Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coil (Exalt) protein AlkB removes various alkyl DNA adducts including N-1-methyladenine (NlmeA) and N3-methylcytosine (N(3)meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N(1)meA and N(3)meC from single-stranded DNA (ssDNA). It can also remove NimeA and N(3)meC from double-stranded DNA by base-flipping. Notably, ssDNA produced during DNA replication and recombination, remains bound to E. coli single-stranded DNA binding protein SSB and it is not known whether AIkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AIkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AIkB could efficiently remove N(3)meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N(3)meC-containing oligonuleotides were used as the substrate, efficiency of AIkB catalyzed reaction was abated compared to SSB-bound DNA substrate of identical length. Truncated SSB containing only the DNA binding domain could also support the stimulation of AlkB activity, suggesting the importance of SSB-DNA interaction for AlkB function. Using 70-mer oligonucleotide containing single N(3)meC we demonstrate that SSB-AIkB interaction promotes faster repair of the methyl DNA adducts. (C) 2018 Elsevier Inc. All rights reserved.