12 research outputs found
Mislocalization of XPF-ERCC1 Nuclease Contributes to Reduced DNA Repair in XP-F Patients
Xeroderma pigmentosum (XP) is caused by defects in the nucleotide excision repair (NER) pathway. NER removes helix-distorting DNA lesions, such as UV–induced photodimers, from the genome. Patients suffering from XP exhibit exquisite sun sensitivity, high incidence of skin cancer, and in some cases neurodegeneration. The severity of XP varies tremendously depending upon which NER gene is mutated and how severely the mutation affects DNA repair capacity. XPF-ERCC1 is a structure-specific endonuclease essential for incising the damaged strand of DNA in NER. Missense mutations in XPF can result not only in XP, but also XPF-ERCC1 (XFE) progeroid syndrome, a disease of accelerated aging. In an attempt to determine how mutations in XPF can lead to such diverse symptoms, the effects of a progeria-causing mutation (XPFR153P) were compared to an XP–causing mutation (XPFR799W) in vitro and in vivo. Recombinant XPF harboring either mutation was purified in a complex with ERCC1 and tested for its ability to incise a stem-loop structure in vitro. Both mutant complexes nicked the substrate indicating that neither mutation obviates catalytic activity of the nuclease. Surprisingly, differential immunostaining and fractionation of cells from an XFE progeroid patient revealed that XPF-ERCC1 is abundant in the cytoplasm. This was confirmed by fluorescent detection of XPFR153P-YFP expressed in Xpf mutant cells. In addition, microinjection of XPFR153P-ERCC1 into the nucleus of XPF–deficient human cells restored nucleotide excision repair of UV–induced DNA damage. Intriguingly, in all XPF mutant cell lines examined, XPF-ERCC1 was detected in the cytoplasm of a fraction of cells. This demonstrates that at least part of the DNA repair defect and symptoms associated with mutations in XPF are due to mislocalization of XPF-ERCC1 into the cytoplasm of cells, likely due to protein misfolding. Analysis of these patient cells therefore reveals a novel mechanism to potentially regulate a cell's capacity for DNA repair: by manipulating nuclear localization of XPF-ERCC1
The active site of the DNA repair endonuclease XPF–ERCC1 forms a highly conserved nuclease motif
XPF–ERCC1 is a structure-specific endonuclease involved in nucleotide excision repair, interstrand crosslink repair and homologous recombination. So far, it has not been shown experimentally which subunit of the heterodimer harbors the nuclease activity and which amino acids contribute to catalysis. We used an affinity cleavage assay and located the active site to amino acids 670–740 of XPF. Point mutations generated in this region were analyzed for their role in nuclease activity, metal coordination and DNA binding. Several acidic and basic residues turned out to be required for nuclease activity, but not DNA binding. The separation of substrate binding and catalysis by XPF–ERCC1 will be invaluable in studying the role of this protein in various DNA repair processes. Alignment of the active site region of XPF with proteins belonging to the Mus81 family and a putative archaeal RNA helicase family reveals that seven of the residues of XPF involved in nuclease activity are absolutely conserved in the three protein families, indicating that they share a common nuclease motif
Biochemical differences between Three Subcell-lines Derived from SV40-Transformed Hamster Lens Cells
Coordination of dual incision and repair synthesis in human nucleotide excision repair
Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1-XPF and 3′ to the lesion by XPG leads to the removal of a lesion-containing oligonucleotide of about 30 nucleotides. The resulting single-stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1-XPF and XPG, we show that the 5′ incision by ERCC1-XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a ‘cut-patch-cut-patch' mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity
Human SNM1A and XPF–ERCC1 collaborate to initiate DNA interstrand cross-link repair
One of the major mammalian DNA interstrand cross-link (ICL) repair pathways is coupled to replication. Here, the 5′–3′ exonuclease activity of SNM1A is found to be critical for ICL repair. Following a 5′ incision of the cross-link by XPF–ERCC1, SNM1A loads at the nick and digests past the ICL, initiating the repair process. Failure of this XPF–ERCC1- and SNM1A-dependent repair pathway causes Mus81-induced replication fork cleavage and double strand breaks, leading to ICL sensitivity
Correction of <i>XPF</i> mutant cell NER defect by microinjection of XPF-ERCC1.
<p>Primary fibroblasts from XFE progeroid patient XP51RO were fused to create homopolykaryons by treatment with inactivated Sendai virus then plated on glass coverslips. Only homopolykaryons were injected with recombinant XPF-ERCC1 protein complex (A) wild-type (B) XPF<sup>R799W</sup>-ERCC1 (C) XPF<sup>R153P</sup>-ERCC1. The cultures were irradiated with 10 J/m<sup>2</sup> UV-C and <sup>3</sup>H-thymidine was added to the culture. UV-induced unscheduled DNA synthesis was detected by autoradiography. Homopolykaryons are indicated with arrows. (D) Histogram indicating the average number of radiographic grains in nuclei injected with each of the recombinant protein complexes and uninjected cells in the same sample. Error bars indicate the standard deviation. N indicates the number of nuclei analyzed in each population. P values for the comparison between injected and uninjected cells were calculated using an unpaired two-tailed Student's t-test.</p
Characteristics of <i>XPF</i> mutant cell lines.
<p>UDS unscheduled DNA synthesis</p><p>*The patient had normal levels of <i>XPF</i> transcript, suggesting one allele encodes a full-length mRNA.</p>♦<p>Mutation could not be confirmed on genomic DNA.</p><p>° Siblings.</p><p>n.d. = not determined</p
Characterization of XPF-YFP and XPF<sup>153</sup>-YFP in CHO cells.
<p>(A) Western blot analysis of XPF-YFP expressed in <i>Xpf</i> mutant cells. XPF-deficient hamster cell line, UV41, was transiently transfected with wild type <i>XPF-YFP</i> or <i>XPF<sup>153</sup>-YFP</i> and the fusion proteins were detected using an antibody against XPF or GFP. C5RO was used as positive control for the XPF blot and as a negative control for the GFP blot. UV41 cells transfected with YFP alone was used as a negative control for XPF blot and as a positive control for GFP blot. (B) Clonogenic survival of wild-type (wt), XPF-deficient CHO cell line UV41, and UV41 transfected with wild type XPF-YFP and XPF<sup>153</sup>-YFP after UV and MMC treatment. Colonies were counted 7–10 days after treatment and results are plotted as mean 3 independent experiments. (C) Subcellular localization of wild type XPF-YFP and XPF<sup>153</sup>-YFP after transient transfection in XPF-deficient the CHO cell line UV41 detected by fluorescence microscopy.</p
Biochemical characterization of XPF<sup>R153P</sup>-ERCC1 and XPF<sup>R799W</sup>-ERCC1 mutants.
<p>(A) Gel filtration profiles from the purification of recombinant XPF-ERCC1, XPF<sup> R153P</sup>-ERCC1 and XPF<sup> R799W</sup>-ERCC1 from baculovirus-infected Sf9 insect cells using a His<sub>6</sub> tag on ERCC1. Aggregated proteins elute at ∼45 ml in the void volume of the column; heterodimeric XPF-ERCC1 elutes at ∼65 ml, corresponding to ∼200 kD, and monomeric ERCC1 elutes at ∼78 ml (∼50kD). (B) SDS PAGE analysis of purified protein complexes. Lane 1, 3 and 5 (D): XPF-ERCC1, XPF<sup> R153P</sup>-ERCC1 and XPF<sup> R799W</sup>-ERCC1, respectively, after purification over NTA-agarose, gel filtration and heparin columns. Lanes 2 and 4 (A) show the proteins present in the fractions eluting at 45 ml in the gel filtration column step of XPF<sup> R153P</sup>-ERCC1 and XPF<sup> R799W</sup>-ERCC1, respectively. (C) Immunodetection of XPF in normal (C5RO) and <i>XPF</i> mutant cells. The star indicates the migration of a cross-reactive band demonstrating equal loading <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000871#pgen.1000871-Niedernhofer2" target="_blank">[32]</a>. (D) Incision activities of XPF-ERCC1, XPF<sup> R153P</sup>-ERCC1 and XPF<sup> R799W</sup>-ERCC1 (200 fmol) on a 5′-<sup>32</sup>P-labeled stem-loop DNA substrate (100 fmol) in the presence of either 0.4 mM MnCl<sub>2</sub> (lanes 2, 4 and 6) or 2 mM MgCl<sub>2</sub> (lanes 3, 5 and 7). Reactions were analyzed on a 15% denaturing polyacrylamide gel. The 46-mer substrate and 9–10-mer products are indicated.</p
Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors
In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position -6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position -19 to +8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.clos