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 (NmeA) and N3-methylcytosine (NmeC) by oxidative demethylation. Previous studies showed that AlkB preferentially removes NmeA and NmeC from single-stranded DNA (ssDNA). It can also remove NmeA and NmeC 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 NmeC adducts inasmuch as DNA length is shorter than 20 nucleotides. However, when longer NmeC-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 NmeC we demonstrate that SSB-AlkB interaction promotes faster repair of the methyl DNA adducts.