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

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  • 15 April 1989
    • journal article
    • research article
    • Vol. 264  (11) , 6355-6363
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.

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