Figure 5. In vitro reconstitution of the repair pathways for εA and αdA residues.

(A) In vitro reconstitution of the long-patch NIR pathway using an oligonucleotide duplex containing single εA residue. A solution of 10 nM of non-labelled 40 mer εA-PP•T oligonucleotide duplex was incubated for 3 h at 37°C in the presence of DNA repair proteins. Lane 1, 20 mer size marker; lane 2, εA-PP•T incubated with all proteins except APE1; lane 3, except FEN1; lane 4, except POLβ; lane 5, except ligase; lane 6, in the presence of all proteins; lane 7, 40 mer size marker. (B) Time dependent formation of the radioactively labelled full-length product during in vitro reconstitution of the BER and NIR pathways initiated by ANPG and APE1, respectively. The 40 mer εA-PP•T and 34 mer αdA•T duplexes were incubated with DNA repair proteins in the presence of [α-³²P]dATP. At defined time intervals, samples were withdrawn to stop the reaction, then the reaction products were separated by denaturing gel electrophoresis and the amounts of of 40 mer and 34 mer products were measured. Full-length product formation: in the BER pathway reconstitution using 40 mer εA-PP•T (upside-down filled triangle); in the NIR pathway reconstitution using 40 mer εA-PP•T (right-side up filled triangle) and 34 mer αdA•T (filled square). For details see Materials and Methods.