5] Purification and assay of type II DNA methylases

Abstract

Publisher Summary This chapter discusses the purification and assay of type II deoxyribonucleic acid (DNA) methylases. Bacterial Type II DNA modification methylases can be readily prepared, because their sequence specificities are well defined and are generally stable monomers. Purification procedures for several Type II modification methylases are available. Generally, purifications have been based upon DNA protection assays. Specific DNA methylases are detected in those chromatographic fractions, which protect bacteriophage DNA from cleavage by a particular restriction endonuclease. A more sensitive method has been successfully used on a commercial scale for the purification of over 20 different DNA methylases. In this assay, a synthetic duplex oligonucleotide is self-ligated (polymerized) to an acid-precipitable form. A specific DNA methylase may then be measured radiometrically by the incorporation of 3 H-labeled S-adeno. It is useful to employ a combination of techniques during a particular DNA methylase purification: (1) electrophoretic protection assays, (2) specific [ 3 H]SAM, polymerized oligonucleotide linker incorporation assays, and (3) less specific [ 3 H]SAM, phage DNA incorporation assays. Generally, the most sensitive and specific linker assay is used in crude or early chromatographic fractions. Partially characterized DNA methylase fractions may then be assayed using less discriminating procedures. The chapter discusses the assay of DNA methylases, including polymerized oligonucleotide linker assay, the trichloroacetic acid (TCA) precipitation of reaction samples, DNA protection assay, and other related concepts. The chapter also presents a summary of DNA methylase purification.