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© 1992 Oxford University Press

research-article

N-Acetylated and deacetylated 4'-fluoro-4-aminobiphenyl and 4-aminobiphenyl adducts differ in their ability to inhibit DNA replication of single-stranded M13 in vitro and of single-stranded {varphi}X174 in Escherichia coli

M.L.M. van de Poll, M.V.M. Lafleur 1, F. Van Gog, H. Vrieling 2 and J.H.N. Meerman 3

Division of Toxicology, Center for Bio-Pharmaceutical Sciences
1Department of Human Genetics of the Free University Amsterdam, The Netherlands
2Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratories, University of Leiden PO Box 9503, 2300 RA Leiden

3To whom all correspondence should be addressed

Calf thymus single-stranded (ss) DNA was modified with the N-sulfate conjugate of N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to yield predominantly N-acetylated adducts of 2-aminofluorene, 4-aminobiphenyl and 4'-fluoro-4-amino-biphenyl respectively to C8 of deoxyguanosine (dG-C8-AAF, dG-C8-AABP and dG-C8-FAABP). The modifed DNAs were used as templates for in vitro DNA synthesis. DNA replication on the randomly primed template was inhibited as compared to control (unmodified) DNA to the same extent by all three types ofadducts, irrespective of whether polymerization was performed by Escherichia coli DNA polymerase I, modified T7 DNA polymerase or Thermus aquaticus (Taq) DNA polymerase. In addition, all three types of adducts completely blocked replication of ss {varphi}X174 in an E.coli host: on average one adduct per DNA molecule was sufficient to inactivate the bacteriophage. Polyacrylamide gel electro-phoresis of DNA fragments synthesized by E.coli DNA polymerase I on FAABP- and AABP-modified ss M13mp9 DNA templates, showed that termination occurred predominantly one nucleotide before (and occasionally opposite) a modified deoxyguanosine in the template. However, the deacetylated adducts, dG-C8-AF, dG-C8-ABP and dG-C8-FABP (obtained by reacting DNA with their N-triftuoroacetyl-{varphi}-acetoxy esters) were frequently bypassed during replication of ss {varphi}X174 in E.coli, though with different efficiencies: 1 out of 7, 1 out of 2 and 1 out of 3 adducts on average respectively caused bacteriophage inactivation. Polyacrylamide gel electrophoresis showed that termination of DNA synthesis occurred at least as frequently opposite as 3' to a modified deoxyguanosine in the template.


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