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Cooperation of HECT-domain ubiquitin ligase hHYD and DNA topoisomerase II-binding protein for DNA damage response.

Ubiquitin ligases define the substrate specificity of protein ubiquitination and subsequent proteosomal degradation. The catalytic sequence was first characterized in the C terminus of E6-associated protein (E6AP) and referred to as the HECT (homologous to E6AP C terminus) domain. The human homologue of the regulator of cell proliferation hyperplastic discs in Drosophila, designated hHYD, is a HECT-domain ubiquitin ligase. Here we show that hHYD provides a ubiquitin system for a cellular response to DNA damage. A yeast two-hybrid screen showed that DNA topoisomerase IIbeta-binding protein 1 (TopBP1) interacted with hHYD. Endogenous hHYD bound the BRCA1 C-terminus domains of TopBP1 that are highlighted in DNA damage checkpoint proteins and cell cycle regulators. Using an in vitro reconstitution, specific E2 (ubiquitin-conjugating) enzymes (human UbcH4, UbcH5B, and UbcH5C) transferred ubiquitin molecules to hHYD, leading to the ubiquitination of TopBP1. TopBP1 was usually ubiquitinated and degraded by the proteosome, whereas X-irradiation diminished the ubiquitination of TopBP1 probably via the phosphorylation, resulting in the stable colocalization of up-regulated TopBP1 with gamma-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.

Pubmed ID: 11714696


  • Honda Y
  • Tojo M
  • Matsuzaki K
  • Anan T
  • Matsumoto M
  • Ando M
  • Saya H
  • Nakao M


The Journal of biological chemistry

Publication Data

February 1, 2002

Associated Grants


Mesh Terms

  • Animals
  • COS Cells
  • Carrier Proteins
  • Catalytic Domain
  • Cercopithecus aethiops
  • DNA Damage
  • DNA-Binding Proteins
  • Dithiothreitol
  • Gene Library
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Muscle, Skeletal
  • Nuclear Proteins
  • Peptide Synthases
  • Phosphorylation
  • Saccharomyces cerevisiae
  • Signal Transduction
  • Substrate Specificity
  • Transfection
  • Ubiquitin
  • Ubiquitin-Protein Ligases
  • X-Rays