A Tripartite Dna-binding Element, Comprised of the Nuclear Localization Signal and Two At-hook Motifs, Mediates the Association of Ledgf/P75 with Chromatin in Vivo.
From: Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Nucleic acids research
- Publish Date: 2006
- ISSN: 1362-4962
- Volume: 34
- Issue: 5
- Pages: 1653-75
- Medium: Internet
- Language: English
- Citation (JAMA): Turlure Fanny, Maertens Goedele, Rahman Shaila, et al. A Tripartite Dna-binding Element, Comprised of the Nuclear Localization Signal and Two At-hook Motifs, Mediates the Association of Ledgf/P75 with Chromatin in Vivo.. Nucleic Acids Res. 2006;34:1653-75
Abstract
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.
Mesh Headings (Keywords): AT-Hook Motifs, Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Binding Sites, Chromatin, Consensus Sequence, DNA, DNA-Binding Proteins, HIV Integrase, Hela Cells, Humans, Molecular Sequence Data, Nuclear Localization Signals, Protein Structure, Tertiary, Transcription Factors
Check for Full Text / PubMed Unique Identifier (PMID): 16549878
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