The HsdR subunit of R.EcoR124II: cloning and over-expression of the gene and unexpected properties of the subunit

• Published in: Nucleic acids research 1997-02, Vol.25 (3), p.503-511
• Main Authors: Zinkevich, V, Popova, L, Kryukov, V, Abadjieva, A, Bogdarina, I, Janscak, P, Firman, K
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Chromatography, Gel; Cloning, Molecular; Gene Expression; Genetic Complementation Test; Plasmids - metabolism; Site-Specific DNA-Methyltransferase (Adenine-Specific) - genetics; Site-Specific DNA-Methyltransferase (Adenine-Specific) - metabolism
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/25.3.503

Type I restriction endonucleases are composed of three subunits, HsdR, HsdM and HsdS. The HsdR subunit is absolutely required for restriction activity; while an independent methylase is composed of HsdM and HsdS 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 activity. Therefore, we have, for the first time, cloned and over-expressed the hsdRgene of the type IC restriction endonuclease EcoR124II. The purified HsdR subunit was found to be a soluble monomeric protein capable 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 bind 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 that the HsdR subunit of a type I restriction endonuclease is capable of independent enzyme activity, and suggests a mechanism for the evolution of the endonuclease from the independent methylase.