Figure 5. ADAAD can hydrolyze ATP using primer 2 as effector DNA.

(A) The primer 2 region of brg1 promoter functions as a repressor. The region was cloned into pGL3 basic vector as well as pGL3 promoter vector. The constructs were transiently transfected into HeLa cells and assayed for luciferase activity after 36 hours. In this experiment the luciferase activity was normalized with respect to Renilla. (B) ADAAD, the bovine homolog of SMARCAL1, hydrolyzes ATP in the presence of primer 2 DNA. 20 nM DNA was incubated with 0.1 μM ADAAD in the presence of ATP for 60 minutes at 37 °C. (C) The K_M for the interaction was calculated for ADAAD-primer 2 DNA interaction by measuring the ATPase activity with increasing DNA concentration. The reactions were incubated at 37 °C for 60 minutes. 0.1 μM ADAAD was used for this experiment. (D) ATPase activity induced by single-strand G, single-strand C, and double-strand GC DNA. 10 nM DNA was incubated with 0.24 μM ADAAD in the presence of ATP for 30 minutes at 37 °C. (E) The K_M for ADAAD-GC DNA was calculated by measuring the ATPase activity with increasing DNA concentration. The reactions were incubated at 37 °C for 30 minutes and 0.24 μM ADAAD was used in the reaction. (F) Comparison of kinetic parameters for the interaction of ADAAD with primer 2 DNA and GC DNA.