Fig. 7: BRCA2 protects and remodels G4-driven stalled replication forks at telomeres.

a Immunofluorescence images of telomere damage. TBI fibroblasts (telomerase-null (mTR^-/-); Brca2^F11/F11; Cre-ER^TM), in which Brca2 depletion can be controlled by the treatment of tamoxifen (4-OHT)⁶⁶ (Supplementary Fig. 16), were serum starved for 16 hours then released. PDS was treated at the point of release when needed. Cells were subjected to immunostaining with anti-TRF1 (red) and anti-γ-H2AX antibodies (green), 4 h post release. Scale bar, 5μm. Inset, enlarged foci marked with dotted square. b Plots representing the number of damaged telomeres per cell from a. Number of cells analyzed (n): Control, n = 126; +shMre11, n = 129; +PDS, n = 129; +PDS + shMre11, n = 131; +4-OHT, n = 121; +4-OHT + shMre11, n = 92; +4-OHT + PDS, n = 130; +4-OHT + PDS + shMre11, n = 137. c Plots representing telomere damage after treatments with (+) or without (−) 1.4 μM Aphidicholin (Aph) and/or 5 μM PDS. Drugs were treated for 16 h then subjected to immunofluorescence with antibodies as in a. Control, n = 39; +Aph, n = 37; +PDS, n = 61; +Aph +PDS, n = 54; +4-OHT, n = 45; +4-OHT + Aph, n = 43; +4-OHT + PDS, n = 38; +4-OHT + Aph +PDS, n = 42. d Rad51 localization at telomeres after the indicated treatments. Red, immunofluorescence with anti-TRF1; green, anti-Rad51. Scale bar, 5 μm. e Rad51-positive telomeres per cell from d. Control, n = 99; +Aph, n = 66; +PDS, n = 78; +Aph +PDS, n = 78; +4-OHT, n = 77; +4-OHT + Aph, n = 53; +4-OHT + PDS, n = 61; +4-OHT + Aph +PDS, n = 45. f Result of EMSA with indicated probes when increasing doses of RAD51 was added and co-incubated with 25 nM of BRCA2OB. g EMSA result when increasing doses of BRCA2OB was added and co-incubated with RAD51. The result is the representative of three independent experiments (f, g). h Model for how BRCA2, RAD51, and MRE11 work with telomeric G4 during replication.