title: Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome.
abstract: 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 gamma-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.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom.
authors.forename: Therina
authors.initials: T
authors.lastname: Theron
authors.forename: Maria I
authors.initials: MI
authors.lastname: Fousteri
authors.forename: Marcel
authors.initials: M
authors.lastname: Volker
authors.forename: Lorna W
authors.initials: LW
authors.lastname: Harries
authors.forename: Elena
authors.initials: E
authors.lastname: Botta
authors.forename: Miria
authors.initials: M
authors.lastname: Stefanini
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Fujimoto
authors.forename: Jaan-Olle
authors.initials: JO
authors.lastname: Andressoo
authors.forename: Jay
authors.initials: J
authors.lastname: Mitchell
authors.forename: Nicolaas G J
authors.initials: NG
authors.lastname: Jaspers
authors.forename: Lisa D
authors.initials: LD
authors.lastname: McDaniel
authors.forename: Leon H
authors.initials: LH
authors.lastname: Mullenders
authors.forename: Alan R
authors.initials: AR
authors.lastname: Lehmann
mesh_terms: D003057:Cockayne Syndrome / Q000150:complications / Q000235:genetics*; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism / Q000528:radiation effects; D006657:Histones / Q000032:analysis; D006801:Humans; D010766:Phosphorylation; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis; D012319:RNA Polymerase II / Q000378:metabolism; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000150:complications / Q000235:genetics*
chemical_list: C468783:H2AX protein, human; D006657:Histones; D011064:Poly Adenosine Diphosphate Ribose; D004247:DNA; D012319:RNA Polymerase II