Fig. 2. Effect of AID expression and UDG expression on DSB-induced mutagenesis.

(A), CAN1 mutation frequency was evaluated as a function of AID expression in wild type cells, DSB-induced cells (CANM, CANM ung1, and CANM pol32), and ung1 cells. The method of Maritz and Jarrett (Maritz and Jarrett, 1978) was used to determine confidence intervals for the median values of mutation frequency (Table II). Mann-Whitney test (Conover, 1976) was performed to calculate the P-values (Table III). The measurements shown are from 3 independent experiments, involving a total of ten replicates for each value. Standard errors are shown (see Tables II and III). (B), Immunoblotting analysis of cell extracts (20 μg), as noted in the Figure using anti-AID antibody. After SDS-PAGE and transfer of separated proteins to a membrane, the membrane was blotted with antibody to AID. Experiments were conducted as described under Materials and Methods. An arrow shows the region of the blot corresponding to the 24-kDa AID monomer. (C), Assays of two cell extracts (CANM and CANM ung1) noted in the Figure for uracil-DNA glycosylase enzymatic activity. Photograph of a phosphorimage is shown, after reaction mixtures were subjected to denaturing gel electrophoresis. Experiments were conducted as described under Materials and Methods. Arrows indicate the migration positions of the ³²P-labeled uracil-containing DNA substrate and the DNA product after uracil removal. The reaction mixtures were incubated without or with purified UDG (lanes 1 and 2, respectively), with cell extracts for wild type UNG1 or deleted UNG1 (CANM or CANM ung1, lanes 3 and 4, respectively), or with cell extracts in lane 4 with purified UDG (CANM ung1 + UDG, lane 5); reaction mixtures analyzed in lanes 1 to 5 were incubated for 5 min. Representative results are shown.