Figure 1.

Excision of 5mC by DME and ROS1 DNA glycosylases. (A) The scheme of base excision by monofunctional or bifunctional DNA glycosylase. Upon encountering a base to remove (i), monofunctional DNA glycosylases catalyze the hydrolysis of an N-glycosidic bond between the base and a ribose sugar creating an AP site (ii). Bifunctional DNA glycosylases possess additional AP lyase activity that catalyzes the scission of the sugar phosphate backbone leaving the 3′-PUA (iii) by the process called β-elimination. Further δ-elimination process generates a 3′-phosphate (iv), which is a blocking lesion for subsequent polymerization. (B) Excision of 5mC by DME and ROS1. Purified MBP-DMEΔ or -ROS1Δ protein (23) was incubated with a radiolabeled 35-mer oligonucleotide duplex containing 5mC. Both 3′-PUA and 3′-phosphate were generated by β- and δ-elimination processes, respectively. The major intermediate formed in early reaction is a 3′-PUA (β-elimination product), and as reaction proceeds, 3′-phosphate (δ-elimination product) begins to accumulate later. (C) Relative amounts of β- and δ-elimination product accumulation. The amounts of every β- and δ-elimination product from the experiments in (B) were measured using the phosphorimager and plotted over time. An arrow indicates the time point when different temperatures (4°C or 65°C) were treated for the experiment in Figure 2. Error bars represent standard deviations from three independent experiments.