Escherichia coli single-stranded DNA binding protein SSB promotes AlkB-mediated DNA dealkylation repair.

Repair of alkylation damage in DNA is essential for maintaining genome integrity. Escherichia coli (E.coli) protein AlkB removes various alkyl DNA adducts including N1-methyladenine (N1 meA) and N3-methylcytosine (N3 meC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes N1 meA and N3 meC from single-stranded DNA (ssDNA). It can also remove N1 meA and N3 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 AlkB can repair methyl adduct present in SSB-coated DNA. Here we have studied AlkB-mediated DNA repair using SSB-bound DNA as substrate. In vitro repair reaction revealed that AlkB could efficiently remove N3 meC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer N3 meC-containing oligonuleotides were used as the substrate, efficiency of AlkB 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 N3 meC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.