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...

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are both rare autosomal recessive disorders with defects in DNA repair. They are usually distinct both clinically and genetically but in rare cases, patients exhibit the clinical characteristics of both diseases concurrently. We report two new phenotypically distinct cases of XP with additional features of CS (xeroderma pigmentosum and Cockayne syndrome crossover syndrome (XP/CS)) carrying an identical mutation (G47R) in the XPD gene within the N terminus of the protein. Both patients had clinical features of XP and CS but only one fulfilled most criteria for diagnosing CS. Unusually, patient 1 developed early skin cancer, in contrast to patient 2, who never developed any malignancies. Cells from both these patients have repair defects typical of xeroderma pigmentosum complementation group D (XPD) cells, but also had the phenotype of uncontrolled DNA breakage found specifically in XPD/CS cells and similarly reduced levels of TFIIH. Despite these similarities between our two patients, their clinical features are quite different and the clinical severity correlates with other cellular responses to ultraviolet irradiation.

The G2 block is a major response of cells to DNA damage and seem to be induced independently of p53 status. It is thought that the G2 block has a protective function and allows cells to repair their DNA. The molecular events involved in the formation of the G2 block therefore are of great interest. We have used pentoxifylline, a potent G2 delay abrogator, to study the expression of an essential component of the mitosis promoting complex (MPF), cyclin B1. Cyclin B1/G2 ratios are used to show that irradiation induces a decrease in cyclin B1 expression and that pentoxifylline restores cyclin B1 expression to control level. This confirms that suppression of cyclin B1 plays a role in the formation of the G2 cell cycle delay, and that elevating cyclin B1 expression is part of the mechanism of action of pentoxifylline on G2 blocked cells.

Mutations in three of the genes encoding the XPB, XPD and TTDA components of transcription factor TFIIH can result in the clinical phenotype of trichothiodystrophy (TTD). Different mutations in XPB and XPD can instead cause xeroderma pigmentosum (XP). The completely different features of these disorders have been attributed to TTD being a transcription syndrome. In order to detect transcriptional differences between TTD and XP cells from the XP-D complementation group, we have compared gene expression profiles in cultured fibroblasts from normal, XP and TTD donors. Although we detected transcriptional differences between individual cell strains, using an algorithm of moderate stringency, we did not identify any genes whose epression was reproducibly different in proliferating fibroblasts from each type of donor.Following UV-irradiation, many genes were up-and down-regulated in all three cell types. The micro-array analysis indicated some apparent differences between the different donor types, but on more detailed inspection, these turned out to be false positives. We conclude that there are minimal differences in gene expression in proliferating fibroblasts from TTD, XP-D and normal donors.Dear Errol I enclose a manuscript entitled "Transcriptional changes in trichothiodystrophy cells" that we would like to be considered for publication in DNAR. This paper reports on a very careful study that we have carried out, using micro-arrays, to look for specific transcriptional differences between TTD fibroblasts and XP-D or normal fibroblasts, both in unirradiated and irradiated cells. We were not able to detect any differences that could be attributed to a TTD-specific genotype. En route our study highlights a number of potential pitfalls that need to be avoided when using such an approach. In particular using a single defective cell line and its corrected derivative might generate data that can be easily misinterpreted.We hope that the results in this paper will be of interest to readers of DNAR.Best regards. strains, using an algorithm of moderate stringency, we did not identify any genes whose expression was reproducibly different in proliferating fibroblasts from each type of donor. Following UV-irradiation, many genes were up-and down-regulated in all three cell types. The micro-array analysis indicated some apparent differences between the different donor types, but on more detailed inspection, these turned out to be false positives. We conclude that there are minimal differences in gene expression in proliferating fibroblasts from TTD, XP-D and normal donors.3