Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties.
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 KM of 14 nM and a kcat of 6.5 min-1 when reacting with pBR322 DNA at 25 degrees C. R.RsrI differs from R.EcoRI 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.
Aiken
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C
Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801.
Gumport
R I
RI
D000595:Amino Acid Sequence; D002384:Catalysis; D002482:Cellulose / Q000031:analogs & derivatives; D002848:Chromatography, DEAE-Cellulose; D004247:DNA / Q000031:analogs & derivatives; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D015246:Deoxyribonuclease EcoRI; D017886:Durapatite; D004789:Enzyme Activation; D006882:Hydroxyapatites; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011094:Polyethyleneimine; D012242:Rhodobacter sphaeroides / Q000201:enzymology*
C033190:DNA-cellulose; D006882:Hydroxyapatites; D011094:Polyethyleneimine; D002482:Cellulose; D004247:DNA; C008881:phosphocellulose; D017886:Durapatite; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI