Restriction endonucleaseRsrI fromRhodobacter sphaeroides, an isoschizomer ofEcoRI: purification and properties

Abstract

We have purified RsrI endonuclease (R·RsrI), an isoschizomer of EcoRI, from Rhodobacter sphaeroides strain 630. The enzyme is homogeneous as judged by polyacrylamide gel electrophoresis and size-exclusion high-performance liquid chromatography. RsrI endonuclease is a dimer over the concentration range of 0.05 to 1.4 mg/ml. The reduced and denatured molecular weight of the enzyme is 30,000 Da. R·RsrI, like R·EcoRI, catalyzes the cleavage of duplex DNA and oligodeoxyribonucleotides between the first two residues of the sequence GAATTC. R·RsrI exhibits a K_M of 14 nM and a kcat of 6.5 min^.1 when reacting with pBR322 DNA at 25°C. R·RsrI differs from REcoRI in its N-terminal amino acid sequence, susceptibility to inhibition by antibodies, sensitivity to N-ethylmaleimide, isoelectric point, state of aggregation at high concentrations, temperature lability, and conditions for optimal reaction. R·RsrI displays a reduction of specificity (“star activity”) under conditions that also relax the specificity of R·EcoRI.