Error-Prone and Error-Free Translesion DNA Synthesis over Site-Specifically Created DNA Adducts of Aryl Hydrocarbons (3-Nitrobenzanthrone and 4-Aminobiphenyl)

Abstract

Aryl hydrocarbons such as 3-nitrobenzanthrone (NBA), 4-aminobiphenyl (ABP), acetylaminofluorene (AAF), benzo(a)pyrene (BaP), and 1-nitropyrene (NP) form bulky DNA adducts when absorbed by mammalian cells. These chemicals are metabolically activated to reactive forms in mammalian cells and preferentially get attached covalently to the N² or C8 positions of guanine or the N⁶ position of adenine. The proportion of N² and C8 guanine adducts in DNA differs among chemicals. Although these adducts block DNA replication, cells have a mechanism allowing to continue replication by bypassing these adducts: translesion DNA synthesis (TLS). TLS is performed by translesion DNA polymerases-Pol η, κ, ι, and ζ and Rev1-in an error-free or error-prone manner. Regarding the NBA adducts, namely, 2-(2'-deoxyguanosin-N²-yl)-3-aminobenzanthrone (dG-N²-ABA) and N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-ABA), dG-N²-ABA is produced more often than dG-C8-ABA, whereas dG-C8-ABA blocks DNA replication more strongly than dG-N²-ABA. dG-N²-ABA allows for a less error-prone bypass than dG-C8-ABA does. Pol η and κ are stronger contributors to TLS over dG-C8-ABA, and Pol κ bypasses dG-C8-ABA in an error-prone manner. TLS efficiency and error-proneness are affected by the sequences surrounding the adduct, as demonstrated in our previous study on an ABP adduct, N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl (dG-C8-ABP). Elucidation of the general mechanisms determining efficiency, error-proneness, and the polymerases involved in TLS over various adducts is the next step in the research on TLS. These TLS studies will clarify the mechanisms underlying aryl hydrocarbon mutagenesis and carcinogenesis in more detail.

Keywords: 3-Nitrobenzanthrone; Aryl hydrocarbon; DNA adduct; Mutation; Translesion DNA synthesis.

Fig. 1

Structures of dG-C8-ABA, dG-N²-ABA, dA-N⁶-ABA, and dG-C8-ABP.

Fig. 2

Methods for the construction of site-specifically dG-C8-ABP-adducted plasmids (A) and the in vivo TLS reporter assay (B). To construct NBA-adducted plasmids, CCTTCCG*TCTCCC is used for dG-N²-ABA and dG-C8-ABA, and CCTTCCA*TCTCCC is applied for dAN⁶-ABA (G* and A* are ABA-adducted bases).

Fig. 3

Rates of TLS over different NBA adducts (A), and frequencies of mutations of the adducted bases (B). The NBA adducts, dG-N²-ABA and dG-C8-ABA are formed in CCTTCCG*TCTCCC, while dA-N⁶-ABA is formed in CCTTCCA*TCTCCC (G* and A* are adducted bases).

Fig. 4

Rates of TLS over ABP adducts formed on guanine in different sequences (A), and frequencies of mutations of the adducted bases (B) in XPA cells and those overexpressing Pol η. dG-C8-ABP is formed in CCG*GAGGC and CCGGAG*GCC (G* is the adducted base), which are codon 248 and 249 sequences, respectively, of the human p53 tumor suppressor gene.