Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus UV-specific endonucleases.

L.K Gordon,W.A Haseltine

doi:10.1016/s0021-9258(19)70242-7

Abstract

A comparison was made of the activity of the UV-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultravilet light-irradiated DNA substrates of defined sequence. The two enzymes cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same, suggesting that the scission of DNA by T4 endonuclease V occurs via the combined actin of a pyrimidine dimer specific DNA glycosylase and an apyrimidinic-apurinic (AP) endonuclease as was recently shown for the M. luteus enzyme. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA.

Highlights

G15TBj at a dose rate of 7.5 J/m2/s and a total dose of 7500 J/m’
M. luteus uv-specific endonuclease was provided by Robert Grafstromand Lawrence Grossman (Johns Hopkins School of Public characterization of the mechanism of action of the M . luteus uv-specific endonuclease has shown that strsacnisdsion occurs by the combined action of two enzymatic activitieast the site of pyrimidine dimers, a pyrimidine dimer DNA glycosylase that cleaves the glycosylic bond between the5’-pyrimidine of the dimer and its corresponding sugar and an apyrimidinicapurinic endonuclease [8,9].Because
T o compare the sites of endonucleolyticcleavage of T4 endonuclease V to thoseof the M . luteus uv-specific endonuclease, a DNA fragment of defined sequence was used as the target for ultraviolet irradiation and subsequeenntdonuclease cleavage

Summary

Introduction

G15TBj at a dose rate of 7.5 J/m2/s and a total dose of 7500 J/m’. The reactions which contained M . luteus uv-specific endonuclease used the G-75 fraction from the purification procedure of Riazuddin and Grossman [4] in a reaction volume of 50 p1 of 0.01 M Tris, pH 7.4, 0.05 M NaCI, and 0.001 M Na2EDTA. Unirradiated DNA, treated with M. luteus uv-specific endonuclease. Lane 2, unirradiated DNA, treated with M. luteus uv-specific endonuclease and hydrolyzed (see "Experimental Procedures" for hydrolysisconditions). Lane 4, unirradiated DNA, treated with T4 endonuclease V and hydrolyzed.

Results

Conclusion