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Molecular and Cellular Biology, December 2008, p. 7402-7413, Vol. 28, No. 24
0270-7306/08/$08.00+0     doi:10.1128/MCB.01108-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Cellular Concentrations of DDB2 Regulate Dynamic Binding of DDB1 at UV-Induced DNA Damage{triangledown}

Sergey Alekseev,1 Martijn S. Luijsterburg,2,{dagger} Alex Pines,3 Bart Geverts,4 Pierre-Olivier Mari,1 Giuseppina Giglia-Mari,1 Hannes Lans,1 Adriaan B. Houtsmuller,4 Leon H. F. Mullenders,3 Jan H. J. Hoeijmakers,1 and Wim Vermeulen1*

Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands,1 Swammerdam Institute for Life Sciences, Nuclear Organization Group, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands,2 Department of Toxicogenetics, Leiden University Medical Center, Einthovenweg 20, P.O. Box 9600, 2300 RC Leiden, The Netherlands,3 Department of Pathology, Josephine Nefkens Institute, Erasmus MC, University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands4

Received 15 July 2008/ Returned for modification 26 August 2008/ Accepted 8 October 2008

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.

* Corresponding author. Mailing address: Department of Cell Biology and Genetics, Medical Genetics Center, Erasmus Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands. Phone: 3110 4087194. Fax: 3110 7044743. E-mail: W.Vermeulen{at}erasmusmc.nl

{triangledown} Published ahead of print on 20 October 2008.

{dagger} Present address: Department of Cell and Molecular Biology, Karolinska Institutet, von Eulers väg 3, S-17177 Stockholm, Sweden.

Molecular and Cellular Biology, December 2008, p. 7402-7413, Vol. 28, No. 24
0270-7306/08/$08.00+0     doi:10.1128/MCB.01108-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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