Sequence context modulation of translesion synthesis at a single N-2-acetylaminofluorene adduct located within a mutation hot spot.

Abstract

Oligonucleotides containing a single N-(deoxyguanosin-8-yl)acetylaminofluorene lesion (dGuo-C8-AAF) at each guanine residue of the sequence (5'-G1G2G3) have been used as templates for in vitro primer extension reactions by several DNA polymerases [Escherichia coli DNA polymerase III holoenzyme, its alpha subunit, DNA polymerase I Klenow fragment proficient (exo+) or deficient (exo-) in its 3' --> 5' exonuclease activity, and Sequenase]. The dGuo-C8-AAF lesion appears to be a strong block for all DNA polymerases: exo+ DNA polymerases stop one nucleotide before encountering the lesion, while partial incorporation opposite the lesion is observed only with enzymes devoid of the exonuclease activity. The efficiency of incorporation across from the adduct depends on both the DNA polymerase and the position of the lesion. When polymerase I Klenow fragment exo- is used, translesion synthesis (TLS) is observed with efficiencies varying according to the position of the adduct (G2 > G1 > G3). Sequencing of the TLS products shows that error-free TLS is observed only when the AAF lesion is bound to G1, while all TLS events occurring at G2- or G3-AAF adducts are mutagenic. The major mutational event is a G deletion (27, 76, and 55% of the events for G1, G2, and G3, respectively), while two-G deletions occur to a lesser extent (17-30%). These results are discussed in view of the slippage model developed for frameshift mutagenesis occurring during translesion synthesis at replication blocking lesions.