The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43°C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.
ASJC Scopus subject areas
- Molecular Biology