(A) Cells were continuously passaged and PDL was calculated using the following equation: PDL = [ln(Nt)-ln(N0*PE)]/ln2. Nt = Number of viable cells counted after passage; N0 = Number of cells seeded prior to passage; PE = plating efficiency. Mutant frequencies were measured for each mismatch repair proficient strain at the indicated PDL (diamonds, Pol εwt/wt; triangles, Pol εwt/exo-). Ten plates for each cell lines were seeded with 2 × 105 cells at each PDL into media containing 6-TG and grown for 12–14 days. Each 6TG-resistant clone was isolated, expanded and the HPRT1 ORF was sequenced. Mutant frequencies were calculated based on the number of unique HPRT1 mutations at each PDL. Pol εwt/wt PDL6.4 MF = 1.8 × 10−6, SEM = 2.7 × 10−6, n = 4; Pol εwt/exo- PDL6.6 MF = 4.1 × 10−6, SEM = 3.1 × 10−6, n = 3, p=0.6003. Pol εwt/wt PDL44.6 MF = 1.1 × 10−6, SEM = 2.3 × 10−6, n = 2; Pol εwt/exo- PDL47.9 MF = 3.2 × 10−6, SEM = 4.7 × 10−6, n = 8, p=0.9066. Pol εwt/wt PDL69 MF = 1.5 × 10−6, SEM = 1.6 × 10−6, n = 5; Pol εwt/exo- PDL71 MF = 3.7 × 10−6, SEM = 2.6 × 10−6, n = 5, p=0.4917. (B) Whole exome sequencing (30 × 106 bp, average 101x coverage) was performed on the indicated cell line at two defined population doubling levels, P0 and P69 or P71, as described in the Methods. P0 for each cell line was used as the matched normal cells to define only those mutations arising during the 70 or 71 population doublings. The fraction of each type of base pair substitution found unique to PDL 69 (for Pol εwt/wt) or PDL 71 (Pol εwt/exo-) was plotted and compared. Fisher’s exact tests were used to calculate p values. p=0.0002 (***p<0.001).