Figure 5.

BER activities in proliferating and post-mitotic Ctrl and A-T neuronal cells. In vitro repair reactions were performed by using whole-cell extracts and as substrate a 32P-labeled circular plasmid containing a single AP site (pGEM-AP) to measure SP-BER (a) or a THF residue (pGEM-THF) for LP-BER (b). The correct insertion of a single lesion in the plasmid molecules was checked by digestion with uracil DNA glycosylase (UDG) followed by endonuclease III (NTH) for pGEM-AP (a), and by AP endonuclease (HAP1) for pGEM-THF (b). Repair kinetics of the AP site (a) or THF residue (b) by proliferating hNPCs and post-mitotic neurons (D15) were measured following incubation of the single lesion-containing plasmids with extracts from Ctrl or A-T defective neuronal cells for increasing periods of time. Repair products were analyzed by agarose gel electrophoresis and the radioactivity of the bands corresponding to nicked (form II) and supercoiled (form I) plasmids was quantified. Repair efficiency is expressed as relative amount of Form I over total radioactivity in each lane. Blue bar, Ctrl cell extracts; red bar, A-T cell extracts. The expression level of SP-BER and LP-BER proteins was evaluated by western blot in proliferating and post-mitotic neuronal cells (c). β-Actin was used as a loading control