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.
Honda
Yoshiomi
Y
Department of Tumor Genetics and Biology and Department of Internal Medicine I, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
Tojo
Masahide
M
Matsuzaki
Kazuhito
K
Anan
Tadashi
T
Matsumoto
Mitsuhiro
M
Ando
Masayuki
M
Saya
Hideyuki
H
Nakao
Mitsuyoshi
M
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002522:Chlorocebus aethiops; D004249:DNA Damage; D004268:DNA-Binding Proteins; D004229:Dithiothreitol / Q000494:pharmacology; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000201:enzymology; D009687:Nuclear Proteins; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D015398:Signal Transduction; D013379:Substrate Specificity; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases; D014965:X-Rays
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D025801:Ubiquitin; C117617:UBR5 protein, human; D044767:Ubiquitin-Protein Ligases; D010453:Peptide Synthases; D004229:Dithiothreitol