Cleavage and methylation of DNA by the restriction endonuclease HinfIII isolated from Haemophilus influenzae Rf

Abstract

HinfIII is a restriction enzyme isolated from Haemophilus influenzae strain Rf. It requires ATP for cleavage and S-adenosyl-methionine for methylation of DNA. This enzyme can be present in two forms: one with AdoMet † † Abbreviation used: AdoMet, S-adenosyl-methionine. bound to it, and a second form free of this cofactor. In the presence of AdoMet and ATP the enzyme cleaves ColE1 DNA molecules once, to produce unit-length linear molecules. The HinfIII endonuclease cleaves at unique sites, though not every site on every molecule is cut. The five HinfIII cleavage sites were mapped relative to the EcoRI restriction endonuclease cleavage site. If AdoMet is omitted from the enzyme reaction mixture, the second form of HinfIII enzyme cleaves ColE1 DNA into several fragments. An average of 6.2 ± 1 methyl groups are transferred to ColE1 DNA from AdoMet. The methyl groups were mapped relative to the HaeIII restriction endonuclease fragments. The position of methylation sites correlates well with the cleavage sites. The restriction activity of the HinfIII enzyme shows some dependence upon the structure of the substrate DNA. The linear molecule of ColE1 DNA is a poorer substrate than the supercoiled molecules. Lambda DNA fragments with molecular weights smaller than approximately 2 × 10 6 are not cleaved by HinfIII enzyme, but since they can be methylated the enzyme is able to recognize the specific sequences on these fragments.