Action of T 4 Endonuclease V on Polydeoxyribonucleotides with Apyrimidinic or Apurinic Sites

Abstract

[³H] Thymine-labeled poly (dA)•poly (dT) carrying many apyrimidinic (AP) sites has been prepared by treating an enzymatically synthesized poly (dA)•poly (dT, dU) with uracil-DNA glycosylase. Incubation of the polymer with a homogeneous preparation of T 4 endonuclease V resulted in conversion of the labeled material into acidsoluble forms. Native DNA with apurinic sites was also cleaved by the enzyme. Single-stranded polymers, poly (dT) carrying AP sites or poly (dT) with thymine dimers, were barely attacked by T 4 endonuclease V. The polymer whose aldehyde moieties at AP sites were reduced to alcoholic forms was not susceptible to the enzyme. The site of endonucleolytic cleavage was determined by using alternating copolymers whose phosphate groups were differentially labeled. The result is consistent with the view that T 4 endonuclease V cleaves a phosphodiester linkage on the 3'-side of AP sites, producing chains terminated at their 3'-ends with base-free deoxyribose and at their 5'-ends with phosphate.