Figure 1.
NCI-318 and MSK-41 pedigrees with RTEL1 mutation and shared risk haplotype.
NCI-318 (A) and MSK-41 (B) pedigrees are shown. Red symbols indicate affected individuals. The pink rectangles indicate the shared haplotype between the pedigrees. Each other colored rectangle indicates a unique haplotype.
Figure 2.
Telomere length is altered in individuals with RTEL1R1264H.
(A) Primary lymphocyte telomeres in family NCI-318 were measured by flow cytometry with fluorescent in situ hybridization (FISH) [3]. The proband is indicated by a triangle, the mother by a circle, and the father by a square. (B) Telomere FISH analysis of MSK-41 hTERT-immortalized fibroblasts revealed extreme telomere length heterogeneity. Quantitation of chromatids lacking detectable telomeric signal is shown. BJ hTERT, a normal hTERT-immortalized fibroblast line, and SaOS-2, an osteosarcoma cell line that relies on recombination-based telomere maintenance (ALT), are presented for comparison. (C) Representative metaphase spreads of MSK-41 and BJ hTERT are shown.
Table 1.
Clinical characteristics of families with RTEL1 mutations.
Figure 3.
RTEL1R1264H affects a putative conserved C4C4 domain.
As displayed on the schematic (representing ENSP00000353332), the RTEL1 mutation is at the C-terminus of the protein, distal to the helicase domain. The affected amino acid is in a putative C4C4 domain. All eight key cysteines and R1264 are conserved in human, orangutan, cattle, and mouse sequences. Higher percent identity at a given amino acid position is indicated by a deeper purple color.
Figure 4.
Inhibiting DNA replication blocks T-circle formation in MSK-41 RTEL1R1264H cells.
(A) Phi29-dependent T-circles in BJ hTERT and MSK-41. (B) Phi29-dependent T-circles in RTEL1 floxed/- MEFs ± Cre, BJ hTERT and MSK-41. (C) Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (D) Dot blot of the Phi29-dependent T-circles in BJ hTERT and MSK-41 ± aphidicolin (APD; 5 µM). (E) Quantification of the fold increase in intensity of Phi29-dependent T-circles in the different cell lines subjected to the indicated treatments. Intensity mean and standard deviation were calculated over two independent experiments; statistical analysis (one-way ANOVA) was calculated with Prism (GraphPad).
Figure 5.
T-circle formation in MSK-41 cells is dependent on SLX4.
(A) Two shRNAs (SLX4-1 and SLX4-2) were used to knockdown SLX4 expression. (B) T-circle formation was measured in the MSK-41 SLX4 knockdown strains relative to MSK-41 with a control shRNA. Mean and standard deviation were calculated from two independent experiments. *P<0.05, **P<0.01 by unpaired two-tailed t-test.
Figure 6.
MSK-41 cells are hypersensitive to DNA damage and experience elevated levels of sister chromatid exchange.
(A) BJ hTERT (blue line) and MSK-41 cells (red line) were treated at the indicated doses of mitomycin C (MMC) for 24 hours, and colony formation was scored 14 days post-treatment. Formation of at least 50 colonies was required at each dose for the experiment to be considered valid. (B) Spontaneous (blue) and MMC-induced (red) sister chromatid exchanges were visualized by Giemsa staining; the number of exchanges per metaphase is shown. Cells were cultured in 20 µM BrdU for 40 hours, with treatment with 25 ng/mL MMC for the final 24 hours.