In Vitro Lesion Bypass Studies of O⁴-Alkylthymidines with Human DNA Polymerase η

Abstract

Environmental exposure and endogenous metabolism can give rise to DNA alkylation. Among alkylated nucleosides, O4-alkylthymidine (O4-alkyldT) lesions are poorly repaired in mammalian systems and may compromise the efficiency and fidelity of cellular DNA replication. To cope with replication-stalling DNA lesions, cells are equipped with translesion synthesis DNA polymerases that are capable of bypassing various DNA lesions. In this study, we assessed human DNA polymerase η (Pol η)-mediated bypass of various O4-alkyldT lesions, with the alkyl group being Me, Et, nPr, iPr, nBu, iBu, (R)-sBu, or (S)-sBu, in template DNA by conducting primer extension and steady-state kinetic assays. Our primer extension assay results revealed that human Pol η, but not human polymerases κ and ι or yeast polymerase ζ, was capable of bypassing all O4-alkyldT lesions and extending the primer to generate full-length replication products. Data from steady-state kinetic measurements showed that Pol η preferentially misincorporated dGMP opposite O4-alkyldT lesions with a straight-chain alkyl group. The nucleotide misincorporation opposite most lesions with a branched-chain alkyl group was, however, not selective, where dCMP, dGMP, and dTMP were inserted at similar efficiencies opposite O4-iPrdT, O4-iBudT, and O4-(R)-sBudT. These results provide important knowledge about the effects of the length and structure of the alkyl group in O4-alkyldT lesions on the fidelity and efficiency of DNA replication mediated by human Pol η.

Replication studies of carboxymethylated DNA lesions in human cells

“…The highly specific misincorporation of thymidine opposite N 3-CMdT is perhaps due to the placement of a carboxyl group at the Watson-Crick hydrogen bonding face, which may foster the base-pairing of the modified nucleobase with thymine (Figure 6 ). The misincorporation of dGMP opposite O 4 -CMdT can be attributed to the alteration of hydrogen bonding property of thymine conferred by O 4 carboxymethylation, as observed previously for this and other O 4 -alkylated thymidine lesions (Figure 6 ) ( 23 , 35 , 36 ).…”

Translesion synthesis ofO4-alkylthymidine lesions in human cells

“…It is also worth discussing the findings made from the present study in the context of recent in-vitro adduct bypass studies with the use of purified DNA polymerases (35). In this vein, O 4 -alkyldT lesions were found to block strongly the primer extension mediated by human Pol κ, Pol ι and Saccharomyces cerevisiae Pol ζ, though these lesions could be readily bypassed by human Pol η in vitro (35).…”

“…In this vein, O 4 -alkyldT lesions were found to block strongly the primer extension mediated by human Pol κ, Pol ι and Saccharomyces cerevisiae Pol ζ, though these lesions could be readily bypassed by human Pol η in vitro (35). These results are largely consistent with the observations made in the present study with a few exceptions.…”

Replicative Bypass of O2-Alkylthymidine Lesions in Vitro

“…In keeping with the findings made for the O 2 -alkyldT lesions in the current study, human Pol η was the only TLS polymerase capable of bypassing all the O 4 -alkyldT lesions and generating full-length extension product in vitro . 30 Additionally, human Pol η and ζ were found to promote the bypass of the O 4 -alkyldT lesions in HEK293T cells. 42 On the other hand, the nature of nucleotide misincorporation mediated by the polymerase is vastly different when bypassing the dT lesions with the same alkyl groups being attached to the O 2 and O 4 positions.…”

“…10 min). 30, 32, 33 The steady-state kinetic parameters (i.e. V max and K m ) for nucleotide incorporation were then determined by plotting V obs versus dNTP concentration and fit to the Michaelis-Menten equation using Origin 6.0 (Origin-Lab, Northampton, MA): 31 $$V_{\text{obs}}=\frac{V_{\text{max}}false[\mathrm{normald}\mathrm{normalN}\mathrm{normalT}\mathrm{normalP}false]}{K_{\mathrm{m}}+false[\mathrm{normald}\mathrm{normalN}\mathrm{normalT}\mathrm{normalP}false]}$$ …”