No abstract

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields -70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct ("13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced AumuD/C strain, both TLS events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.Most mutagens and carcinogens react with the bases of DNA by forming covalent adducts that interfere with DNA metabolism if left unrepaired. Repair pathways remove these lesions: excision repair removes damaged bases or nucleotides prior to replication and post-replication repair fills in gaps formed in newly synthesized DNA strands when damaged DNA is replicated. These two major repair pathways are believed to be essentially error-free and to contribute about equally to cell survival. The effect of DNA adducts on replication has been analyzed in in vitro assays using damaged single-stranded DNA templates and purified DNA polymerases (1-6). Studies of templates containing a single adduct have shown that some adducts are absolute blocks for in vitro DNA synthesis. For example, there is no in vitro trans-lesion synthesis (TLS) by most DNA polymerases through N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) (1), an adduct formed at the C8 position of guanine by the rodent hepatocarcinogen N-2-acetylaminofluorene (AAF) (7). However, in vivo doublestranded plasmids containing AAF adducts can replicate in excision-repair deficient hosts in an error-free or error-prone (mutagenic) manner (8-11). These observations confirm the fact that there are efficient mechanisms that rescue a blocked replication fork in vivo (12). Two such pathways are TLS involving a modified replisome and damage avoidance (DA) mechanisms that use ...

No abstract

To study the mechanisms of mutagenesis by the carcinogen N-acetyl-2-aminofluorene (AAF), we determined by DNA sequencing the spectrum of mutations in the Escherichia coli lacI gene induced by the ultimate metabolite N-acetoxy-N-acetyl-2-aminofluorene, using an E. coli derivative with increased permeability to this compound. Several different classes of mutations were recovered, including base substitutions (11%), single-base frameshifts (11%), double-base frameshifts (22%), deletions (21%), duplications (2%) and 'spontaneous hot-spot' mutations [26%; the gain (19%) or loss (7%) of TGGC at the sequence TGGCTGGCTGGC]. Among the base substitutions, both transitions and transversions occurred. The single-base frameshifts were all the loss of a base. The double-base frameshifts represented the loss of a (GpC) or (ApC) dinucleotide from alternating (GpC)n or (ApC)n sequences. The deletion, duplication and hot-spot mutations showed relative G,C-richness at their endpoints, suggesting that AAF-induced lesions at or near the endpoints promoted their occurrence. Taken together, the data are consistent with several previous findings on AAF mutagenesis, extending in particular the importance of frameshift mutagenesis at alternating purine-pyrimidine sequences and establishing deletion and duplication mutations as an important consequence of treatment with this carcinogen.