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First published online 17 July 2007
doi: 10.1242/jcs.008367

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Dynamic in vivo interaction of DDB2 E3 ubiquitin ligase with UV-damaged DNA is independent of damage-recognition protein XPC

Martijn S. Luijsterburg^1,*, Joachim Goedhart^2,*, Jill Moser^3,*, Hanneke Kool³, Bart Geverts⁴, Adriaan B. Houtsmuller⁴, Leon H. F. Mullenders³, Wim Vermeulen⁵ and Roel van Driel^1,

¹ Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Nuclear Organisation Group, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
² Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, Section of Molecular Cytology and Center for Advanced Miscroscopy, University of Amsterdam, Kruislaan 316, 1098 SM Amsterdam, The Netherlands
³ Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, PO Box 9600, 2300 RC Leiden, The Netherlands
⁴ Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands
⁵ Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands

Author for correspondence (e-mail: van.driel{at}science.uva.nl)

Accepted 30 May 2007

Damage DNA binding protein 2 (DDB2) has a high affinity for UV-damaged DNA and has been implicated in the initial steps of global genome nucleotide excision repair (NER) in mammals. DDB2 binds to CUL4A and forms an E3 ubiquitin ligase. In this study, we have analyzed the properties of DDB2 and CUL4A in vivo. The majority of DDB2 and CUL4A diffuse in the nucleus with a diffusion rate consistent with a high molecular mass complex. Essentially all DDB2 binds to UV-induced DNA damage, where each molecule resides for 2 minutes. After the induction of DNA damage, DDB2 is proteolytically degraded with a half-life that is two orders of magnitude larger than its residence time on a DNA lesion. This indicates that binding to damaged DNA is not the primary trigger for DDB2 breakdown. The bulk of DDB2 binds to and dissociates from DNA lesions independently of damage-recognition protein XPC. Moreover, the DDB2-containing E3 ubiquitin ligase is bound to many more damaged sites than XPC, suggesting that there is little physical interaction between the two proteins. We propose a scenario in which DDB2 prepares UV-damaged chromatin for assembly of the NER complex.

Key words: DNA repair, Chromatin, Live cell imaging, Nuclear organization, Nucleotide excision repair

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