Figure 6.

CTC1 is required for efficient replication and efficient restart of stalled replication fork at telomeres. (A) Left, examples of the fragile telomere phenotype observed in CTC1 null MEFs and WT MEFs treated with 0.2 μM aphidicolin. Arrows point to fragile telomeres. (B) Quantification of fragile telomeres observed in (A). (C) Summary of telomere replication in CTC1^F/F and CAG-Cre^ER; CTC1^F/F MEFs treated with or without 4-hrdroxytamoxifen (4-HT) to delete CTC1. Two-tailed t-test was used to calculate statistical significance. n≥4, Error bars: s.e.m. (D) Examples of CO-FISH images of partial metaphase spreads from WT (PD 49), CTC1^−/− (PD14) and CTC1^−/− (PD58) MEFs. Red signals: G-strand telomeres, green signals: C-strand telomeres. Red arrowheads point to loss of lagging-strand telomeres and green arrowheads point to loss of leading-strand telomeres. (E) Quantification of signal intensity of leading- and lagging-strand telomeres in (D). Two independent cell lines were analysed, and a minimum of 35 metaphases and 1500 chromosomes were scored per genotype and passage. Mean values were derived from two cell lines. The two-tailed t-test was used to calculate statistical significance. Error bars: s.e.m. (F) Distribution of telomere signals on CsCl gradient after releasing from HU treatment. BrdU pulse-labelled telomere DNA following HU treatment was separated on CsCl gradient. Fractions were collected from the bottom of the gradient and slot-blot was used to quantitate the amount of telomeres in each fraction. Telomere signals from telomeres with density ≥1.755 g/ml are amplified in the insert. (G) Percentage of BrdU-containing replicated telomeres. One-tailed t-test was used to calculate statistical significance. n=3. Error bars: s.e.m.