Use of single-turnover kinetics to study bulky adduct bypass by T7 DNA polymerase.

The mechanism by which T7 DNA polymerase (exo-) bypasses N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) adducts was studied by single-turnover kinetics. These adducts are known to be mutagenic in several cell types, and their bypass was studied in the framework of understanding how they promote mutations. Synthetic primer/templates were made from a template sequence containing a single guanine, to which the adducts were covalently attached, and one of three primers whose 3' ends were various distances from the adduct in the annealed substrates. Upon approaching the site of either adduct, the polymerase was found to add nucleotides as rapidly as to unmodified primer/templates, until just opposite the lesion. The incorporation rate of dCTP (at 100 microM) opposite AF-dG or AAF-dG was approximately 5 x 10(4)- and 4 x 10(6)-fold slower, respectively, than incorporation at the same position into an unmodified primer/template. The polymerase dissociated from the sites of the adducts at approximately the same rate that it dissociated from unmodified DNA. Correct nucleotide incorporation was favored both opposite and immediately after AF-dG. However, at both positions, dATP was the most rapidly misincorporated nucleotide. Misincorporation of dATP was more rapid than correct nucleotide incorporation both opposite and immediately after AAF-dG. These results are discussed in terms of the effects of AF and AAF adducts in vivo.

[1]  T. Krugh,et al.  Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics. , 1993, Biochemistry.

[2]  K. Johnson.  Conformational coupling in DNA polymerase information transfer. , 1992, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[3]  I. Lambert,et al.  Carcinogen-induced frameshift mutagenesis in repetitive sequences. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. J. Culp,et al.  Replication of acetylaminofluorene-adducted plasmids in human cells: spectrum of base substitutions and evidence of excision repair. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[5]  L. Romano,et al.  Accurate in vitro translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorene-modified oligonucleotide. , 1991, Carcinogenesis.

[6]  A. Grollman,et al.  Isolation and characterization of oligodeoxynucleotides containing dG-N2-AAF and oxidation products of dG-C8-AF. , 1991, Carcinogenesis.

[7]  Smita S. Patel,et al.  Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. , 1991, Biochemistry.

[8]  Smita S. Patel,et al.  Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction. , 1991, Biochemistry.

[9]  K. Johnson,et al.  An induced-fit kinetic mechanism for DNA replication fidelity: direct measurement by single-turnover kinetics. , 1991, Biochemistry.

[10]  J. Wang,et al.  Role of DNA polymerase 3'----5' exonuclease activity in the bypass of aminofluorene lesions in DNA. , 1990, Carcinogenesis.

[11]  J. Mccormick,et al.  Mutations induced by aminofluorene-DNA adducts during replication in human cells. , 1989, Carcinogenesis.

[12]  D. Patel,et al.  NMR and computational characterization of the N-(deoxyguanosin-8-yl)aminofluorene adduct [(AF)G] opposite adenosine in DNA: (AF)G[syn].A[anti] pair formation and its pH dependence. , 1989, Biochemistry.

[13]  P. Koehl,et al.  Single adduct mutagenesis: strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[14]  P. Koehl,et al.  Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot. A new probe for frameshift mutagenesis. , 1989, Journal of molecular biology.

[15]  D. Johnson,et al.  Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct. , 1987, The Journal of biological chemistry.

[16]  M. Bichara,et al.  DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity. , 1985, Journal of molecular biology.

[17]  D. O'Connor,et al.  Site-specifically modified oligodeoxyribonucleotides as templates for Escherichia coli DNA polymerase I. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[18]  R. Fuchs.  DNA binding spectrum of the carcinogen N-acetoxy-N-2-acetylaminofluorene significantly differs from the mutation spectrum. , 1984, Journal of molecular biology.

[19]  M. Bichara,et al.  Carcinogen-induced mutation spectrum in wild-type, uvrA and umuC strains of Escherichia coli. Strain specificity and mutation-prone sequences. , 1984, Journal of molecular biology.

[20]  J. Gangloff,et al.  A quenched-flow apparatus which allows the measurement of the kinetics of a reaction in one stroke. , 1984, Journal of biochemical and biophysical methods.

[21]  E. Seeberg,et al.  pBR322 plasmid DNA modified with 2‐acetylaminofluorene derivatives: transforming activity and in vitro strand cleavage by the Escherichia coli uvrABC endonuclease. , 1984, The EMBO journal.

[22]  R. Fuchs,et al.  Different levels of induction of RecA protein in E. coli (PQ 10) after treatment with two related carcinogens. , 1983, Nucleic acids research.

[23]  R. Fuchs,et al.  Repair and mutagenesis of plasmid DNA modified by ultraviolet irradiation or N-acetoxy-N-2-acetylaminofluorene. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[24]  R. Fuchs,et al.  Hot spots of frameshift mutations induced by the ultimate carcinogen N- acetoxy-N-2-acetylaminofluorene , 1981, Nature.

[25]  M. Ptak,et al.  Conformation of acetylaminofluorene and aminofluorene modified guanosine and guanosine derivatives. , 1980, Biochemical and biophysical research communications.

[26]  R. Santella,et al.  Circular dichroism and proton magnetic resonance studies of dApdG modified with 2-aminofluorene and 2-acetyl-aminofluorene , 1980 .

[27]  F. Beland,et al.  Sensitivity of the conformation of deoxyguanosine to binding at the C-8 position by N-acetylated and unacetylated 2-aminofluorene. , 1980, Carcinogenesis.

[28]  E. Sage,et al.  Discrimination by antibodies between local defects in DNA induced by 2‐aminofluorene derivatives , 1979, FEBS letters.

[29]  C. Spelt,et al.  Differential excision from DNA of the C-8 deoxyguanosine reaction products of N-hydroxy-2-aminofluorene and N-acetoxy-N-acetyl-2-aminofluorene by endonuclease S1 from Aspergillus oryzae. , 1979, Cancer letters.

[30]  J. Lefèvre,et al.  Comparative orientation of the fluorene residue in native DNA modified by N-acetoxy-N-2-acetylaminofluorene and two 7-halogeno derivatives. , 1976, Biochemistry.

[31]  R. Fuchs,et al.  Physical studies on deoxyribonucleic acid after covalent binding of a carcinogen. , 1972, Biochemistry.

[32]  R. Fuchs,et al.  Changes of stability and conformation of DNA following the covalent binding of a carcinogen , 1971, FEBS letters.