Figure 6.

Ddk1 requires free ends for activity in vitro. (A) Wild-type or catalytically inactive (D251N K253A) Ddk1 were incubated with linear 5′-labelled single-stranded 44DNA substrate or its circularized form. Indicated ratios of enzyme to substrate were analysed. Reactions were performed in standard conditions using an optimal concentration of magnesium (5.12 mM) or manganese (0.16 mM) and resolved by 12% denaturing PAGE. The nature of the substrates was controlled using exonuclease Exo I (NEB) and endonuclease DNAse I (Roche). (B) Ddk1 activity tested using 5′-labelled 44RNA–DNA–RNA substrate. To control the activity of Ddk1, reactions with single-stranded 44DNA or 44RNA were performed. Standard conditions were used. Products were resolved using 15% urea–PAGE. An RNA ladder and DNA ladder, as well as a reference RNA fragment (20RNA), were also resolved. NP-no protein.