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Molecular and Cellular Biology, September 2005, p. 8368-8378, Vol. 25, No. 18
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.18.8368-8378.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Transcription-Associated Breaks in Xeroderma Pigmentosum Group D Cells from Patients with Combined Features of Xeroderma Pigmentosum and Cockayne Syndrome

Therina Theron,^1, Maria I. Fousteri,^2, Marcel Volker,^1,2, Lorna W. Harries,^1, Elena Botta,³ Miria Stefanini,³ Mitsuo Fujimoto,⁴ Jaan-Olle Andressoo,⁵ Jay Mitchell,⁵ Nicolaas G. J. Jaspers,⁵ Lisa D. McDaniel,⁶ Leon H. Mullenders,² and Alan R. Lehmann^1*

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom,¹ MGC Department of Toxicogenetics, Leiden University Medical Centre, Wassenaarseweg 72, 2333AL-Leiden, The Netherlands,² Istituto di Genetica Molecolare CNR, 27100 Pavia, Italy,³ Department of Dermatology, Jichi Medical School, Minami-kawachi-machi, Japan,⁴ MGC Department of Cell Biology and Genetics, P.O. Box 1738, Erasmus University MC, 3000 DR Rotterdam, The Netherlands,⁵ Department of Pathology, CY1.107, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Texas 75390-8840⁶

Received 7 March 2005/ Returned for modification 13 April 2005/ Accepted 15 June 2005

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either -H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.

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* Corresponding author. Mailing address: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom. Phone: 44-1273 678120. Fax: 44-1273 678121. E-mail: a.r.lehmann{at}sussex.ac.uk.

Present address: Department of Research and Innovation, University of Cape Town, Rondebosch 7701, South Africa.

These authors contributed equally to the work.

Present address: Molecular Genetics, Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, United Kingdom.

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Molecular and Cellular Biology, September 2005, p. 8368-8378, Vol. 25, No. 18
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.18.8368-8378.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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