Abstract
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.
Highlights
The ubiquitin-proteosome proteolytic system is involved in a variety of fundamental cell regulations including gene expression, stress response, DNA repair, and cell cycles [1,2,3,4,5,6]
topoisomerase II-binding protein 1 (TopBP1) shows the presence of BRCA1 C terminus (BRCT) domains, which are found in many molecules involved in DNA damage response and checkpoint-mediated repair including BRCA1, p53-binding protein 1 (53BP1), Nijmegen breakage syndrome 1 (NBS1), x-ray repair crosscomplementing 1 (XRCC1), DNA ligases 3 and 4, and poly(ADP-ribose) polymerase (PARP) (16 –19)
Interaction of hHYD with TopBP1—To identify a protein factor that interacts with hHYD, we performed a yeast twohybrid screen of HeLa and skeletal muscle cDNA libraries utilizing the region of amino acids 2520 –2798, which contains the HECT domain and its N-terminal portion of hHYD as bait (Fig. 1A)
Summary
E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; hHYD, human hyperplastic discs; TopBP1, DNA topoisomerase II-binding protein 1; BRCT, BRCA1 C-terminus; E6AP, E6-associated protein; HECT, homologous to E6AP C-terminus; Gy, gray; PBS, phosphate-buffered saline; HA, hemagglutinin; ATP␥S, adenosine 5Ј-O-(thiotriphosphate). We focused on characterizing the human HECT-domain protein, a counterpart of the regulator of cell proliferation and the putative tumor suppressor hyperplastic discs in Drosophila melanogaster, termed hHYD. TopBP1 shows the presence of BRCA1 C terminus (BRCT) domains, which are found in many molecules involved in DNA damage response and checkpoint-mediated repair including BRCA1, p53-binding protein 1 (53BP1), Nijmegen breakage syndrome 1 (NBS1), x-ray repair crosscomplementing 1 (XRCC1), DNA ligases 3 and 4, and poly(ADP-ribose) polymerase (PARP) (16 –19). Our observations demonstrated that hHYD regulates TopBP1 for the DNA damage response
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