WT ATRIP cDNA but not cDNA encoding p.Arg760* ATRIP complements the G2/M checkpoint defect in CV1720 cells, and p.Arg760*ATRIP impairs ATR–ATRIP protein interaction.

<p>A) Analysis of the G2/M checkpoint defect in CV1720 cells following expression of ATRIP cDNA. G2/M checkpoint arrest was examined 2 h post exposure to 5 Jm⁻² UV. 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>, WT cells showed proficient checkpoint arrest whilst DK0064 (ATR–SS) and CV1720 (patient) cells are unable to undergo arrest. Expression of WT ATRIP cDNA restored the ability of CV1720 (patient) and DK0064 (ATR–SS) to undergo checkpoint arrest but this was not observed following transfection of cDNA encoding R760* ATRIP. Significantly, expression of ATRIP R760* did not impair checkpoint arrest in WT cells verifying that it does not exert a dominant negative impact. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#s2" target="_blank">Results</a> represent the mean and SD of three experiments. WT cells were GM2188. ATR–SS represents DK0064 and patient, CV1720. B) R760* ATRIP impairs ATR–ATRIP interaction. Crude lysates were prepared from HEK293T cells and either mock transfected (lane1), transfected with HA-tagged WT <i>ATRIP</i> cDNA (lane2), or R760* <i>ATRIP</i> cDNA (lane3) (generating p.Arg760* ATRIP protein) together with <i>ATR</i> cDNA. The extracts were immunoprecipitated with agarose-conjugated rabbit anti-HA-tag antibody (MBL). Interaction with ATR was examined by immunoblotting with ATR antibodies (left panel). Immunoblotting using the HA-tag (ATRIP; right panel) verified expression of the appropriately sized ATRIP in the samples. 33% of the crude lysate was loaded; IP, immunoprecipitate.</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

Patients 27-4BI and 19-8BI have reduced ATR and ATRIP expression and mutations in ATR.

<p>A) Photographs of patient included with informed consent of parent. B) Cell extracts (50 µg) from LBLs derived from WT (IM257), patient 27-4BI or patient 19-8BI were immunoblotted using the indicated antibodies. Reduced expression of ATR was observed in both patients. 27-4BI also had reduced ATRIP expression. C) Structure of ATR showing the site of the mutations identified and the UME domain. D) The UME domain is conserved between species and the methionine residue within this domain is conserved in yeast.</p

LBLs from patient 27-4BI and 19-8BI showed impaired ATR–dependent damage responses.

<p>A) 27-4BI cells were examined for their ability to activate G2/M checkpoint arrest at 4 h following exposure to 7 Jm⁻² UV. In contrast to WT cells (GM2188), no significant arrest was observed in 27-4BI cells. The checkpoint response to ionizing radiation, which is ATM rather than ATR dependent, was normal. B) LBLs derived from patients 27-4BI and 19-8BI were examined for their ability to phosphorylate the indicated ATR substrates at 1 h following exposure to 0.5 mM HU. WT represents IM257. 27-4BI and control LBLs have a similar cell cycle profile demonstrating that the lack of ATR substrate phosphorylation cannot be attributed to the lack of S phase cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002945#pgen.1002945.s002" target="_blank">Figure S2</a>).</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

Clinical features of ATR/ATRIP–deficient patients.

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

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