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Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation.

ABSTRACT: The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA repair (NER) pathway. XPD is a superfamily-2 5'-3' helicase containing an iron-sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a short DNA fragment, thus revealing the polarity of the translocated strand and providing insights into how the enzyme achieves its 5'-3' directionality. Accompanied by a detailed mutational and biochemical analysis of taXPD, we define the path of the translocated DNA strand through the protein and identify amino acids that are critical for protein function.

SUBMITTER: Kuper J

PROVIDER: S-EPMC3261550 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Functional and structural studies of the nucleotide excision repair helicase XPD suggest a polarity for DNA translocation.

Kuper Jochen J Wolski Stefanie C SC Michels Gudrun G Kisker Caroline C

The EMBO journal 20111111 2

The XPD protein is a vital subunit of the general transcription factor TFIIH which is not only involved in transcription but is also an essential component of the eukaryotic nucleotide excision DNA repair (NER) pathway. XPD is a superfamily-2 5'-3' helicase containing an iron-sulphur cluster. Its helicase activity is indispensable for NER and it plays a role in the damage verification process. Here, we report the first structure of XPD from Thermoplasma acidophilum (taXPD) in complex with a shor ...[more]

PMID: 22081108

Similar Datasets

Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD.

Project description:DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an alpha-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster-containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.

| S-EPMC2435149 | biostudies-literature

Mechanism of lesion verification by the human XPD helicase in nucleotide excision repair.

Project description:In nucleotide excision repair (NER), the xeroderma pigmentosum D helicase (XPD) scans DNA searching for bulky lesions, stalls when encountering such damage to verify its presence, and allows repair to proceed. Structural studies have shown XPD bound to its single-stranded DNA substrate, but molecular and dynamic characterization of how XPD translocates on undamaged DNA and how it stalls to verify lesions remains poorly understood. Here, we have performed extensive all-atom MD simulations of human XPD bound to undamaged and damaged ssDNA, containing a mutagenic pyrimidine (6-4) pyrimidone UV photoproduct (6-4PP), near the XPD pore entrance. We characterize how XPD responds to the presence of the DNA lesion, delineating the atomistic-scale mechanism that it utilizes to discriminate between damaged and undamaged nucleotides. We identify key amino acid residues, including FeS residues R112, R196, H135, K128, Arch residues E377 and R380, and ATPase lobe 1 residues 215-221, that are involved in damage verification and show how movements of Arch and ATPase lobe 1 domains relative to the FeS domain modulate these interactions. These structural and dynamic molecular depictions of XPD helicase activity with unmodified DNA and its inhibition by the lesion elucidate how the lesion is verified by inducing XPD stalling.

| S-EPMC9262607 | biostudies-literature

DNA translocation mechanism of an XPD family helicase.

Project description:The XPD family of helicases, that includes human disease-related FANCJ, DDX11 and RTEL1, are Superfamily two helicases that contain an iron-sulphur cluster domain, translocate on ssDNA in a 5'-3' direction and play important roles in genome stability. Consequently, mutations in several of these family members in eukaryotes cause human diseases. Family members in bacteria, such as the DinG helicase from Escherichia coli, are also involved in DNA repair. Here we present crystal structures of complexes of DinG bound to single-stranded DNA (ssDNA) in the presence and absence of an ATP analogue (ADP•BeF3), that suggest a mechanism for 5'-3' translocation along the ssDNA substrate. This proposed mechanism has implications for how those enzymes of the XPD family that recognise bulky DNA lesions might stall at these as the first step in initiating DNA repair. Biochemical data reveal roles for conserved residues that are mutated in human diseases.

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Structure of the DNA repair helicase XPD.

Project description:The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

| S-EPMC3326533 | biostudies-literature

Structural basis of TFIIH activation for nucleotide excision repair.

Project description: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.

| S-EPMC6599211 | biostudies-literature

Structural basis of TFIIH activation for nucleotide excision repair

Project description: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.

2019-07-02 | PXD013947 | Pride

XPD DNA nucleotide excision repair gene polymorphisms associated with DNA repair deficiency predict better treatment outcomes in secondary acute myeloid leukemia.

Project description:Pharmacogenetic studies in DNA repair pathway have consistently demonstrated correlations between the XRCC1 Arg399Gln, XPD Lys751Gln and XPD Asp312Gln genotypes, previously associated with suboptimal DNA repair, and differential cancer treatment outcomes. We evaluated these polymorphisms and XPD haplotypes in adult de novo (n=214) and secondary (n=79) acute myeloid leukemia (AML) patients treated with cytarabine and anthracycline chemotherapy. Genotyping was performed by MALDI-TOF mass spectrometry. Logistic and proportional hazards regression models were used to evaluate relationships. Differential responses were observed in secondary, but not de novo, AML. Among secondary AML patients, the odds of achieving complete remission (CR) were higher for the XPD 312Asn/Asn (OR= 11.23; 95% CI, 2.23-56.63) and XPD 751Gln/Gln (OR= 7.07; 95% CI, 1.42-35.18) genotypes. The XPD diplotypes were coded as the combination of two of the following haplotypes: haplotype A=(Lys)751A/(Asp) 312G; B=(Gln)751C/(Asn)312A; C=(Lys)751A/(Asn)312A; and D=(Gln)751C/(Asp)312G. The BB diplotype was associated with CR attainment [OR=18.31; 95% CI: 2.08-283.57] and longer survival [HR=0.31; 95% CI: 0.14-0.73] compared to the referent AA diplotype. The XPD 751 CC, 312GA, 312AA genotypes and the XPD DC diplotype were also associated with longer overall survival (OS).Thus, XPD codon 312 and 751 variant genotypes and haplotypes containing at least one variant allele may predict better treatment responses. If validated, these findings could support stratification of chemotherapy in secondary AML.

| S-EPMC3049908 | biostudies-literature

Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair.

Project description:Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP-bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre-incision complex of UvrB and the damaged DNA is formed by insertion of a flexible beta-hairpin between the two DNA strands.

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Prokaryotic nucleotide excision repair.

Project description:Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation.

| S-EPMC3578354 | biostudies-literature

Mechanism of DNA loading by the DNA repair helicase XPD.

Project description: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.

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