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Published online 28 June 2004. doi:10.1083/jcb.200401056
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 166, Number 1, 27-36
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Article

 DNA damage stabilizes interaction of CSB with the transcription elongation machinery

Vincent van den Boom1, Elisabetta Citterio1, Deborah Hoogstraten1, Angelika Zotter1, Jean-Marc Egly4, Wiggert A. van Cappellen2, Jan H.J. Hoeijmakers1, Adriaan B. Houtsmuller3, and Wim Vermeulen1

1 Department of Cell Biology and Genetics, Medical Genetic Cluster
2 Department of Endocrinology and Reproduction, Medical Genetic Cluster
3 Department of Pathology, Josephine Nefkens Institute, Erasmus MC Rotterdam, 3000 DR Rotterdam, Netherlands
4 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/Université Louis Pasteur, 67404 Illkirch Cedex, C.U. de Strasbourg, France

Address correspondence to Wim Vermeulen, Medical Genetic Cluster, Erasmus MC, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel.: 31-10-408-7194. Fax: 31-10-408-9468. email: w.vermeulen{at}erasmusmc.nl; or Adriaan B. Houtsmuller, Dept. of Pathology, Josephine Nefkens Institute, Erasmus MC Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, Netherlands. Tel.: 31-10-408-8456. email: a.houtsmuller{at}erasmusmc.nl


Abstract
The Cockayne syndrome B (CSB) protein is essential for transcription-coupled DNA repair (TCR), which is dependent on RNA polymerase II elongation. TCR is required to quickly remove the cytotoxic transcription-blocking DNA lesions. Functional GFP-tagged CSB, expressed at physiological levels, was homogeneously dispersed throughout the nucleoplasm in addition to bright nuclear foci and nucleolar accumulation. Photobleaching studies showed that GFP-CSB, as part of a high molecular weight complex, transiently interacts with the transcription machinery. Upon (DNA damage-induced) transcription arrest CSB binding these interactions are prolonged, most likely reflecting actual engagement of CSB in TCR. These findings are consistent with a model in which CSB monitors progression of transcription by regularly probing elongation complexes and becomes more tightly associated to these complexes when TCR is active.

Key Words: Cockayne syndrome; GFP; photobleaching studies; TCR

V. van den Boom and E. Citterio contributed equally to this work.

E. Citterio's current address is IFOM-FIRC Institute of Molecular Oncology, via Adamello 16, 20139 Milano, Italy.

Abbreviations used in this paper: 6-4PP, 6-4 photoproduct; CPD, cyclo-butane pyrimidinedimer; CS, Cockayne syndrome; FLIP, fluorescence loss in photobleaching; GGR, global genome repair; RTS, reconstituted transcription system; TCR, transcription-coupled repair; WCE, whole cell extracts.


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