Cytotoxic and mutagenic properties of O4-alkylthymidine lesions in Escherichia coli cells

Abstract

Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O(4)-alkylthymidine (O(4)-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O(4)-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O(4)-alkyldT lesions and examined their impact on DNA replication in Escherichia coli cells. We showed that the replication past all the O(4)-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of the O(4)-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O(4)-alkyldT lesions, Ogt was able to repair O(4)-MedT and, to a lesser extent, O(4)-EtdT and O(4)-nPrdT, but not other O(4)-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O(4)-alkyldT lesions and their recognition by the E. coli replication machinery.

Scheme 1.

Structures of the O⁴-alkyldT lesions examined in the present study.

Scheme 2.

Syntheses of phosphoramidite building blocks of O⁴-alkylthymidine^α. ^αReagents and conditions: (a) ROH, DBU, r.t., 10 h; (b) TBAF, THF, r.t., 1 h; (c) DMTr-Cl, DMAP, pyridine, r.t., 10 h; (d) 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite, DIEA, CH₂Cl₂, 1 h.

Figure 1.

Native PAGE (30%) for monitoring the bypass efficiencies and mutation frequencies of O⁴-alkyldT in SOS-induced wild-type (WT) AB1157 Escherichia coli cells. (A) Sequential restriction enzyme digestion for the selective labeling of the strand initially bearing the lesion or the complementary strand. ‘SAP’ and ‘PNK’ designate shrimp alkaline phosphatase and T4 polynucleotide kinase, respectively. (B) Gel image showing the 13-mer and 10-mer products released from the bottom-strand (opposite to lesion-containing strand) of the PCR products of the progeny of the competitor genome and the control or lesion-carrying genome, where 10mer A, 10mer C, 10mer G and 10mer T represent the [5′-³²P]-labeled standard ODNs 5′-AATTATAGCN-3′, with ‘N’ being A, C, G and T, respectively. (C) Gel image showing the 13-mer and 10-mer products released from the top-strand (lesion-containing strand) of the PCR products of the progeny of the competitor genome and the control or lesion-carrying genome, where 10mer A, 10mer C, 10mer G and 10mer T represent the [5′-³²P]-labeled standard ODNs 5′-GGCGMGCTAT-3′, with ‘M’ being A, C, G and T, respectively.

Figure 2.

The bypass efficiencies (A) and mutation frequencies (B) of the O⁴-alkyldT lesions in AB1157 Escherichia coli strains that are proficient in translesion synthesis or deficient in Pol II, Pol IV, Pol V or all three SOS-induced DNA polymerases. The data represent the means and standard deviations of results from three independent replication experiments.

Figure 3.

The bypass efficiencies (A) and and mutation frequencies (B) of the O⁴-alkyldT lesions in SOS-induced Escherichia coli cells that are proficient in O⁶-alkylguanine-DNA alkyltransferases or deficient in Ogt (ΔOgt), both Ada and Ogt (ΔAda, ΔOgt), or Ada, AlkB and Ogt (ΔAda, ΔAlkB, ΔOgt). The data represent the means and standard deviations of results from three independent replication experiments.