Site‐Specific N²‐dG DNA Adducts: Formation, Synthesis, and TLS Polymerase‐Mediated Bypass

Abstract

The N2‐position of deoxyguanosine (dG) in DNA is susceptible to modification by various damaging agents. These modifications (lesions or adducts) can stall the DNA replication by replicative polymerases, and if the common DNA repair pathways do not remove them, it can result in genomic instability. This generally leads to the death or oncogenic transformation of the cell. An important mechanism to deal with this problem is Translesion synthesis (TLS), a bypass mechanism, which involves the tolerance of DNA damage by low‐fidelity DNA polymerases (TLS polymerases). To understand the accuracy of TLS polymerases, a chemical biology approach is required. In this review, we discuss the reliable methods to synthesize specific N2‐dG DNA adducts and how they are tolerated by bacterial TLS polymerase Pol IV and its mammalian orthologue hpol κ. Molecular insights on the accurate bypass of these adducts emerge from the primer extension assays, X‐ray crystallographic, and molecular modeling studies are reviewed here.

Translesion Synthesis across the N2-Ethyl-deoxyguanosine Adduct by Human PrimPol

“…In this work, several N 2 -adducts of guanine were studied with PrimPol: N 2 -Et-dG, N 2 -Ib-dG, N 2 -Bn-dG, N 2 -Naph-dG, N 2 -Anth-dG, N 2 -Pyre-dG, and LG (Figure ). The formation and synthesis of these DNA adducts have been discussed previously. , …”

“…The formation and synthesis of these DNA adducts have been discussed previously. 23,47 To evaluate the ability of human PrimPol to synthesize past N 2 -dG adducts, primer extension reactions in the presence of Mg 2+ (10 mM) or Mn 2+ (1 mM) ions and 400 nM PrimPol were carried out. In the running start assay, PrimPol incorporated dNMPs opposite N 2 -ethyl-dG (+5 position upstream of the primer) but could not extend DNA synthesis beyond the lesion in Mg 2+ -reactions (Figure 2, lane 3).…”

“…The exocyclic N 2 -atom of guanine is well known to form stable DNA adducts upon reactions with multiple carcinogenic agents from the metabolites of aromatic amines, polycyclic aromatic hydrocarbons, − and aldehydes − Since the interaction of DNA polymerases with the minor groove of DNA is essential for its activity, N 2 -dG adducts, depending on their size and shape, can disrupt contacts of DNA polymerases with DNA and block the replication. ,− Many unrepaired bulky N 2 -dG adducts present a strong block to high-fidelity replicative DNA polymerases. , In contrast, a few DNA polymerases (e.g., Pol κ, Pol η, and Pol ι) efficiently bypass N 2 -dG adducts in vitro ,− via TLS DNA synthesis. In this work, we analyzed the DNA polymerase activity of PrimPol on DNA templates with seven N 2 -dG lesions of varying steric bulkiness and chemical nature (Figure ).…”

DNA Polymerase II Supports the Replicative Bypass of N2-Alkyl-2′-deoxyguanosine Lesions in Escherichia coli Cells

“…Exposure to endogenous and exogenous genotoxic agents can lead to the formation of various DNA lesions, many of which block replicative DNA polymerases and require TLS polymerases for their replicative bypass . A large body of literature revealed the roles of B- and Y-family polymerases in modulating the cytotoxic and mutagenic properties of various DNA lesions. , In this vein, Pol IV and Pol V were found to participate in error-free TLS and induce a −1 frameshift mutation at the site of an N 2 -dG adduct of benzo­[ a ]­pyrene; Pol II, however, is involved in bypassing the bulky N 2 -dG adduct of acetylaminofluorene and elicits a −2 frameshift mutation at the lesion site. , In addition, previous in vitro biochemical studies showed that purified Pol IV preferentially inserts a dCMP opposite N 2 -Et-dG, N 2 - i Bu-dG, and N 2 -CE-dG in template DNA, , though the kinetic parameters for nucleotide insertions opposite simple N 2 -alkyl-dG lesions were not measured. It will be important to determine, in the future, the efficiencies and fidelities of Pol II- and Pol IV-catalyzed nucleotide incorporation opposite the N 2 -alkyl-dG lesions.…”

“…18,19 In this vein, Pol IV and Pol V were found to participate in error-free TLS and induce a −1 frameshift mutation at the site of an N 2 -dG adduct of benzo[a]pyrene; Pol II, however, is involved in bypassing the bulky N 2 -dG adduct of acetylaminofluorene and elicits a −2 frameshift mutation at the lesion site. 20,21 In addition, previous in vitro biochemical studies showed that purified Pol IV preferentially inserts a dCMP opposite N 2 -Et-dG, N 2 -iBu-dG, and N 2 -CE-dG in template DNA, 11,22 though the kinetic parameters for nucleotide insertions opposite simple N 2 -alkyl-dG lesions were not measured. It will be important to determine, in the future, the efficiencies and fidelities of Pol II-and Pol IV-catalyzed nucleotide incorporation opposite the N 2 -alkyl-dG lesions.…”