Cytotoxic and mutagenic properties of minor-groove O2-alkylthymidine lesions in human cells

Jun Wu,Pengcheng Wang,Lin Li,Changjun You,Yinsheng Wang

doi:10.1074/jbc.ra118.003133

Jun Wu, Pengcheng Wang + Show 3 more

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https://doi.org/10.1074/jbc.ra118.003133

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Abstract

Endogenous metabolism, environmental exposure, and cancer chemotherapy can lead to alkylation of DNA. It has been well documented that, among the different DNA alkylation products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are inefficiently repaired. In the present study, we examined how seven O2-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu, or sBu, are recognized by the DNA replication machinery in human cells. We found that the replication bypass efficiencies of these lesions decrease with increasing length of the alkyl chain, and that these lesions induce substantial frequencies of T→A and T→G mutations. Replication experiments using isogenic cells deficient in specific translesion synthesis (TLS) DNA polymerases revealed that the absence of polymerase η or polymerase ζ, but not polymerase κ or polymerase ι, significantly decreased both the bypass efficiencies and the mutation frequencies for those O2-alkyldT lesions carrying a straight-chain alkyl group. Moreover, the mutagenic properties of the O2-alkyldT lesions were influenced by the length and topology of the alkyl chain and by TLS polymerases. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of O2-alkyldT lesions, and illustrate the roles of TLS polymerases in replicative bypass of these lesions in human cells.

Highlights

DNA is intrinsically unstable, where it may undergo spontaneous deamination or depurination under physiological conditions [1]
Similar to what we observed in E. coli cells [20], the O2-alkyldT lesions inhibit strongly DNA replication in mammalian cells, with the blockage effect increasing with the size and branching of the alkyl groups (Fig. 4a)
We found that the bypass efficiencies for all the O2-alkyldT lesions carrying a straight chain alkyl group were significantly reduced in cells depleted of Pol ␩ or Pol ␨ (Fig. 4a), suggesting that these two translesion synthesis (TLS) polymerases play important roles in bypassing these lesions in vivo

Summary

Introduction

DNA is intrinsically unstable, where it may undergo spontaneous deamination or depurination under physiological conditions [1]. Among the plethora of alkylated DNA damage products, minor-groove O2-alkylthymidine (O2-alkyldT) lesions are known to be poorly repaired In this respect, the POB and pyridylhydroxybutyl derivatives of guanine and thymine could be detected in various tissues of rats exposed with tobacco-derived N-nitrosamines [11,12,13,14]. Tissues of rats treated with DNA ethylating agents displayed much higher levels of O2-EtdT than O6-EtdG [15,16,17], and lymphocyte DNA of smokers exhibited elevated levels of O2-EtdT relative to that of nonsmokers [18] Together, these findings suggest that the minor-groove O2-alkyldT lesions are recalcitrant to repair [15,16,17]; it is important to examine how these lesions are recognized by DNA replication machinery

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