Ada protein- and sequence context-dependent mutagenesis of alkyl phosphotriester lesions in Escherichia coli cells

Alkyl phosphotriester (alkyl-PTE) lesions are frequently induced in DNA and are resistant to repair. Here, we synthesized and characterized methyl (Me)- and n-butyl (nBu)-PTEs in two diastereomeric configurations (S_p and R_p) at six different flanking dinucleotide sites, i.e. XT and TX (X = A, C, or G), and assessed how these lesions impact DNA replication in Escherichia coli cells. When single-stranded vectors contained an S_p-Me-PTE in the sequence contexts of 5'-AT-3', 5'-CT-3', or 5'-GT-3', DNA replication was highly efficient and the replication products for all three sequence contexts contained 85-90% AT and 5-10% TG. Thus, the replication outcome was largely independent of the identity of the 5' nucleotide adjacent to an S_p-Me-PTE. Furthermore, replication across these lesions was not dependent on the activities of DNA polymerases II, IV, or V; Ada, a protein involved in adaptive response and repair of S_p-Me-PTE in E. coli, however, was essential for the generation of the mutagenic products. Additionally, the R_p diastereomer of Me-PTEs at XT sites and both diastereomers of Me-PTEs at TX sites exhibited error-free replication bypass. Moreover, S_p-nBu-PTEs at XT sites did not strongly impede DNA replication, and other nBu-PTEs displayed moderate blockage effects, with none of them being mutagenic. Taken together, these findings provide in-depth understanding of how alkyl-PTE lesions are recognized by the DNA replication machinery in prokaryotic cells and reveal that Ada contributes to mutagenesis of S_p-Me-PTEs in E. coli.