Ada protein? and sequence context?dependent mutagenesis of alkyl phosphotriester lesions inEscherichia colicells

Alkyl phosphotriester (alkyl-PTE) lesions are frequently induced in DNA and are resistant to repair. Here, we synthesized and characterized methyl (Me)- andn-butyl (nBu)-PTEs in two diastereomeric configurations (S(p)andR(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 inEscherichia colicells. When single-stranded vectors contained anS(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 anS(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 ofS(p)-Me-PTE inE. coli, however, was essential for the generation of the mutagenic products. Additionally, theR(p)diastereomer of Me-PTEs atXT sites and both diastereomers of Me-PTEs at TXsites exhibited error-free replication bypass. Moreover,S-p-nBu-PTEs atXT sites did not strongly impede DNA replication, and othernBu-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 ofS(p)-Me-PTEs inE. coli.