An investigation into the mutability of clustered DNA damage in E. coli

Full text: DNA clustered damage is the signature lesion induced by ionising radiation. Clustered damage is characterised by two or more DNA lesions occurring within two to three helical turns of the DNA. Recent studies in vitro have shown that clustered DNA damage is repaired less efficiently than isolated lesions due to the close proximity of other lesions. We have now used an in vivo protocol to assess the repairing of clustered DNA damage and its mutability in wild type and repair deficient mutants of E. coli. Synthetic 40mer oligonucleotides containing a BsmA1 restriction site were designed to include specifically altered bases that mimic the type of lesions caused by ionising radiation (8-oxoguanine and abasic sites (AP)). Plasmid constructs containing oligonucleotides with differently co-ordinated clustered damages were used to transform the repair deficient mutants for formamidopyrimidine DNA glycosylase (Fpg) and mutY glycosylase. Following a 16hr incubation at 37 deg C, retrieval of plasmid DNA and subsequent digestion with BsmA1, differential fragment patterns were found for repaired and mis-repaired DNA through agarose gel electrophoresis. The quantification of the amount of DNA in each fragment showed that even in the wild type bacteria the proximity of an AP site, up to 5 base pairs from the 8-oxoguanine on the opposite strand, caused the percentage of mis-repair to increase 2-3 fold compared with that of the 8-oxoguanine alone. These results were mirrored in the double mutant (fpg /mutY), where the differences were far more striking, with 35-40% of the cluster damages being mis-repaired. The results for the single mutants provided clear indication that mutY performs a more influential role than fpg when processing this particular type of clustered damage. It is proposed that the stalled repair of these types of clustered DNA damage results in the damage sites being processed during replication where the replication polymerases are error prone