Title

Mutagenicity of N-(deoxyguanosin-8-yl)-1-aminopyrene, the major DNA adduct formed by 1-nitropyrene, in mammalian cells

Date of Completion

January 2008

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

N-(deoxyguanosin-8-yl)-1-aminopyrene (AP-dG) is the major DNA adduct formed in a cell following metabolism of 1-nitropyrene, a carcinogenic nitro-aromatic compound found in grilled food and environmental pollutants. AP-dG is reported to induce base substitutions, predominantly G-to-T transversions, DNA polymerase stalling, and frameshift mutations in Escherchia coli. The effects of AP-dG in mammalian cells have been the focus of my studies. The significance of this research is to determine the effect of sequence context on the mutagenicity of AP-dG in mammalian cells using a site-specific approach and to establish the involvement of translesional synthesis DNA polymerases processing of an AP-dG adduct in vitro. ^ A short fragment of DNA containing the adduct was chemically synthesized in three non-repetitive CGC, CGT, and mCGT sequences, where mC denotes 5-methylcytosine. The latter two sequences contained the local DNA sequence of the p53 codon 273, a mutational hotspot found in many types of human cancers. In simian kidney (COS-7) cells, the isolated progeny DNA from the AP-dG modified CGC sequence had shown an overall targeted mutation frequency of 4.8%, where the predominant mutation was a G-to-T transversion at the site originally occupied by AP-dG. The CGT sequence had given rise to several mutations with a frequency of 6.8%, in which the major mutations were G-to-T and G-to-A, both occurring at the AP-dG site. The mCGT sequence exhibited mutations similar to that of the unmethylated sequence; however, the frequency of targeted base substitutions (10.6%) in the mCGT sequence was significantly higher. In human embryonic kidney (293T/17) cells, the DNA from the CGT sequence contained several targeted mutations with a frequency of 5.1%, in which the major mutations were G-to-T and G-to-A. The mCGT sequence exhibited similar types of mutations, albeit the G-to-A transitions were more than 50% higher to that in the unmethylated sequence, but G-to-C transversions were also detected at a significant level. The AP-dG adduct in all three sequence contexts resulted in various mutations in both simian and human kidney cells. Although targeted G-to-T substitution was the major type of mutation in each case, a significant frequency of G-to-A transitions also occurred in the CGT and mCGT sequences. In summary, AP-dG was found to be mutagenic in simian and human kidney cells inducing primarily base substitutions, even though the mutational types and frequencies may be dependent upon the sequence context and the methylation status of the adjacent 5' cytosine. ^ Finally, in vitro studies with the Y Family of mammalian DNA polymerases eta (Pol η), iota (Pol ι), and kappa (Pol κ) were performed to determine their involvement in translesion synthesis of AP-dG. An AP-dG site-specifically modified 26mer oligonucleotide was annealed to a 32P labeled 12mer primer positioned three bases 3' to the AP-dG site. The TLS polymerase was added to the reaction mixture and incubated at varying time points from 30 sec to 2 h. The reaction products were analyzed on a denaturing polyacrylamide gel. Pol ι was able to incorporate a base opposite AP-dG but could not continue beyond the adduct site. However, Pol η and κ were able to yield full length extension products suggesting that they might be involved in translesion synthesis of AP-dG. Further studies will be needed to determine the error-free and error-prone bypass of Pol η and κ. ^