A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed ‘unscheduled DNA synthesis (UDS)’, is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis

Identification of the first ATRIP-deficient patient and novel mutations in ATR define a clinical spectrum for ATR-ATRIP Seckel Syndrome

A homozygous mutational change in the Ataxia-Telangiectasia and RAD3 related (ATR) gene was previously reported in two related families displaying Seckel Syndrome (SS). Here, we provide the first identification of a Seckel Syndrome patient with mutations in ATRIP, the gene encoding ATR-Interacting Protein (ATRIP), the partner protein of ATR required for ATR stability and recruitment to the site of DNA damage. The patient has compound heterozygous mutations in ATRIP resulting in reduced ATRIP and ATR expression. A nonsense mutational change in one ATRIP allele results in a C-terminal truncated protein, which impairs ATR-ATRIP interaction; the other allele is abnormally spliced. We additionally describe two further unrelated patients native to the UK with the same novel, heterozygous mutations in ATR, which cause dramatically reduced ATR expression. All patient-derived cells showed defective DNA damage responses that can be attributed to impaired ATR-ATRIP function. Seckel Syndrome is characterised by microcephaly and growth delay, features also displayed by several related disorders including Majewski (microcephalic) osteodysplastic primordial dwarfism (MOPD) type II and Meier-Gorlin Syndrome (MGS). The identification of an ATRIP-deficient patient provides a novel genetic defect for Seckel Syndrome. Coupled with the identification of further ATR-deficient patients, our findings allow a spectrum of clinical features that can be ascribed to the ATR-ATRIP deficient sub-class of Seckel Syndrome. ATR-ATRIP patients are characterised by extremely severe microcephaly and growth delay, microtia (small ears), micrognathia (small and receding chin), and dental crowding. While aberrant bone development was mild in the original ATR-SS patient, some of the patients described here display skeletal abnormalities including, in one patient, small patellae, a feature characteristically observed in Meier-Gorlin Syndrome. Collectively, our analysis exposes an overlapping clinical manifestation between the disorders but allows an expanded spectrum of clinical features for ATR-ATRIP Seckel Syndrome to be define

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

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

UV-sensitive syndrome (UVSS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma1, 2, 3, 4. Despite mild clinical features, cells from individuals with UVSS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER)2, 4, 5, which removes DNA damage in actively transcribed genes6. Three of the seven known UVSS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively)7, 8. The remaining four individuals with UVSS, one of whom is described for the first time here, formed a separate UVSS-A complementation group1, 9, 10; however, the responsible gene was unknown. Using exome sequencing11, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UVSS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders

Identification of mutational changes in <i>ATRIP</i> in CV1720.

<p>A) Upper panel shows primer pairs used to distinguish cDNA products encompassing or lacking exon 2. Lower panel shows RT-PCR products obtained using the primers shown in the upper panel. RT-PCR from patient CV1720 generated a smeared product with a defined band of 458 bp, as observed in WT cells, and a weaker band of 325 bp. The latter band was not detected using cDNA from WT cells (MRC5). A similar single 458 bp band was obtained using the same primers with cDNA derived from a distinct wild type cell line (GM2188; data not shown). (B) Sequencing of the RT-PCR products derived from WT (MRC5) and patient (CV1720) cells. A double sequence pattern at the exon 2–3 boundary is observed using patient CV1720 cDNA. C) Selective quantitative amplification of the WT or 2278C>T <i>ATRIP</i> alleles. Primers located in <i>ATRIP</i> exon 12 and 13 were designed to selectively amplify the WT (c.2278C) (P1 and P3C) versus the mutated (c.2278C>T) (P2 and P3C) alleles. The WT PCR product is shown in blue and the c.2278C>T PCR product in red. The exon 12 mutated allele is only observed in the patient and mother cDNA whilst the WT allele is observed in the patient, mother and father cDNA although the level is reduced in the patient and mother. D) qRT-PCR analysis of <i>ATRIP</i> splicing variants from patient CV1720 and parental cells. qRT-PCR analysis of the level of the normally spliced (encompassing exons 1-2-3) and the aberrantly spliced (Δexon2) <i>ATRIP</i> cDNA in the patient and parent cells. PCR primers were designed at the exon2-exon3 or exon1-exon3 boundaries to selectively amplify the splicing variants. Transcripts from <i>HPRT1</i> were used as a quantification control. The correctly spliced transcript from the paternal allele of the patient (wild type c.2278C, blue fraction in the cumulative bar labelled, ‘patient’, at the left panel) was estimated to be ∼25% of the normal level. (E) The mis-spliced paternal allele is subject to nonsense mediated mRNA decay (NMD). Cycleave-qPCR confirmed that the <i>ATRIP</i> c.2278C>T mutant allele was expressed exclusively in the patient and the mother. The <i>ATRIP</i> exon12-13 fragment was amplified with PCR primers P7/P8 as shown in the figure. A set of fluorescent probes were used to distinguish the WT versus c.2278C>T allele (probe1 and probe2, respectively). In the patient, the paternal mRNA transcript level (emerald lines) is low because of NMD (top left). Puromycin treatment eliminated the NMD and the paternal transcript level returned to the normal level. In all panels WT represented MRC5, patient was CV1720 and parents were as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen-1002945-g001" target="_blank">Figure 1A</a>.</p

CV1720 cells show impaired ATR–dependent DNA damage responses.

<p>A) WT, DK0064 (ATR–SS), CV1720 (patient), CV1780 (patient's mother) and CV1783 (patient's father) cells were exposed to 5 Jm⁻² UV and the mitotic index (MI) assessed 2 h post exposure. A greater than two fold decrease in mitotic index is observed in WT and both paternal cell lines but not in DK0064 (ATR–SS) or CV1720 (patient) cells. B) Cells were exposed to 5 mM HU for 2 h and the percentage of p-H2AX (γ-H2AX) positive cells assessed by immunofluorescence. Note that HU causes pan nuclear p-H2AX formation rather than defined foci as observed after exposure to ionising radiation. Thus, the percentage of γ-H2AX positive cells was scored. C) Cells were exposed to UV (5 Jm⁻²) and subjected to Western Blotting (WB) using p-Chk1 (p-Ser317) antibodies at 2 h. Chk1 expression was shown to be similar in WT and patient cells (lower panel). D) Cells were exposed to 3 mM HU for 2 h and whole cell extracts analysed by WB using FANCD2 antibodies. The ubiquitylation of FANCD2, detectable by a product with reduced mobility, is diminished in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. E) Cells were exposed to 5 mM HU and examined for the percentage of cells showing >5 53BP1 foci at 2 h post exposure. 53BP1 foci formation is reduced in DK0064 (ATR–SS) and CV1720 cells compared to WT cells. F–I) The indicated cells were processed by WB using ATRIP or ATR antibodies. MCM2 was used as a loading control. F shows the analysis of a range of protein levels for accurate comparison. CV1720 (patient) cells show markedly reduced ATR and ATRIP protein levels. G shows that both parental lines have approximately half the level of ATR and ATRIP compared to two WT cell lines. DK0064 (ATR–SS) and CV1720 cells, in contrast, have more dramatically reduced ATR and ATRIP protein levels. 50 ug protein was loaded. WT in all panels was GM2188. Patient, mother and father were as shown in panel A. H and I show the quantification of ATRIP and ATR protein levels from at least three independent WB experiments.</p

Clinical features of ATR/ATRIP–deficient patients.

<p>NR; not recorded. NA; not assessed.</p

MGS and Seckel syndrome patient phenotypes.

*<p>standard deviations from the age-related normal population mean, NA = not assessed.</p><p>MGS data from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Bicknell1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Bicknell2" target="_blank">[14]</a><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-Guernsey1" target="_blank">[33]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945-deMunnik1" target="_blank">[34]</a>.</p