Structural basis of TFIIH activation for nucleotide excision repair.

Nucleotide excision repair (NER) is the major DNA repair pathway that removes UV-induced and bulky DNA lesions. There is currently no structure of NER intermediates, which form around the large multisubunit transcription factor IIH (TFIIH). Here we report the cryo-EM structure of an NER intermediate containing TFIIH and the NER factor XPA. Compared to its transcription conformation, the TFIIH structure is rearranged such that its ATPase subunits XPB and XPD bind double- and single-stranded DNA, consistent with their translocase and helicase activities, respectively. XPA releases the inhibitory kinase module of TFIIH, displaces a 'plug' element from the DNA-binding pore in XPD, and together with the NER factor XPG stimulates XPD activity. Our results explain how TFIIH is switched from a transcription to a repair factor, and provide the basis for a mechanistic analysis of the NER pathway

Mechanism of DNA loading by the DNA repair helicase XPD

Funding: Welcome Trust Programme Grant [WT091825MA to M.F.W., J.H.N.]; Wellcome Trust [099149/Z/12/Z]; Royal Society Wolfson Merit Award (to M.F.W., J.H.N.). Funding for open access charge: Wellcome Trust [WT091825MA].The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5′ to 3′ helicase with an essential iron–sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.Publisher PDFPeer reviewe

DNA repair: Disorders

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Hydrogen peroxide induced genomic instability in nucleotide excision repair-deficient lymphoblastoid cells

Copyright @ 2010 Gopalakrishnan et al; licensee BioMed Central Ltd.Background The Nucleotide Excision Repair (NER) pathway specialises in UV-induced DNA damage repair. Inherited defects in the NER can predispose individuals to Xeroderma Pigmentosum (XP). UV-induced DNA damage cannot account for the manifestation of XP in organ systems not directly exposed to sunlight. While the NER has recently been implicated in the repair of oxidative DNA lesions, it is not well characterised. Therefore we sought to investigate the role of NER factors Xeroderma Pigmentosum A (XPA), XPB and XPD in oxidative DNA damage-repair by subjecting lymphoblastoid cells from patients suffering from XP-A, XP-D and XP-B with Cockayne Syndrome to hydrogen peroxide (H2O2). Results Loss of functional XPB or XPD but not XPA led to enhanced sensitivity towards H2O2-induced cell death. XP-deficient lymphoblastoid cells exhibited increased susceptibility to H2O2-induced DNA damage with XPD showing the highest susceptibility and lowest repair capacity. Furthermore, XPB- and XPD-deficient lymphoblastoid cells displayed enhanced DNA damage at the telomeres. XPA- and XPB-deficient lymphoblastoid cells also showed differential regulation of XPD following H2O2 treatment. Conclusions Taken together, our data implicate a role for the NER in H2O2-induced oxidative stress management and further corroborates that oxidative stress is a significant contributing factor in XP symptoms. Resistance of XPA-deficient lymphoblastoid cells to H2O2-induced cell death while harbouring DNA damage poses a potential cancer risk factor for XPA patients. Our data implicate XPB and XPD in the protection against oxidative stress-induced DNA damage and telomere shortening, and thus premature senescence.This research is supported by the Defence Innovative Research Programme, Defence Science and Technology Agency, Singapore (POD: 0613592) and the Academic Research Fund, Ministry of Education, Singapore (T206B3108). Supported in part by a grant from British Council, PMI2 Connect (Grant Number: RC134)

ATP-Stimulated, DNA-Mediated Redox Signaling by XPD, a DNA Repair and Transcription Helicase

Using DNA-modified electrodes, we show DNA-mediated signaling by XPD, a helicase that contains a [4Fe-4S] cluster and is critical for nucleotide excision repair and transcription. The DNA-mediated redox signal resembles that of base excision repair proteins, with a DNA-bound redox potential of ~80 mV versus NHE. Significantly, this signal increases with ATP hydrolysis. Moreover, the redox signal is substrate-dependent, reports on the DNA conformational changes associated with enzymatic function, and may reflect a general biological role for DNA charge transport

Transcriptional changes in trichothiodystrophy 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 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 microarray 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

Adverse Effects of Trichothiodystrophy DNA Repair and Transcription Gene Abnormalities on Human Fetal Development

The effects of DNA repair and transcription genes in human prenatal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10^-6^) recessive disorder caused by mutations in genes involved in the nucleotide excision repair (NER) pathway and in transcription. Based on our clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offspring (N=24) with respect to abnormalities in their antenatal and neonatal periods to pregnancies resulting in their unaffected siblings (N=18), accounting for correlation, and to population reference values. Significantly higher incidence of several severe gestational complications was noted in TTD-affected pregnancies. Gestational complications were noted in nearly all pregnancies resulting in TTD-affected offspring with _XPD_ and _TTDN1_, but not _TTD-A_, gene mutations. Abnormal placental development may explain the constellation of observed complications; therefore, we hypothesize that some TTD genes play an important role in normal placental and fetal development. We investigated this hypothesis by analyzing the expression patterns of TTD genes. Expression of _TTDA_ was strongly negatively correlated (r=-0.7,P<0.0001) with gestational age, while _XPD, XPB_ and _TTDN1_ were consistently expressed from 14 to 40 weeks gestation. *Conclusion:* Our results indicate an important role for _XPD, XPB_ and _TTDN1_ gene products during normal human placental and fetal development

Transcription as a Threat to Genome Integrity

Genomes undergo different types of sporadic alterations, including DNA damage, point mutations, and genome rearrangements, that constitute the basis for evolution. However, these changes may occur at high levels as a result of cell pathology and trigger genome instability, a hallmark of cancer and a number of genetic diseases. In the last two decades, evidence has accumulated that transcription constitutes an important natural source of DNA metabolic errors that can compromise the integrity of the genome. Transcription can create the conditions for high levels of mutations and recombination by its ability to open the DNA structure and remodel chromatin, making it more accessible to DNA insulting agents, and by its ability to become a barrier to DNA replication. Here we review the molecular basis of such events from a mechanistic perspective with particular emphasis on the role of transcription as a genome instability determinant

An Extended Network of Genomic Maintenance in the Archaeon Pyrococcus abyssi Highlights Unexpected Associations between Eucaryotic Homologs.

In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes