Article

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

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.
Journal of Biological Chemistry (impact factor: 4.77). 03/2002; 277(5):3599-605. DOI:10.1074/jbc.M104347200 pp.3599-605
Source: PubMed

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.

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Keywords

BRCA1 C-terminus domains
 
catalytic sequence
 
cell cycle regulators
 
cell proliferation hyperplastic discs
 
cellular response
 
DNA damage checkpoint proteins
 
DNA topoisomerase IIbeta-binding protein 1
 
E6-associated protein
 
Endogenous hHYD
 
gamma-H2AX nuclear foci
 
HECT-domain ubiquitin ligase
 
protein ubiquitination
 
specific E2
 
stable colocalization
 
subsequent proteosomal degradation
 
substrate specificity
 
ubiquitin system
 
ubiquitination
 
up-regulated TopBP1
 
yeast two-hybrid screen