ENZYMATIC ANALYSIS OF ISOMERIC TRITHYMIDYLATES CONTAINING ULTRAVIOLET LIGHT-INDUCED CYCLOBUTANE PYRIMIDINE DIMERS .1. NUCLEASE P1-MEDIATED HYDROLYSIS OF THE INTRADIMER PHOSPHODIESTER LINKAGE

Abstract

Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3''-phosphodiester group in the 5''-isomer (d-T < p > TpT) but was totally inactive toward the 3''-isomer (d-TpT < p > T). In contrast, calf spleen phosphodiesterase only operated on the 3''-isomer by cleaving the 5''-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5'' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3'' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T < p > TpT as with d-TpT < p > T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5''-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT < p > T. Both phosphate groups in d-T < p > TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T < p > TpT with nuclease P1, however, generated the novel compound dT < > d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.