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This raised the possibility that these helicases use the FeS cluster to detect oxidative stress, or even to detect DNA damage directly, as has been proposed for other FeS-containing DNA repair proteins Boal et al. The observation that the FeS domain is found in a variety of helicases with different functions suggests strongly that the role of the cluster is not to detect specific forms of DNA damage, but rather to function as a generic component of the helicase. This supports the theory that the FeS cluster in these helicases is a structural feature that stabilizes a small domain that physically separates the DNA duplex strands.

We have confirmed this prediction by over-expressing the S. Consistent with the hypothesis that the FeS cluster has a purely structural role, these organisms appear to have evolved an alternative domain structure that obviates the requirement for the cluster. The complex spectrum of phenotypes arising from mutation of the xpd gene in humans has been the source of much discussion and debate.

XP-causing mutations are the simplest to understand, manifesting as NER-defective but transcription-normal in eukaryotic systems.


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It has therefore not been possible to disentangle the mutations that target the helicase activity directly from those which destabilize protein: This discrepancy has been explained as a consequence of the subtle interplay between NER and transcriptional defects in TTD patients, with the suggestion that the rapid cell death and early senescence caused by TTD is protective against cancer Andressoo et al. Since the archaeal XPD is fully functional in the absence of protein partners, it is a useful model system in which to deconvolute the role of mutations.

A priori , one can make three predictions: Secondly, mutations that destabilise the XPD protein structure directly, rather than disrupt complex formation, are unlikely to be conserved but should give a detectable phenotype. Finally, mutations that inactivate the helicase activity whilst preserving the stability of TFIIH should target conserved residues in the archaeal enzyme and reduce the archaeal helicase activity. As described above, bioinformatic comparisons and biochemical assays of mutant proteins confirm all three of these predictions. Our data are in broad agreement with the work of Egly and co-workers Dubaele et al.

These studies demonstrated that the SR and RW mutations do not compromise binding to p44 in vitro , consistent with our data. Given the clustering of these 3 residues, their distance from other residues known to interact with p44 and the XP phenotype of the RW mutation, which has no transcriptional defect associated with it Dubaele et al. Potentially, the introduction of a bulky tryptophan residue at this position has a subtle effect on human XPD stability.

In this respect they resemble XP rather than CS mutations. Mutations targetting human XPD thus give rise to a broad spectrum of pathologies, but also to a range of severity of clinical symptoms. For example, it is notable that the clinical severity of the RW mutation is classed as severe, whilst that of the RQ mutation is moderate Botta et al. This correlates with our observation that the former mutation has a more deleterious effect on ssDNA binding affinity than the latter Figure 4D.

Furthermore, patients with identical mutations in XPD can have quite different clinical symptoms, emphasising the complexity of the disorders that can arise in humans Fujimoto et al.

Structure of the DNA repair helicase XPD

The structure suggests strongly that helicase polarity is determined by the direction of translocation rather than the nucleic acid binding orientation. Finally, the structure allows the many naturally-occurring human mutations of XPD, and the three overlapping diseases that can result, to begin to be understood at a molecular level. For crystallization trials, the xpd gene from Sulfolobus tokodaii str. The cloned gene contained a TEV protease cleavable N-terminal 6x histidine tag. After removal of the tag, the purified XPD had one extra glycine at the N-terminus.

The XPD protein was expressed in E. This utilized two steps of nickel affinity chromatography intervened by a TEV cleavage step to remove the tag and followed by gel filtration chromatography in buffer containing 10 mM Tris pH 7. The selenomethionine variant of XPD was expressed using the methionine biosynthesis inhibition method Guerrero et al. The protein was expressed and purified as described previously Rudolf et al.

Site-directed mutant forms of the protein were constructed using the Quikchange protocol Stratagene , sequenced fully to ensure sequence integrity and purified as for the wild-type enzyme.

The oligonucleotide sequences used to construct the mutants are available from the corresponding author on request. ATPase and helicase assays were carried out as described previously Rudolf et al. Diffraction data were collected at Diamond beamline I03 in two passes. A high resolution pass to a resolution of 2. A low resolution pass to a resolution of 3. A data set to 2. The SeMet crystal was prepared in the same manner as the native crystal for data collection. The phases were transferred to the native data and extended to 2.

Simulated annealings were performed using CNS Brunger et al. Validation was performed using the Molprobity server Davis et al.

XPD phenotypes

The excitation pathlength was 10 nm, the emission pathlength 2 nm and excitation slitwidth 5 nm. Anisotropy and total fluorescence intensity were measured in parallel following each protein addition and the effects of dilution were corrected. Each protein titration was repeated in triplicate. Data were fitted to the equation below using KaleidaGraph Synergy Software:. Crystallographic statistics for data collected from crystals of native and selenomethionine-labelled XPD.

We thank Michal Zawadzki for technical help, and Catherine Botting for mass spectrometry services, which are funded in St Andrews by the Wellcome Trust.

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Thanks to John Tainer for communicating data prior to publication. National Center for Biotechnology Information , U. Author manuscript; available in PMC Apr Malcolm F White, tel. The publisher's final edited version of this article is available at Cell. See other articles in PMC that cite the published article. Open in a separate window. Structure of XPD A. Biochemical characterisation of selected mutations of XPD A. Towards a molecular understanding of xpd mutation phenotypes The complex spectrum of phenotypes arising from mutation of the xpd gene in humans has been the source of much discussion and debate.

Experimental procedures Expression and purification of XPD from Sulfolobus tokodaii For crystallization trials, the xpd gene from Sulfolobus tokodaii str. Data were fitted to the equation below using KaleidaGraph Synergy Software: Supplementary Material 1 Click here to view. The cancer-free phenotype in trichothiodystrophy is unrelated to its repair defect. Analysis of mutations in the XPD gene in Italian patients with trichothiodystrophy: Am J Hum Genet.

XPD - Wikipedia

Two individuals with features of both xeroderma pigmentosum and trichothiodystrophy highlight the complexity of the clinical outcomes of mutations in the XPD gene. Click on genes, proteins and metabolites below to link to respective articles. From Wikipedia, the free encyclopedia. Chromosome 19 human [1]. ERCC2 excision repair cross-complementing rodent repair deficiency, complementation group 2 xeroderma pigmentosum D ". Annual Review of Genetics. American Journal of Human Genetics. Journal of Biomedical Science.

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