Mutagenicity of the 1-Nitropyrene-DNA Adduct N-(Deoxyguanosin-8-yl)-1-aminopyrene in Mammalian Cells

Abstract

The mutagenesis of the major DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene (C8-AP-dG) formed by 1-nitropyrene was compared with the analogous C8-dG adducts of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) in simian kidney (COS-7) cells. The DNA sequence chosen for this comparison contained 5′-CCATCGCTACC-3′ that has been used for solution NMR investigations. The structural and conformational differences among these lesions are wellestablished (Patel et al., 1998). Accordingly, we found a notable difference in the viability of the progeny, which showed that the AAF adduct was most toxic and the AF adduct was least toxic with the AP adduct exhibiting intermediate toxicity. However, analysis of the progeny showed that translesion synthesis was predominantly error-free. Only low level mutations (<3%) were detected with G→T as the dominant type of mutation by all three DNA adducts. When C8-AP-dG was evaluated in a repetitive 5′-CGCGCG-3′ sequence, higher mutational frequency (~8%) was observed. Again, G→T was the major type of mutations in simian kidney cells, even though in bacteria CpG deletions predominate in this sequence (Hilario et al, 2002). Mutagenesis of C8-AP-dG in a 12-mer containing the local DNA sequence around codon 273 of the p53 tumor suppressor gene, where the adduct was located at the second base of this codon, was also investigated. In this 5′-GTGCGTGTTTGT-3′ site, the mutations were slightly lower but not very different from the progeny derived from the 5′-CGCGCG-3′ sequence. However, the mutational frequency increased by more than 50% when the 5′ C to the adduct was replaced with a 5-methylcytosine (5-MeC). With a 5-MeC, the most notable change in mutation was the enhancement of G→A, which occurred 2.5-times relative to a 5′ C. The C8-AP-dG adduct in codon 273 dodecamer sequence with a 5′ C or 5-MeC was also evaluated in human embryonic kidney (293T) cells. Similar to COS cells, targeted mutations doubled with a 5-MeC 5′ to the adduct. Except for an increase in G→C transversions, the results in 293T were similar to that in COS cells. We conclude that C8-AP-dG mutagenesis depends on the type of cell in which it is replicated, the neighboring DNA sequence, and the methylation status of the 5′ C.

DNA polymerase ζ limits chromosomal damage and promotes cell survival following aflatoxin exposure

“…The vectors were a single-stranded pMS2 carrying a sitespecific AFB 1 -Fapy-dG adduct and an undamaged pSP189 that served as an internal control. Both vectors can be replicated in HEK293T cells (28,29). Following replication in the HEK293T cells, the relative efficiency of replication was determined by The relative efficiencies of replication of single-stranded pMS2 vectors containing a site-specific AFB 1 -Fapy-dG adduct in HEK293T cells that were depleted for Rev3L by siRNA or treated with a control scrambled siRNA.…”

Mutagenicity of a Model DNA-Peptide Cross-Link in Human Cells: Roles of Translesion Synthesis DNA Polymerases

“…26,27 Control plasmids containing a dG, 7-deaza-dG, or 7-(2,3-dihydroxyprop-1-yl)-7-deaza-dG were also generated in a similar manner. The HEK 293T cells were grown to ~90% confluency and transfected with 50 ng of construct in 6 µL of Lipofectamine cationic lipid reagent (Invitrogen, Carlsbad, CA).…”

Mechanistic Studies of the Bypass of a Bulky Single-base Lesion Catalyzed by a Y-family DNA Polymerase

“…To better understand the mutagenic potential of 1-NP-induced DNA damage, a single-base lesion, dG AP , was placed specifically in a GC-rich region of a synthetic DNA template. Regions composed of repetitive DNA sequences, such as those in oncogenes, have been shown previously to induce more mutations than non-repetitive regions (33,49,50). The mechanistic basis of the bypass of this bulky dG AP lesion catalyzed by Dpo4 was comprehensively investigated using pre-steady-state kinetic methods.…”