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

The UV-sensitive V-H1 cell line has a T461 substitution mutation in the Wal ker A box in both alleles of XPD and lacks DNA helicase activity. We charac terized three partial revertants that curiously display intermediate IN cyt otoxicity (2- to 2.5-fold) but normal levels,of UV-induced hprt mutations. In revertant RH1-26, the efficient removal of pyrimidine (6-4) pyrimidone p hotoproducts from both strands of hprt suggests that global-genomic nucleot ide excision repair is normal, but the pattern of cyclobutane pyrimidine di mer removal suggests that transcription-coupled repair (TCR) is impaired. T o explain the intermediate UV survival and lack of RNA synthesis recovery i n RH1-26 after 10 J of UV/m(2), We propose a defect in repair-transcription coupling, i.e., the inability of the cells to resume or reinitiate transcr iption after the first TCR event within a transcript. All three revertants carry an R658H suppressor mutation, in one allele of revertants RH1-26 and RH1-53 and in both alleles of revertant RH1-3. Remarkably, the R658H mutati on produces the clinical phenotype of trichothiodystrophy (TTD) in several patients who display intermediate UV sensitivity. The XPDR658H TTD protein, like XPDT461/R658H, is codominant when, overexpressed in V-H1 cells and pa rtially complements their UV sensitivity. Thus, the suppressing R658H subst itution must restore helicase activity to the inactive XPDT461 protein. Bas ed on current knowledge of helicase structure, the intragenic reversion mut ation may partially compensate for the T461 mutation by perturbing the XPD structure in a way that counteracts the effect of this mutation. These find ings have implications for understanding the differences between xeroderma pigmentosum and TTD and illustrate the value of suppressor genetics for stu dying helicase structure-function relationships.