Abstract
We have previously analyzed the effects of site-specific N-2-acetylaminofluorene (AAF) adducts on the efficiency and frameshift fidelity of SV40-based DNA replication in a human cell extract (Thomas, D. C., Veaute, X., Kunkel, T. A., and Fuchs, R. P. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7752-7756). Here we use two sets of substrates to examine the probability of replication termination and error-free and error-prone bypass of AAF adducts. The substrates contained site-specific adducts at one of three guanines in a NarI sequence (5'-GGCGCC-3') placed within the lacZ alpha reporter gene and located on the template for either leading or lagging strand replication. The presence of the adduct at any position strongly reduces the efficiency of a single round of replication in a HeLa cell extract. Product analysis reveals preferential replication of the undamaged strand and termination of replication of the damaged strand occurring one nucleotide before incorporation opposite either a leading or lagging strand adduct. Products resistant to restriction endonuclease cleavage at the adducted site were generated in amounts consistent with 16-48% lesion bypass during replication. Most of this bypass was error-free. However, two-nucleotide deletion errors were detected in the replication products of DNA containing an AAF adduct in either the leading or lagging strand, but only when present at the third guanine position. Collectively, the data suggest that the replication apparatus in a HeLa cell extract generates a template-primer slippage error at an AAF adduct once for every 30-100 bypass events.
Highlights
For several years, we have studied the effects of an encounter between the replication fork and the major adduct of a known carcinogen, N-2-acetylaminofluorene (AAF) located at one template position
When a ColE1-derived plasmid is replicated in E. coli, sitespecific AAF adducts induced both Ϫ1 and Ϫ2 frameshift mutations at a 20-fold higher frequency when the adduct is located on the lagging strand as compared to the leading strand (Veaute and Fuchs, 1993)
Measurement of Adduct Removal in the Extract—To more accurately quantitate lesion bypass efficiency and replication fidelity, we examined the extent of repair of AAF adducts by the same HeLa cell extract used for replication
Summary
Vol 270, No 36, Issue of September 8, pp. 21226 –21233, 1995 Printed in U.S.A. Frequency and Fidelity of Translesion Synthesis of Site-specific N-2-Acetylaminofluorene Adducts during DNA Replication in a Human Cell Extract*. We examined the efficiency and frameshift fidelity of replication of DNA containing a sitespecific AAF adduct present at one of the three guanine positions in the NarI sequence, each on the leading strand template relative to the first fork to encounter the adduct (Thomas et al, 1994). For this study we constructed two sets of four DNA substrates, each containing the SV40 replication origin and either no damage or a single, site-specific AAF adduct at one of the three guanines in a NarI sequence In both sets of substrates, the adducts are located asymmetrically relative to the origin of replication, such that they will be encountered first by either the leading or lagging strand replication apparatus. After replication in a human cell extract, we performed product analyses to quantitatively compare the probabilities of replication termination, error-free bypass and sequence context-dependent, error-prone bypass
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