Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence: A slipped frameshift intermediate model for dinucleotide deletion

Pablo Hilario, Shixiang Yan, Brian E. Hingerty, Suse Broyde, Ashis K. Basu

Research output: Contribution to journalArticle

Abstract

In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 (8)-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.

Original languageEnglish (US)
Pages (from-to)45068-45074
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number47
DOIs
StatePublished - Nov 22 2002

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1-nitropyrene
Mutagenesis
Guanine
Atrial Natriuretic Factor
Mutation Rate
Repair
DNA Polymerase III
Salmonella
DNA Mismatch Repair
Nucleic Acid Repetitive Sequences
Mutagens
Salmonella typhimurium
Hydrogen Bonding
Metabolism
Escherichia coli
Assays
Hydrogen bonds
Nucleotides
Stabilization
1,6-dinitropyrene

ASJC Scopus subject areas

  • Biochemistry

Cite this

Comparative mutagenesis of the C8-guanine adducts of 1-nitropyrene and 1,6- and 1,8-dinitropyrene in a CpG repeat sequence : A slipped frameshift intermediate model for dinucleotide deletion. / Hilario, Pablo; Yan, Shixiang; Hingerty, Brian E.; Broyde, Suse; Basu, Ashis K.

In: Journal of Biological Chemistry, Vol. 277, No. 47, 22.11.2002, p. 45068-45074.

Research output: Contribution to journalArticle

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abstract = "In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 (8)-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7{\%} CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0{\%} two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1{\%} by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.",
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AU - Hilario, Pablo

AU - Yan, Shixiang

AU - Hingerty, Brian E.

AU - Broyde, Suse

AU - Basu, Ashis K.

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N2 - In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 (8)-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.

AB - In the Ames Salmonella typhimurium reversion assay 1,6- and 1,8-dinitropyrenes (1,6- and 1,8-DNPs) are much more potent mutagens than 1-nitropyrene (1-NP). Genetic experiments established that certain differences in the metabolism of the DNPs, which in turn result in increased DNA adduction, play a role. It remained unclear, however, if the DNP adducts, N-(guanin-8-yl)-1-amino-6 (8)-nitropyrene (Gua-C8-1,6-ANP and Gua-C8-1,8-ANP), which contain a nitro group on the pyrene ring covalently linked to the guanine C8, are more mutagenic than the major 1-NP adduct, N-(guanin-8-yl)-1-aminopyrene (Gua-C8-AP). In order to address this, we have compared the mutation frequency of the three guanine C8 adducts, Gua-C8-AP, Gua-C8-1,6-ANP, and Gua-C8-1,8-ANP in a CGCG*CG sequence. Single-stranded M13mp7L2 vectors containing these adducts and a control were constructed and replicated in Escherichia coli. A remarkable difference in the induced CpG deletion frequency between these adducts was noted. In repair-competent cells the 1-NP adduct induced 1.7% CpG deletions without SOS, whereas the 1,6- and 1,8-DNP adducts induced 6.8 and 10.0% two-base deletions, respectively. With SOS, CpG deletions increased up to 1.9, 11.1, and 15.1% by 1-NP, 1,6-, and 1,8-DNP adducts, respectively. This result unequivocally established that DNP adducts are more mutagenic than the 1-NP adduct in the repetitive CpG sequence. In each case the mutation frequency was significantly increased in a mutS strain, which is impaired in methyl-directed mismatch repair, and a dnaQ strain, which carries a defect in proofreading activity of the DNA polymerase III. Modeling studies showed that the nitro group on the pyrene ring at the 8-position can provide additional stabilization to the two-nucleotide extrahelical loop in the promutagenic slipped frameshift intermediate through its added hydrogen-bonding capability. This could account for the increase in CpG deletions in the M13 vector with the nitro-containing adducts compared with the Gua-C8-AP adduct itself.

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