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

Abstract

The UV-sensitive V-H1 cell line has a T46I substitution mutation in the Walker A box in both alleles of XPD and lacks DNA helicase activity. We characterized three partial revertants that curiously display intermediate UV cytotoxicity (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 photoproducts from both strands of hprt suggests that global-genomic nucleotide excision repair is normal, but the pattern of cyclobutane pyrimidine dimer removal suggests that transcription-coupled repair (TCR) is impaired. To explain the intermediate UV survival and lack of RNA synthesis recovery in 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 transcription 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 mutation produces the clinical phenotype of trichothiodystrophy (TTD) in several patients who display intermediate UV sensitivity. The XPD(R658H) TTD protein, like XPD(T46I/R658H), is codominant when overexpressed in V-H1 cells and partially complements their UV sensitivity. Thus, the suppressing R658H substitution must restore helicase activity to the inactive XPD(T46I) protein. Based on current knowledge of helicase structure, the intragenic reversion mutation may partially compensate for the T46I mutation by perturbing the XPD structure in a way that counteracts the effect of this mutation. These findings have implications for understanding the differences between xeroderma pigmentosum and TTD and illustrate the value of suppressor genetics for studying helicase structure-function relationships.

FIG. 1

UV survival based on colony-forming ability of revertants and parental cell lines. Cell lines tested were WT V79 (○), V-H1 (□), and the phenotypic revertants RH1-3 (⋄), RH1-26 (▵), and RH1-53 (▿). Lines intersecting the abscissa indicate data points below the axis. Each survival curve represents the average of two experiments. Error bars represent SEMs.

FIG. 2

UV-induced hprt mutations based on thioguanine-resistant cells for WT, mutant, and revertant cell lines. (A) Mutations were induced by irradiation with 254-nm UV light in parental V79 (○), V-H1 (□), RH1-3 (◊), and RH1-26 (▵) cell lines. Data for V-H1 were taken from reference . The spontaneous mutation frequencies that were subtracted were as follows: WT, 0.4 × 10⁻⁵; V-H1, 1.8 × 10⁻⁵; RH1-3, 1.6 × 10⁻⁵; and RH1-26, 1.0 × 10⁻⁵. (B) Survival curves measured immediately after UV exposure for the cultures shown in panel A.

FIG. 3

Rates of RNA synthesis after UV irradiation. Values are expressed relative to those for untreated cells following irradiation with 10 J/m². V79 (○) and RH1-26 (□) cells were tested. Error bars represent SEMs.

FIG. 4

Removal of 6-4 PPs from the hprt gene after UV irradiation with 30 J/m². (A) WT cells, transcribed strand (●) and nontranscribed strand (○). (B) RH1-26, transcribed strand (▪) and nontranscribed strand (□). Error bars represent SEMs.

FIG. 5

Partial restoration of in vitro NER activity in revertant RH1-26. Cell extracts were incubated with a 140-bp duplex DNA containing a cholesterol moiety in the middle of the substrate. The substrate was internally labeled with ³²P 5 bp 5′-ward of the adduct on the same strand of DNA. Reactions were performed at 30°C for 60 min. After protein removal, the substrate was separated from the product by denaturing PAGE and visualized by film autoradiography.

FIG. 6

Evidence that a suppressing mutation resides in an xpd allele. (A) Fifty micrograms of soluble protein from each extract was resolved by SDS-PAGE, and XPD protein was identified by Western blotting using an affinity-purified anti-XPD polyclonal antibody. In some lanes, the XPD protein appears as a doublet, for reasons not understood. (B) UV survival curves for cells of the WT (○), V-H1 (□), revertant RH1-53 (◊), V-H1 transfected with XPD^WT (●), V-H1 transfected with XPD^RH1-53 (⧫), and RH1-53 transfected with XPD^T46I (▪). Each survival curve represents the average of two experiments. Error bars represent SEMs.

FIG. 7

Analysis of the XPD intragenic suppressor mutation(s) in revertants. (A) The positions of the conserved helicase motifs in XPD, as described by Koonin for the Rad3 helicase family (23), are shown. The original mutation in the V-H1 cell line is a homozygous substitution in the Walker A box of motif I (GT₄₆GKT) of the protein that results in a T46I change. The suppressing mutation in all three revertant cell lines changed arginine at position 658 to histidine. Arginine 658 is positioned on the immediate N-terminal side of motif VI. (B) HhaI digestion of the genomic PCR products from WT and V-H1 DNAs produces DNA fragments of 416, 241, 183, 74, and 40 bp. In the revertant DNA, the suppressing mutation eliminates an HhaI site. (C) Genomic DNAs from V-H1 and the revertants were isolated, and a portion of the XPD gene corresponding to nucleotide positions 13113 to 14066 was PCR amplified and purified. PCR products were digested with HhaI and resolved by PAGE. The 40-bp bands are faint. The image was black-white inverted for ease of viewing.

FIG. 8

UV survival of V-H1 clones overexpressing XPD^R658H cDNA. (A) Fifty micrograms of soluble protein extract from each cell line resolved by SDS-PAGE and probed for XPD using anti-XPD antibody from a rabbit as described in Materials and Methods. (B) UV survival curves for V-H1 (dotted line), RH1-53 (dashed line, no symbols), WT (solid line, no symbols), and VH-1/R658H transformant clone 30 (▾), clone 22 (▴), clone 23 (▵) and clone 28 (▿). The curves for V-H1, RH1-53, and WT are taken from Fig. 6.