Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli

Abstract

DNA from bacteriophage T7 was used to monitor the capacity of gently lysed extracts of E. coli to perform repair resynthesis after UV irradiation. Purified DNA damaged by up to 100 J/m2 of UV radiation was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. Incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original MW. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional to the number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymrease I since it does not occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.