THE HSDR SUBUNIT OF R-CENTER-DOT-ECOR124II - CLONING AND OVER-EXPRESSION OF THE GENE AND UNEXPECTED PROPERTIES OF THE SUBUNIT

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS, The HsdR subunit is absolutely required for restrictio n activity; while an independent methylase is composed of HsdM and Hsd S subunits. DNA cleavage is associated with a powerful ATPase activity during which DNA is translocated by the enzyme prior to cleavage, The presence of a Walker type I ATP-binding site within the HsdR subunit suggested that the subunit may be capable of independent enzymatic act ivity, Therefore, we have, for the first time, cloned and over-express ed the hsdR gene of the type IC restriction endonuclease EcoR124II. Th e purified HsdR subunit was found to be a soluble monomeric protein ca pable of DNA- and Mg2+-dependent ATP hydrolysis. The subunit was found to have a weak nuclease activity both in vivo and in vitro, and to bi nd plasmid DNA; although was not capable of binding a DNA oligoduplex. We were also able to reconstitute the fully active endonuclease from purified M EcoR124I and HsdR. This is the first clear demonstration th at the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evoluti on of the endonuclease from the independent methylase.