Termination of in vitro DNA synthesis at AAF adducts in the DNA

Moore, Peter D.; Rabkin, Samuel D.; Strauss, Bernard S.

doi:10.1093/nar/8.19.4473

Cited by 39 publications

(21 citation statements)

References 15 publications

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“…Adducts of AAF are also blocks to DNA synthesis (9) and, as previously reported (5), the principal termination bands for pol I are found (Fig. 2 than a 3' hydroxyl terminus, we conclude that the two types of termination pattern we have observed are due to the addition of an extra nucleotide opposite the guanyl-AAF adduct when synthesis is catalyzed by AMV reverse transcriptase.…”

supporting

confidence: 89%

“…DNA synthesis by either the E. coli DNA polymerase I large (Klenow) fragment (pol I) or the E. coli DNA polymerase III holoenzyme (pol III) is blocked by the presence of UV-induced pyrimidine dimers (6)(7)(8) or ofadducts ofN-acetylaminofluorene (AAF) (9) in the DNA template. pol I terminates synthesis one nucleotide before the site ofa pyrimidine dimer or guanine that has reacted with AAF (5).…”

mentioning

confidence: 99%

“…The preparation of DNA and the construction of templates have been described (5,9). Analysis on agarose gels showed the templates to be intact, suggesting little or no breakage at the sites of either AAF adducts or pyrimidine dimers in the DNA.…”

mentioning

confidence: 99%

“…A maximum estimate for the frequency ofbypass can be obtained from a determination of the relative amounts of termination at successive lesions. When termination is at-a guanyl-AAF adduct, for example, the proportion ofchains terminating up to position n in a sequence containing N guanines is (1 _ Pn)/(l -pN), where P is the probability of proceeding past a guanine, as determined from the number of AAF residues per guanine in the molecule (9). The best fit of the data from five of our experiments (9) is to a curve having 100% termination at each lesion, but the standard deviation (11-14% of the mean for the first six (16), and all the positions of termination occur at or near these sites.…”

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confidence: 99%

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Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

Moore¹,

Bose²,

Rabkin³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

165

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In vitro DNA synthesis on a OX174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol l) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase a from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3'-5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase a preparations. On N-acetylaminofluorene templates, pol I, pol Ill, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase a-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern ofpol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3'--5' exonuclease activity associated with many polymerases.Induced mutagenesis is a process that occurs when the DNA synthetic machinery ofa cell approaches a damaged nucleotide. In Escherichia coli, an inducible mechanism, the SOS repair pathway, plays a crucial role in mutagenesis (1, 2). In some way, the process of bypassing a lesion results in mutation. Because in vivo experiments are difficult to interpret mechanistically, we have adapted an in vitro system-4X174 DNA primed by a single restriction fragment-as a useful model system for DNA synthesis. Using this model, others (3, 4) have carried out a transfectional analysis of the production of mutation, and we have determined the biochemical nature of the molecules synthesized on damaged templates (5).DNA synthesis by either the E. coli DNA polymerase I large (Klenow) fragment (pol I) or the E. coli DNA polymerase III holoenzyme (pol III) is blocked by the presence of UV-induced pyrimidine dimers (6-8) or ofadducts ofN-acetylaminofluorene (AAF) (9) in the DNA template. pol I terminates synthesis one nucleotide before the site ofa pyrimidine dimer or guanine that has reacted with AAF (5). There are two distinct mechanisms that might be responsible for termination; (i) either the enzyme is unable to insert a nucleotide opposite a damaged base or (ii) an inserted nucleotide is rapidly removed by the 3'---5' exonuclease editing function associated with the polymerase. The second of these explanations is favored by Villani et aL (8), who have suggested that inhibition ofexonuclease activity could permit a polymerase to bypass a lesion in the DNA. This would provide a simple mechanism for the inducible error-prone repair believed to be responsible for the bulk of mutagenesis following UV irradiation (1, 2). A test of this hypothesis would be to distinguish the contributions of polymerase and nuclease functions in the process of termin...

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supporting

confidence: 89%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

Moore¹,

Bose²,

Rabkin³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

165

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“…Nonacetylated adducts, however, were less effective: on average two to seven adducts were needed for complete inactivation. In addition, termination of in vitro replication by DNA polymerase I (Klenow fragment) of single stranded M13mp9 DNA modified with dG-C8-AAF, dG-C8-FAABP, and dG-C8-AABP adducts always occured before a modified base, whereas replication of M13 DNA modified with dG-C8-AF, dG-C8-FABP, and dG-C8-ABP terminated before as well as opposite an adduct (32,33). These data suggest that all dG-C8-acetylarylamine adducts may block DNA replication equally effectively and that dG-C8-arylamine adducts are less effective.…”

Section: Blocking Of Dna Replication By Dg-c8-acetylarylamine and Dg-mentioning

confidence: 99%

Metabolic activation routes of arylamines and their genotoxic effects.

Meerman

Poll

1994

Environ Health Perspect

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Two different types of DNA adducts are formed from many aromatic amines by bioactivation: N-acetylated and nonacetylated, arylamine DNA adducts. It has become clear from experiments using N-acetyl-2-aminofluorene and 2-aminofluorene adducts to C8 of deoxyguanosine that these two types of adducts may have different effects on DNA structure and DNA replication. We have determined blocking of DNA replication by various other N-acetylarylamine and arylamine deoxyguanosine adducts. It was found that the N-acetyl group in general is required for blocking of DNA replication; the nature of the aromatic moiety seems to be of minor importance. Little information is available on the genotoxic effects of these adducts in mammalian cells in vivo. We have tried to get more insight in this by investigating the clastogenicity, the initiation of preneoplastic cells, and the promotional effects of various aromatic amines from which different ratios of N-acetylarylamine DNA adducts to arylamine DNA adducts are formed in the rat liver. Our results show that formation of N-acetylarylamine adducts to C8 of deoxyguanosine in the liver is correlated with clastogenicity and hepatic promoting effect. Initiation capacities, however, seem to be correlated with formation of nonacetylated, arylamine adducts. Mechanisms by which formation of N-acetylarylamine DNA adducts may generate a promoting effect in the liver are discussed. -Environ Health Perspect 102(Suppl 6): 153-159 (1994)

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Mutagenesis from a Chemical Perspective: Nucleic Acid Reactions, Repair, Translation, and Transcription

Singer

1982

Molecular and Cellular Mechanisms of Mutagenesis

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Termination of in vitro DNA synthesis at AAF adducts in the DNA

Cited by 39 publications

References 15 publications

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

Metabolic activation routes of arylamines and their genotoxic effects.

Mutagenesis from a Chemical Perspective: Nucleic Acid Reactions, Repair, Translation, and Transcription

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