The HsdR subunit of R.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 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.
Zinkevich
V
V
Biophysics Laboratories, School of Biological Sciences, University of Portsmouth, St Michael's Building, White Swan Road, Portsmouth PO1 2DT, UK.
Popova
L
L
Kryukov
V
V
Abadjieva
A
A
Bogdarina
I
I
Janscak
P
P
Firman
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015870:Gene Expression; D005816:Genetic Complementation Test; D010957:Plasmids / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics / Q000378:metabolism
C072994:EcoR124 modification methylase type I; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D000251:Adenosine Triphosphatases