Electron Microscopic Studies on Substrate Specificity of T4 Excision Repair Endonuclease

Abstract

FOLLOWING infection of a suitable host by bacteriophage T4, a phage-specific endonuclease that makes single-strand breaks in ultraviolet-irradiated DNA^1,2 is produced. This enzyme is presumably involved in pyrimidine dimer excision, since a mutant phage T4ν₁, which does not produce detectable enzyme, is abnormally sensitive to ultraviolet light and fails to excise dimers from its DNA³. This endonuclease has been extensively purified⁴. Previous in vitro studies utilizing sedimentation velocity of DNA in sucrose density gradients showed ultraviolet dose-dependent reduction in sedimentation velocity of ultraviolet-irradiated T4 and T7 DNA following incubation with the purified enzyme. The sedimentation velocity of unirradiated DNA was not detectably reduced. Furthermore, the average number of enzymatically-induced nicks per molecule correlated well with the calculated number of dimers per molecule⁴. These studies suggested that the only substrate sites in ultraviolet-irradiated DNA were pyrimidine dimers. Unirradiated DNA was unaffected by the enzyme and it was concluded that such DNA either does not contain areas of distortion in the secondary structure such as might be produced by pyrimidine dimers, or if such distortions are present they are not recognized as substrate sites by the endonuclease.