Figure 1.

E. coli Nei and Fpg proteins incise at m^N4,5C in DNA. (a) Structure of 5′-labelled dsDNA substrates for the determination of DNA glycosylase activity at modified cytosines (X) placed against different opposing bases (Y). Stars (*) indicate nuclease-resistant phosphorothioate bonds; [Cy3], cyanine3 fluorophore. (b) Nei exhibits activity towards m^N4,5C (but not C, m^N4C or m⁵C) when placed opposite G in DNA. Nei (8.3 pmol) was incubated with the DNA substrate (1 pmol) at 37°C for 1 h (final volume, 20 µl). (c) Nei exhibits the highest activity towards m^N4,5C when opposite G in DNA. For experimental details, see electronic supplementary material, figure S3. The average (±s.d.) of four independent experiments is presented. (d) Fpg exhibits the highest activity towards m^N4,5C when placed opposite C, with lower activity opposite T, in DNA. DNA substrate (1 pmol) was incubated alone (lanes 1–5) or with Fpg (13 pmol; lanes 6–9) at 37°C 1 h. U · G-DNA (60 nt; 1 pmol) incubated without (lane 10) and with hUNG, the latter followed by NaOH/heat treatment (lane 11), was used as negative and positive control for active hUNG, respectively, which was used to convert U · G-DNA into AP-DNA to demonstrate active Fpg (i.e. lyase activity; lane 12). Reaction products were analysed by denaturing PAGE at 200 V for 2 h. The average (±s.d.) of three independent experiments is presented in (d)(ii). (Online version in colour.)