Fig. 6: EXD2-deficient U2OS cells are synthetic sick upon depletion of factors that promote BIR-mediated ALT DNA synthesis. | Nature Communications

Fig. 6: EXD2-deficient U2OS cells are synthetic sick upon depletion of factors that promote BIR-mediated ALT DNA synthesis.

From: Pathway choice in the alternative telomere lengthening in neoplasia is dictated by replication fork processing mediated by EXD2’s nuclease activity

Fig. 6

a Colony formation assays in WT U2OS and EXD2−/− cells treated with siRNA targeting BLM, SLX4 or control siRNA (Luciferase). Surviving fraction relative to control is quantified (n = 4 biologically independent samples examined over 2 independent experiments, statistical significance was determined by two-sided student’s t-test, bars represent +/−SEM). b Colony formation assays in WT U2OS and EXD2−/− cells treated with siRNA targeting DNA2 or control siRNA. Surviving fraction relative to control is quantified (n = 6 biologically independent samples examined over 3 independent experiments, statistical significance was determined by two-sided student’s t-test, bars represent +/−SEM). c Colony formation assay in WT U2OS and EXD2−/− cells treated with siRNA targeting POLD3 or control siRNA, as indicated. Surviving fraction relative to control is quantified (n = 8 biologically independent samples examined over 2 independent experiments, statistical significance was determined by two-sided student’s t-test, bars represent +/−SEM). Source data are provided as a source data file. d Model: WT U2OS cells restart stalled replication forks at the telomere by HR-mediated fork restart or employ RAD52-dependent break induced replication upon replication fork collapse/cleavage (MUS81-independent). RAD52 acts as a strand annealing factor to promote POLD3-dependent conservative DNA synthesis. In the absence of EXD2, excessive replication fork regression mediated by SMARCAL1 leads to fork degradation and fork collapse by MUS81-dependent nucleolytic cleavage. This results in a fork conformation that is preferentially processed by the RAD52-independent arm of the ALT BIR mechanism, whereby an as-yet unidentified annealing factor promotes POLD3-dependent conservative DNA synthesis resulting in poor telomere elongation and the presence of T-SCEs. In the absence of factors that promote BIR-mediated ALT synthesis, collapsed replication forks at the telomere in EXD2-deficient cells cannot be efficiently repaired.

Back to article page