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 twohybrid screen showed that DNA topoisomerase II-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 ␥-H2AX nuclear foci in DNA breaks. These results demonstrated that hHYD coordinated TopBP1 in the DNA damage response.The ubiquitin-proteosome proteolytic system is involved in a variety of fundamental cell regulations including gene expression, stress response, DNA repair, and cell cycles (1-6). Ubiquitination includes a cascade of three classes of enzymes, the ubiquitin-activating enzyme E1, 1 the ubiquitin-conjugating enzyme Ubc or E2, and the ubiquitin ligase E3. After activation of a ubiquitin by E1, E2 and E3 cooperate to catalyze the formation of a multiubiquitin chain on a protein substrate. The E3 enzymes or the protein complexes with a ligase activity are believed to target the substrate for selective ubiquitination and a subsequent turnover by a large protease complex, the 26 S proteosome. There are two distinct groups of human ubiquitin ligases, the HECT domain E3 enzymes, including E6-associated protein (E6AP) and NEDD4, and the RING finger E3 enzymes, including SCF (Skp1-cullin-F box), VBC (VHL-Elongin B-Elongin C), APC (anaphase-promoting complex) and other single RING finger proteins such as c-Cbl and MDM2 (7). The HECT sequence of ϳ200 amino acids is highly conserved in the C-terminal catalytic domain of family members from yeast to mammals. The HECT-domain protein forms a thiol ester bond with a ubiquitin at the active cysteine residue and transfers the ubiquitin directly to the substrate (8 -10), whereas RING finger proteins form complexes containing an E2 enzyme, which facilitate ubiquitination of the substrate. However, the overall mechanisms by which these ubiquitin ligases recognize the target proteins remain to be elucidated.In the present study, 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 Drosophil...