Fig 3. ALC1 induces chromatin relaxation around TOPI-cc.
(A) (Left) Schematic representation of the ALC1 domain. Glu-165 on ALC1 was altered by Gln to inhibit the ATPase activity of ALC1. (Right) The ALC1-ATPase dead mutant (ALC1-/E165Q) and the ALC1-/- cells showed a similar sensitivity to CPT. Error bars represent standard deviations from three independent experiments. (B) Nucleosome assembly in close proximity to TOPI-cc. 5 ×107 wild-type and ALC1-/- cells were pulse-labeled with BrdU for 10 min followed by treatment with CPT (20 μM) for 15 min. Nuclei were prepared and treated with 5, 10, and 20 units/ml MNase for 5 min. DNA was resolved in 2% agarose gel and stained with ethidium bromide (EtBr) (upper panel), transferred onto a membrane, and detected with an anti-BrdU antibody (lower panel). Blue box indicates quantified band corresponding to mononucleosome. P-value was calculated by Student’s t-test (*P<0.05).