FIGURE 7.

hnRNP-U enhances NEIL1-initiated repair. A, effect of hnRNP-U on NEIL1-initiated SN-BER in vitro. Repair of 5-OHU in DNA was analyzed using a 5-OHU-containing duplex oligonucleotide substrate (top) and purified proteins as indicated. SN-BER was measured by incorporation of [³²P]dCMP in a reaction containing 50 fmol each of NEIL1, polynucleotide kinase/phosphatase (PNKP), and DNA polymerase β (Polβ) and 100 fmol of DNA ligase IIIα (LigIIIα). Increasing amounts (10, 25, and 50 fmol) of hnRNP-U were included in lanes 3–5. Error bars indicate S.D. B, repair of 5-OHU in a plasmid with proteins isolated from NEIL1 IP. FLAG-NEIL1 was immunoprecipitated from HEK293 cells, with or without pretreatment with treatment with GO (50 ng/ml, 30 min) or transfection with hnRNP-U siRNA (see Fig. 8B for the extent of depletion) or appropriate controls as indicated. Error bars indicate S.D. C, repair of a 5-OHU residue incorporated in a plasmid (top) was compared for various extracts normalized for the FLAG level using Western analysis.