Evidence for a Rad18-Independent Frameshift Mutagenesis Pathway in Human Cell-Free Extracts

Bypass of replication blocks by specialized DNA polymerases is crucial for cell survival but may promote mutagenesis and genome instability. To gain insight into mutagenic sub-pathways that coexist in mammalian cells, we examined N-2-acetylaminofluorene (AAF)-induced frameshift mutagenesis by means of SV40-based shuttle vectors containing a single adduct. We found that in mammalian cells, as previously observed in E. coli, modification of the third guanine of two target sequences, 5'-GGG-3' (3G) and 5'-GGCGCC-3' (NarI site), induces –1 and –2 frameshift mutations, respectively. Using an in vitro assay for translesion synthesis, we investigated the biochemical control of these events. We showed that Pol eta, but neither Pol iota nor Pol zeta, plays a major role in the frameshift bypass of the AAF adduct located in the 3G sequence. By complementing PCNA-depleted extracts with either a wild-type or a non-ubiquitinatable form of PCNA, we found that this Pol eta-mediated pathway requires Rad18 and ubiquitination of PCNA. In contrast, when the AAF adduct is located within the NarI site, TLS is only partially dependent upon Pol eta and Rad18, unravelling the existence of alternative pathways that concurrently bypass this lesion

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

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