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. 2004 Jun;24(11):4710–4719. doi: 10.1128/MCB.24.11.4710-4719.2004

FIG. 5.

FIG. 5.

BLM inhibits CAF-1-mediated chromatin assembly coupled to DNA repair. (A) Addition of BLM inhibits CAF-1 activity. Each lane contains 75 ng of UV-irradiated circular pBluescript plasmid DNA, as indicated above the figure. Purified CAF-1 complex is present in lanes 2 to 7 but not in lane 1 which serves as a negative control. The concentration of recombinant BLM was 10 nM in lane 3 and 25 nM in lanes 4 to 6. In lanes 5 and 6, BLM protein was incubated with 1 μl of anti-BLM antibody before being added to the chromatin assembly reaction mix. Lane 7 is a control of anti-BLM antibody. The positions of the relaxed/nicked (Ir, II) and supercoiled (I) DNA are indicated on the right. (B) An excess of CAF-1 holoenzyme complex restores BLM-mediated inhibition of chromatin assembly in vitro. A 75-ng portion of UV-irradiated plasmid DNA was used in each lane; 25 nM BLM was added in lanes 1, 3 and 4, but not in lane 2. CAF-1 was present only in lanes 3 and 4, at 0.5 and 4 nM, respectively. The positions of the relaxed/nicked (Ir, II) and supercoiled (I) DNA are indicated on the right. (C) Inhibition of CAF-1 activity is BLM specific. Bacterial helicase UvrD does not inhibit CAF-1-mediated chromatin assembly coupled to DNA repair (lane 3). Lanes 1 and 2 are negative and positive controls, respectively.