Restoration of nucleotide excision repair in a helicase-deficient XPD mutant from intragenic suppression by a trichothiodystrophy mutation.

The nucleotide excision repair (NER) pathway in eukaryotic cells is required for the error-free removal from DNA of lesions such as pyrimidine (6-4) pyrimidone photoproducts (PPs), cyclobutane pyrimidine dimers (CPDs), and bulky chemical adducts (1, 9, 37, 48). The molecular mechanism of NER is a complex process requiring about 25 proteins in humans. NER can be subdivided into global-genomic repair and transcription-coupled repair (TCR). These subpathways differ with respect to damage recognition and the rate at which some forms of DNA damage are repaired. For many lesions, including CPDs, the global-genomic pathway operates in all parts of the genome but acts relatively slowly. In this pathway, the XPA, RPA, and XPC-hHR23B proteins participate in damage recognition (41, 61, 62). Removal of CPDs and chemical adducts during TCR occurs more rapidly in the transcribed strand of active genes (30). Repair is initiated when an RNA polymerase II elongation complex is blocked by a lesion and requires the CSA and CSB proteins but not XPC-hHR23B (9). In both repair pathways, repair or transcription factor TFIIH is required to unwind the DNA duplex surrounding the lesion to facilitate incision by the XPG and XPF-ERCC1 single-strand or double-strand junction-specific endonucleases (10, 11, 34, 35, 38). This unwinding reaction absolutely requires the 3′ to 5′ and 5′ to 3′ helicase activities of the TFIIH subunits XPB/ERCC3 (28) and XPD/ERCC2 (42, 66), respectively. Along with its role in NER, TFIIH (reviewed in reference 15) is necessary for promoter opening during basal transcription initiation by RNA polymerase II. This nine-member protein complex has several enzymatic activities, including a cyclin-dependent kinase activity comprising subunits Cdk7, cyclin H, and Mat1. The XPB and XPD subunits of TFIIH possess single-stranded DNA-dependent helicase-associated ATPase activities. Interestingly, the helicase activity of only XPB is required for transcription. Although XPD is required for transcription, its role appears structural rather than catalytic (7, 16, 32, 51). A K48R mutation in the canonical GKS/T ATP binding motif of XPD or the Rad3 homolog abolishes repair but does not compromise transcription (43, 66). Mutations in the XPD/ERCC2 gene result in three distinct diseases (reviewed in references 3, 5, and 48): cancer-prone xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (CS; a developmental disorder), and trichothiodystrophy (TTD), which involves neurological and developmental abnormalities distinct from those of CS. Unlike the situation with most other XP genes, XPD mutations exhibit variable and complex clinical phenotypes due to the fact that XPD functions in both transcription and repair. Mutations in XPD resulting in simple XP appear to affect only the NER pathway and probably result from defective helicase activity. XPD-CS mutations show a more complex phenotype that includes defective TCR of oxidative lesions from ionizing radiation and hydrogen peroxide and increased mutations from an 8-oxo-7,8-dihydroguanine lesion in the transcribed strand in a shuttle vector (26). Mechanistically, TTD defects have been ascribed to an altered enzyme structure that reduces stability and/or interaction with other TFIIH members, thereby impairing transcription initiation (4, 7, 15). However, it is becoming apparent in the case of some XPD mutants that the clinical phenotypes require a more complex explanation than either lack of activity or lack of native XPD conformation. For example, it was reported that the R683W XPD mutant has a reduced interaction with the TFIIH p62 subunit, which normally stimulates XPD helicase activity (7). It is clear that understanding the structure-function relationships of wild-type (WT) and mutant XPD proteins will increase insight into the molecular bases of these diseases. A step in this direction was taken with the isolation of the hamster V-H1 mutant (68) and its partially UV-resistant revertants (69). The T46I substitution mutation in both alleles of V-H1 resides in helicase motif I (21), which contains an ATP binding motif found in all helicases (12). V-H1 cells are about sixfold more UV sensitive to killing than parental cells (69) and are defective in XPD helicase activity (19). In this study, we characterize three phenotypic revertants by demonstrating that these revertants have partially restored the NER activity of XPD. In each revertant, the suppressing mutation(s) was found to be R658H. Remarkably, this substitution is identical to the mutation seen in three UV-sensitive TTD patients. These results suggest that TTD mutations do in fact change the structure of XPD and that suppressor genetics is a useful tool for studying the gross structural features of the XPD helicase.