XPG Function Research Articles

Xpg limits the expansion of haematopoietic stem and progenitor cells after ionising radiation.

Reduced capacity of genome maintenance represents a problem for any organism, potentially causing premature death, carcinogenesis, or accelerated ageing. Strikingly though, loss of certain genome stability factors can be beneficial, especially for the maintenance of tissue stem cells of the intestine and the haematopoietic system. We therefore screened for genome stability factors negatively impacting maintenance of haematopoietic stem cells (HSC) in the context of ionising radiation (IR). We found that in vivo knock down of Xeroderma pigmentosum, complementation group G (Xpg) causes elevation of HSC numbers after IR treatment, while numbers of haematopoietic progenitors are elevated to a lesser extent. IR rapidly induces Xpg both on mRNA and on protein level. Prevention of this induction does not influence activation of the checkpoint cascade, yet attenuates late checkpoint steps such as induction of p21 and Noxa. This causes a leaky cell cycle arrest and lower levels of apoptosis, both contributing to increased colony formation and transformation rates. Xpg thus helps to adequately induce DNA damage responses after IR, thereby keeping the expansion of damaged cells under control. This represents a new function of Xpg in the response to IR, in addition to its well-characterized role in nucleotide excision repair.

182. Characterization and AAV-Mediated Correction of Xeroderma Pigmentosum-Cockayne Syndrome (XP/CS)

Cockayne Syndrome (CS) is caused by mutations in several genes that encode proteins involved in DNA repair. These include traditional CS proteins (CSA/CSB), as well as several Xeroderma Pigmentosum (XP) proteins (including XPG). While traditional CS is characterized by neurodegeneration and other clinical manifestations that result in an overall phenotype of premature aging, the typical XP phenotype is photosensitivity and skin cancer susceptibility. However, XPG presents with one of two different phenotypes, depending on the individual mutations: photosensitivity alone (XP) or photosensitivity + CS (XP/CS). Although the more severe XP/CS phenotype correlates well with mutations that result in XPG truncation, a function for this protein that explains the neurological deficit has yet to be described. XPG is involved in nuclear DNA repair as part of a complex that includes CSA and CSB. As CSA and CSB have also been shown to play a role in mitochondrial DNA (mtDNA) repair, it is likely, though unproven, that XPG is also involved in mtDNA repair. Insufficient mtDNA repair in neurons would ultimately lead to mitochondrial dysfunction and cause progressive neurodegeneration. The objective of this project is two-fold: 1) to characterize the two different phenotypes observed in XPG patients through examination of patient cells representing the XP as well as the XP/CS phenotype and compare them to healthy and CSA cells in order to identify a novel role for XPG that would explain the neurological deficit that occurs when the protein is truncated, and 2) to develop adeno-associated virus (AAV) mediated gene therapy for XPG and CSA and thus, overexpress these genes in an attempt to further characterize their function in healthy and patient-derived human cells. Cellular and mitochondrial function, sensitivity to oxidative and UV-induced stress, and general cell health and morphology are all being evaluated in healthy, XPG, and CSA patient-derived fibroblasts. Preliminary studies demonstrate reduced ATP production through oxidative phosphorylation in fibroblasts from patients with an XP/CS or CS phenotype as compared to those patients displaying XP alone or healthy controls. Scratch tests revealed motility and expansion deficiencies in XP/CS and CS cells as compared to XP or healthy controls, suggesting a potential role for mitochondria in these processes. AAV-XPG and AAV-CSA constructs have been generated and are being tested for their potential to correct cellular dysfunction. We are reprogramming a subset of these cells into induced pluripotent stem cells that will then be differentiated into neurons for further mitochondrial analyses and treatment studies. These studies will determine whether XPG function correlates with mitochondrial function in an in vitro human model of disease and generate preclinical data to justify further investigations into gene therapy strategies for this devastating syndrome.

Repair pathway crosstalk in genome stability maintenance through BRCA1/2‐mediated homologous recombination (352.1)

XPG is an endonuclease required for nucleotide excision repair with important non‐enzymatic roles in base excision repair (BER) and transcription‐coupled repair (TCR). XPG enzymatic defects cause cancer‐prone xeroderma pigmentosum (XP), while truncating mutations cause the progeroid disorder Cockayne syndrome (CS). We now reveal an unexpected XPG role in homologous recombination (HR). XPG loss elevates the DNA damage response and causes double‐strand break (DSB) formation with pronounced genomic instability. XPG knockdown (KD) cells have impaired recovery from replicative stress, and DNA fiber analysis reveals inability to restart stalled replication forks. XPG interacts directly with the HR proteins RAD51, BRCA2, and PALB2 in a stable complex, and XPG KD results in their reduced chromatin loading and in decreased HR at site‐specific DSBs. Placing its role in HR upstream of BRCA2, XPG KD also reduces single‐strand annealing and increases chromatin‐bound BRCA1 and BRCA1 foci. Thus, loss of XPG function impairs replication fork maintenance by HR and may be important in BRCA‐associated cancers. It also accelerates aging, leading to cellular senescence or apoptosis and to severe progeria in XP‐G/CS patients and a mouse model. Whether this accelerated aging is related to XPG roles in TCR, BER, or HR remains to be determined.

Yeast RAD2, a homolog of human XPG, plays a key role in the regulation of the cell cycle and actin dynamics

SummaryMutations in the human XPG gene cause Cockayne syndrome (CS) and xeroderma pigmentosum (XP). Transcription defects have been suggested as the fundamental cause of CS; however, defining CS as a transcription syndrome is inconclusive. In particular, the function of XPG in transcription has not been clearly demonstrated. Here, we provide evidence for the involvement of RAD2, the Saccharomyces cerevisiae counterpart of XPG, in cell cycle regulation and efficient actin assembly following ultraviolet irradiation. RAD2 C-terminal deletion, which resembles the XPG mutation found in XPG/CS cells, caused cell growth arrest, the cell cycle stalling, a defective α-factor response, shortened lifespan, cell polarity defect, and misregulated actin-dynamics after DNA damage. Overexpression of the C-terminal 65 amino acids of Rad2p was sufficient to induce hyper-cell polarization. In addition, RAD2 genetically interacts with TPM1 during cell polarization. These results provide insights into the role of RAD2 in post-UV irradiation cell cycle regulation and actin assembly, which may be an underlying cause of XPG/CS.

Retraction for Nouspikel et al., A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: Implications for a second XPG function

GENETICS. For the article “A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: Implications for a second XPG function,” by Thierry Nouspikel, Philippe Lalle Steven A. Leadon Priscilla K. Cooper and Stuart G. Clarkson, which appeared in issue 7, April 1, 1997, of Proc Natl Acad Sci USA (94:3116–3121), the editors wish to note that Steven Anthony Leadon has submitted a letter to PNAS that states, “I have recently had the opportunity to review some of the raw data used for Figure 6 in this paper in the above-referenced publication and it is clear that the data as reported in this figure cannot be relied upon. Therefore, I request that you retract Figure 6 of this paper.” Fig. 6 is hereby retracted.

Recognition of RNA Polymerase II and Transcription Bubbles by XPG, CSB, and TFIIH: Insights for Transcription-Coupled Repair and Cockayne Syndrome

Loss of a nonenzymatic function of XPG results in defective transcription-coupled repair (TCR), Cockayne syndrome (CS), and early death, but the molecular basis for these phenotypes is unknown. Mutation of CSB, CSA, or the TFIIH helicases XPB and XPD can also cause defective TCR and CS. We show that XPG interacts with elongating RNA polymerase II (RNAPII) in the cell and binds stalled RNAPII ternary complexes in vitro both independently and cooperatively with CSB. XPG binds transcription-sized DNA bubbles through two domains not required for incision and functionally interacts with CSB on these bubbles to stimulate its ATPase activity. Bound RNAPII blocks bubble incision by XPG, but an ATP hydrolysis-dependent process involving TFIIH creates access to the junction, allowing incision. Together, these results implicate coordinated recognition of stalled transcription by XPG and CSB in TCR initiation and suggest that TFIIH-dependent remodeling of stalled RNAPII without release may be sufficient to allow repair.

Severe growth retardation and short life span of double-mutant mice lacking Xpa and exon 15 of Xpg

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause an early onset of Cockayne syndrome (CS) in some patients (XP-G/CS) with characteristics, such as growth retardation and a short life span. In the previous studies, we generated four Xpg mutant mice with two different C-terminal truncations, null, or a base substitution mutation to identify the protein region that causes the onset of CS, and found that the CS-causing mutations, null or a deletion of the last 360 amino acids, completely inhibited the NER activity of mouse XPG (Xpg), but the non-CS-causing mutations, XpgD811A (base substitution that eliminates the nuclease activity of Xpg) or XpgΔex15 (deletion of the exon 15 corresponding to the last 183 amino acids), resulted in the retention of residual NER activity. To understand why mutations that completely eliminate the NER activity of Xpg cause CS but those that abolish the nuclease activity without totally eliminating the NER activity of Xpg do not result in CS, we made a series of Xpg mutant mice with Xpa-null mutant allele and found that mice with the non-CS-causing deletion mutation ( XpgΔex15) exhibited the CS phenotype when XPA was also absent but the base substitution mutation ( XpgD811A) that eliminated the Xpg nuclease activity did not. These results indicate that Xpg has a second function, beside NER, and that the disruption of this second function (deletion of the last 183 amino acids) when combined with an NER defect causes CS. When we compared amino acid sequences corresponding to the exon 15 of Xpg, a significant homology was conserved among vertebrates, but not in Drosophila and Saccharomyces cerevisiae. These observations suggest that the second function of XPG may be conserved only in vertebrates and CS symptoms may occur in its absence.

Identification of the XPG region that causes the onset of Cockayne syndrome by using Xpg mutant mice generated by the cDNA-mediated knock-in method.

In addition to xeroderma pigmentosum (XP), mutations in the human XPG gene cause early onset of Cockayne syndrome (CS) in some patients (XPG/CS). The CS-causing mutations in such patients all produce truncated XPG proteins. To test the hypothesis that the CS phenotype, with characteristics such as growth retardation and a short life span in XPG/CS patients, results from C-terminal truncations, we constructed mutants with C-terminal truncations in mouse XPG (Xpg) (from residue D811 to the stop codon [XpgD811stop] and deletion of exon 15 [Xpg Delta ex15]). In the XpgD811stop and Xpg Delta ex15 mutations, the last 360 and 183 amino acids of the protein were deleted, respectively. To generate Xpg mutant mice, we devised the shortcut knock-in method by replacing genomic DNA with a mutated cDNA fragment (cDNA-mediated knock in). The control mice, in which one-half of Xpg genomic DNA fragment was replaced with a normal Xpg cDNA fragment, had a normal growth rate, a normal life span, normal sensitivity to UV light, and normal DNA repair ability, indicating that the Xpg gene partially replaced with the normal cDNA fragment retained normal functions. The XpgD811stop homozygous mice exhibited growth retardation and a short life span, but the Xpg Delta ex15 homozygous mice did not, indicating that deletion of the last 360 amino acids results in the CS phenotype but deletion of the last 183 amino acids does not. The XpgD811stop homozygous mice, however, exhibited a slightly milder CS phenotype than did the Xpg null mutant mice, indicating that the XpgD811stop protein still retains some Xpg function that affects the severity of the CS phenotype.

Xeroderma Pigmentosum Group G with Severe Neurological Involvement and Features of Cockayne Syndrome in Infancy

We describe a premature, small for gestational age infant girl with micropthalmia, bilateral congenital cataracts, hearing impairment, progressive somatic and neurodevelopmental arrest, and infantile spasms. She presented a massive photosensitive reaction with erythema and blistering after minimal sun exposure, which slowly gave place to small skin cancers. Her skin fibroblasts were 10-fold more sensitive than normal to UV exposure due to a severe deficiency in nucleotide excision repair. By complementation analysis, the patient XPCS4RO was assigned to the very rare xeroderma pigmentosum (XP) group G (XP-G). One allele of her XPG gene contained a 526C-->T transition that changed Gln-176 to a premature UAG stop codon. Only a minor fraction of XPG mRNA was encoded by this allele. The second, more significantly expressed XPG allele contained a 215C-->A transversion. This changed the highly conserved Pro-72 to a histidine, a substitution that would be expected to seriously impair the 3' endonuclease function of XPG in nucleotide excision repair. In cases suspected of having XP and/or early-onset Cockayne syndrome, extensive DNA repair studies should be performed to reach a correct diagnosis, thereby allowing reliable genetic counseling and prenatal diagnosis.

Conserved Residues of Human XPG Protein Important for Nuclease Activity and Function in Nucleotide Excision Repair

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

Binding and Dissociation of Cytochrome c to and from Membranes Containing Acidic Phospholipids

Membrane association and detachment of cytochrome c (cyt c) in millisecond to second time domain were investigated by stopped-flow fluorescence spectroscopy monitoring the efficiency of energy transfer from a pyrene-fatty acid containing phospholipid derivative, 1-palmitoyl-2-[10-(pyren-1-yl)-decanoyl]-sn-glycero-3-phosphoglyce rol (PPDPG, mole fraction X = 0.01) to the heme of the cyt c. Large unilamellar liposomes composed of egg phosphatidylcholine (eggPC) with varying content of the acidic phospholipid phosphatidylglycerol (eggPG) were employed. Unexpectedly, the rate of binding of cyt c to membranes was attenuated upon increasing the mole fraction of the acidic phospholipid (XPG). For example, at 50 microM phospholipid and 5 microM cyt c, when XPG was increased from 0.20 to 0.40 the half-time for the single-exponential decay in fluorescence increased from 4.7 to 8.6 ms. A similar observation was made for the membrane binding of another cationic protein, histone H1. We suggest that the formation of cooperative hydrogen-bonded networks by deprotonated and protonated PG in the vesicle surface retards the binding of cyt c to the liposome surface. However, once formed, the complex of cyt c with acidic phospholipids is stabilized by increasing XPG. Accordingly, significantly prolonged half-times of dissociation of cyt c from liposomes by NaCl, ATP, and different cationic proteins are measured upon increasing XPG. Differences between the latter cationic membrane binding ligands most likely reflect the varying relative contributions of hydrophobicity and Coulombic forces to their attachment to liposomes. Our data on the release and binding of cyt c to liposomes as a function of XPG and in the presence of ATP also provide the first direct experimental evidence for multiple lipid binding sites in cyt c.

Defective transcription-coupled repair of oxidative base damage in Cockayne syndrome patients from XP group G.

In normal human cells, damage due to ultraviolet light is preferentially removed from active genes by nucleotide excision repair (NER) in a transcription-coupled repair (TCR) process that requires the gene products defective in Cockayne syndrome (CS). Oxidative damage, including thymine glycols, is shown to be removed by TCR in cells from normal individuals and from xeroderma pigmentosum (XP)-A, XP-F, and XP-G patients who have NER defects but not from XP-G patients who have severe CS. Thus, TCR of oxidative damage requires an XPG function distinct from its NER endonuclease activity. These results raise the possibility that defective TCR of oxidative damage contributes to the developmental defects associated with CS.

A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: Implications for a second XPG function

A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: Implications for a second XPG function