the overall affinity of LEDGF/p75 for human chromatin.

Journal Article.  9091 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

Users without a subscription are not able to see the full content. Please, subscribe or login to access all content.