Abstract
A sensitive method for the assay of Ca2+,Mg2+-dependent endonuclease was developed. The assay procedure is composed of two parts: (i) microscale endonuclease digestion of highly polymerized calf thymus DNA and (ii) the quantification of DNA breaks by measuring the activation of poly(ADP-ribose) polymerase, which is known to be activated proportionally to the number of nicks and ends of DNA added in the reaction mixture. This method was approximately 105-fold more sensitive than a conventional DNase assay detecting acid-soluble DNA formation and, thus, the activity of 20 to 100 fg of purified bull seminal Ca2+,Mg2+-dependent endonuclease could be reliably measured. Ca2+and Mg2+requirements and the response to histone H2B of the endonuclease were also demonstrated by this method. Using this method, the assay of a very small amounts of Ca2+,Mg2+-dependent endonuclease in crude extracts of calf thymus chromatin was possible. This method may be applied to other types of endonucleases by modifying the mixture for endonuclease reaction.
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