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.Xeroderma pigmentosum (XP), trichothiodystrophy (TTD), and Cockayne syndrome (CS) are genetic disorders, all associated with defects in nucleotide excision repair (NER) of DNA damage. Defects in XP have been assigned to eight complementation groups (XP-A through -G and variant) corresponding to proteins involved in different stages of the NER process. The XP-D complementation group is of particular interest and complexity, because mutations in the XPD gene can result in any of at least five different clinical phenotypes (23). XP and TTD are relatively frequent outcomes of XPD mutations, whereas the combined features of XP and CS, XP and TTD, or cranio-oculofacial-skeletal syndrome are rare outcomes (7,16,23). XPD protein is a subunit of transcription factor TFIIH, which is a multifunctional protein required for NER, basal transcription by RNA polymerase I (17) and II (10), and transcriptional activation (21, 27). It is likely that the complexity of the clinical outcomes arises because different mutations affect these various functions differentially. Thus, XP is thought to result if the mutation affects NER but has little effect on transcription. Conversely, TTD is thought to be the result of transcriptional deficiencies. In support of this hypothesis, each mutation site is specific for a particular disorder (37). Thus, for example, many XP patients with the XP clinical phenotype have a mutation at Arg683, whereas no TTD patients have this mutation. Conversely R112H and R722W are mutations found in several TTD patients but not in any XP patients (23). Furthermore, a mouse generated with the R722W mutation had many of the features of TTD (11).At the start of this study, only two...
