Figure 4.

NEIL1’s CID is required for efficient repair of oxidized DNA lesions. (A) Repair of the 5-OHU-containing plasmid was monitored by the incorporation of ³²P-dTMP and analysis of a 32 nt Nt.BstNB1 restriction repaired fragment after denaturing gel electrophoresis [18,38]. (B) In vitro reconstitution of NEIL1-intitiated SN-BER with purified proteins (10 and 50 fmol WT NEIL1 [lanes 2 and 3] or N311mutant [lanes 4 and 5] and 50 fmol each of PNKP, Polβ, LigIIIα and XRCC1). 5'-³²P-labeled 32 and 20-mer oligos were used as size markers (lane 6). The histogram shows quantitation of repair. (C-E) FLAG-N311 IP, with reduced DNA glycosylase activity compared to FLAG-WT NEIL1 IP (D), was extremely deficient in overall repair (E). The DNA glycosylase activity was measured with 5-OHU-duplex oligo and complete repair with the plasmid substrate. FLAG levels in stably expressing cells were compared by Western analysis (C).