Abstract
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.
Highlights
Translesion synthesis (TLS) is a regulated and coordinated process in which the replacement of the high fidelity replicative DNA polymerase by one or several specialized TLS polymerases allows replication through DNA blocking lesions that have escaped repair
We present data showing that –1 and –2 frameshift mutations are observed in mammalian cells transfected with SV40-based shuttle vectors monomodified on the third guanine of each target DNA sequence
We clearly show that whereas Pol eta, along with Rad18 and monoubiquitinated PCNA, fully mediate the bypass of the AAF adduct located within the 3G sequence, these proteins only partially contribute to the bypass across the same lesion within the NarI site, unravelling a yet uncharacterized Rad18-independent TLS pathway
Summary
Translesion synthesis (TLS) is a regulated and coordinated process in which the replacement of the high fidelity replicative DNA polymerase by one or several specialized TLS polymerases allows replication through DNA blocking lesions that have escaped repair. In addition in E. coli, AAF adducts trigger hot spots (i) of –1 frameshift mutations when located at the third G in monotonous runs of G bases (5’-GGG-3’ 5’-GG-3’) and (ii) of – 2 frameshift mutations in regions of alternating GC base pairs, further referred to as the NarI site (5’-GGCGCC-3’ 5’-GGCC-3’) [17] Within these two particular DNA sequence contexts, insertion of a cytosine residue opposite the lesion increases the likelihood of forming slipped mispairs that, when elongated, give rise to frameshift products. We clearly show that whereas Pol eta, along with Rad and monoubiquitinated PCNA, fully mediate the bypass of the AAF adduct located within the 3G sequence, these proteins only partially contribute to the bypass across the same lesion within the NarI site, unravelling a yet uncharacterized Rad18-independent TLS pathway
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