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100 nmol/L Cy5-labeled star DNA was digested by 500 nmol/L DraIII.
Cy5-labeled canonical or star DNA was digested by DraIII as described in Supplementary EXPERIMENTAL.
To decipher the DraIII star activity cleavage mode, 39-bp duplex star DNA was used for time course analysis.
100 nmol/L of the canonical or star DNA was incubated with 500 nmol/L of DraIII WT in NEBuffer 4 at 37°C for 1 h.
100 nmol/L of star DNA and 500 nmol/L DraIII WT (or 100 nmol/L of canonical DNA and 10 nmol/L DraIII WT) were incubated in NEBuffer 4 at 37°C.
The 16-nt single strand product (5′-Cy5-catgatccccCATGTT) represents nicking at the top canonical half site and the 26-nt single strand product (5′-Cy5-atggtagagacgcttttttgCACAAC) represents nicking at the bottom star half site (Fig. 2A, star DNA; Fig. 2C).
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Prime STAR GXL DNA polymerase (TaKaRa) was used for reaction.
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