In Vitro Replication Studies of Carboxymethylated DNA Lesions with Saccharomyces cerevisiae Polymerase η

Abstract

Humans are exposed to N-nitroso compounds (NOCs) both endogenously and exogenously from a number of environmental sources, and NOCs are both mutagenic and carcinogenic. After metabolic activation, some NOCs can induce carboxymethylation of nucleobases through a diazoacetate intermediate, which could give rise to similar p53 mutations as those seen in human gastrointestinal cancers. It was previously found that the growth of polymerase η-deficient human cells was inhibited by treatment with azaserine, a DNA carboxymethylation agent, suggesting the importance of this polymerase in bypassing the azaserine-induced carboxymethylated DNA lesions. In the present study, we examined how carboxymethylated DNA lesions, which included N6-carboxymethyl-2′-deoxyadenosine (N6-CMdA), N4-carboxymethyl-2′-deoxycytidine (N 4-CMdC), N3-carboxymethylthymidine (N3-CMdT) and O4-carboxymethylthymidine (O4-CMdT), perturbed the efficiency and fidelity of DNA replication mediated by Saccharomyces cerevisiae polymerase η (pol η). Our results from steady-state kinetic assay showed that pol η could readily bypass and extend past N6-CMdA, and incorporated the correct nucleotides opposite the lesion and its neighboring 5′ nucleoside with high efficiency. By contrast, the polymerase could bypass N4-CMdC inefficiently, with substantial misincorporation of dCMP followed by dAMP, though pol η could extend past the lesion with high fidelity and efficiency when dGMP was incorporated opposite the lesion. On the other hand, yeast pol η experienced great difficulty in bypassing O4-CMdT and N3-CMdT and the polymerase inserted preferentially the incorrect dGMP opposite these two DNA lesions; the extension step, nevertheless, occurred with high fidelity and efficiency when the correct dAMP was opposite the lesion, as opposed to the preferentially incorporated incorrect dGMP. The above results suggest that these lesions may contribute significantly to diazoacetate-induced mutations and those in p53 gene observed in human gastrointestinal tumors.

Kinetic analysis of bypass of abasic site by the catalytic core of yeast DNA polymerase eta

“…Inactivation of Pol Á in S. cerevisiae and humans results in a increased UVinduced mutagenesis [15]. Pol Á is also capable of bypassing a broad spectrum of other bulky DNA lesions with varying degrees of efficiency and fidelity [6,[16][17][18][19].…”

Kinetic analysis of bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by the catalytic core of yeast DNA polymerase η

“…DNA replication is generally performed by DNA polymerases with high fidelity. In most cases, the progress of the polymerases is impeded upon encounter of endogenous or exogenous DNA damage [1][2]. Reactive oxygen species are a major source of DNA damage.…”

“…Compared to replicative DNA polymerases, TLS polymerases have relatively larger active sites, allowing them to accommodate mismatched base pairs and bulky DNA lesions at the cost of a lower fidelity [1, 2]. …”

“…Inactivation of Pol η in S. cerevisiae and humans leads to increased UV-induced mutagenesis [12]. For example, a variant form (V) of xeroderma pigmentosum, characterized by an elevated incidence of sunlight-induced skin cancers, was reported due to lack of Pol η in humans [2]. In addition to CPDs, Pol η can also bypass many other DNA lesions with varying degrees of efficiency and fidelity [2, 13–15].…”

“…For example, a variant form (V) of xeroderma pigmentosum, characterized by an elevated incidence of sunlight-induced skin cancers, was reported due to lack of Pol η in humans [2]. In addition to CPDs, Pol η can also bypass many other DNA lesions with varying degrees of efficiency and fidelity [2, 13–15]. When encountering bulky adducts (e.g., dG-C8-AAF and cisplatin-GG lesions) human and yeast Pol η can incorporate nucleotides opposite the sites at reduced rates [1, 16, 17].…”