Figure 7.
Targeting of BLM to the chromatin in the absence of RNF8/RNF168 decreases HR. (A, E) Expression of chromatin targeted BLM fusions. (A) Untagged BLM (WT), H2AX-BLM (WT) or MDC1 FHA-BLM (WT) (cloned in pIRES vector) (E) AcGFP-tagged BLM (WT), H2AX-BLM (WT) or MDC1 FHA-BLM (WT) (cloned in AcGFP-N1 vector) were expressed in 293T cells. Western analysis was carried out with antibodies against (A) BLM (A300-110A) and hsp90 and (E) GFP and hsp90. (B, C) Chromatin targeted BLM rescues the hyper-recombinogenic phenotype of cells not expressing either RNF8 or RNF168. (B) U2OS shRNF8 or (C) U2OS shRNF168 cells were grown with or without Dox treatment. The cells were either transfected with pBHRF construct alone or along with BLM, MDC1 FHA-BLM or H2AX-BLM. Forty eight hours post transfection, the host cell reactivation assays were carried out after treatment with HU for the final 16 h. The GFP/BFP ratio was determined as the readout for the extent of HR. (D) Lack of BLM ubiquitylation prevents its anti-recombinogenic function. A-15/BS cells were transfected with pBHRF. In BS cells, co-transfection was carried out with either wild-type BLM or the BLM (3K-R) mutant. Forty eight hours post transfection, the host cell reactivation assays were carried out after treatment with HU for the final 16 h. The GFP/BFP ratio was determined as the readout for the extent of HR. (F) Direct chromatin targeting BLM rescues the loss of ubiquitylation machinery. (Top) BLM fusions (MDC1 FHA-EGFP-BLM and H2AX-EGFP-BLM) were transiently transfected into U2OS shRNF8 cells in the presence of Dox. Transfection was also carried out in parallel with BLM (WT). The cells were treated with HU. EGFP-BLM expression of the fusion proteins was tracked. Nuclei were stained by DAPI. Scale 5 μM. (Bottom) The percentage of cells with nucleolar BLM was determined from the above experiment.
Source data for this figure is available on the online supplementary information page.
