Mutagenic TLS through AAF within the 3G sequence or the NarI site in COS-7 and XP12RO cells.

<p>Frequencies of frameshift mutations were expressed as the ratio of the targeted mutant blue colonies to total transformants.</p

Analysis of Rad18 dependence of the G-AAF bypass in HCT116 cell-free extracts.

<p>HCT116 cell-free extracts, wild-type (WT) and Rad18−/− (20 mg), were incubated 30 min at 37°C in the presence of 10 fmoles of AAF-modified substrates either at the 3G sequence or at the NarI site in a final volume of 6.25 ml, as indicated. The samples were analysed by electrophoresis through a 8% denaturing polyacrylamide gel. L-1 is a product generated if synthesis is blocked one nucleotide before and opposite the lesion, respectively. TLS0 and TLS-1 or –2 are products from TLS via non-slipped and slipped intermediates, respectively.Quantitative analysis of experiments with two independent extracts are presented.</p

Analysis of Ub-PCNA dependence of G-AAF bypass in MRC5 cell-free extracts.

<p>Mock depleted (M) or PCNA depleted MRC5 cell extracts (20 mg) were incubated 30 min at 37°C in the presence of 10 fmoles of unmodified or modified substrates in a final volume of 6.25 ml, as indicated. Recombinant wild-type (WT) or mutated K164R PCNA (60 ng) was added to the reactions, as indicated. Aliquotes of the samples were analysed either by Western blot with an anti-PCNA antibody (panel A) or by 8% denaturing polyacrylamide gel electrophoresis (panel B). L-1 is a product generated if synthesis is blocked one nucleotide before the lesion. TLS0 and TLS-1 or –2 are products from TLS via non-slipped and slipped intermediates, respectively. FL are Full Length products.</p