Purification, properties and determinations of recognition sequence and cleavage site of restriction endonuclease from "Agrobacterium gelatinovorum" IAM 12617, a marine bacterium (AgeI).
A new restriction endonuclease, designated as AgeI, was purified from cell-free extracts of a marine bacterium, "Agrobacterium gelatinovorum" IAM 12617 by streptomycin treatment, ammonium sulfate fractionation, combined column chromatographies on heparin-Sepharose CL-6B and DEAE-Sepharose CL-6B and FPLC on Mono Q (HR 5/5) and Superose 12 (HR 10/30). The purified enzyme was homogeneous on SDS-polyacrylamide gel disc electrophoresis and free from other phosphatase and exonuclease activities on ligation-recutting test. The relative molecular mass of the enzyme was 24,000 daltons by SDS-polyacrylamide gel disc electrophoresis. The gel filtration using Superose 12 (HR 10/30) gave the same calculation (23,000 daltons). These data indicated that the enzyme is a monomer. The isoelectric point of the enzyme was 6.5. The purified enzyme cleaved lambda and Ad2 DNAs at 10 or more and 5 sites, respectively. However, the purified enzyme did not cleave SV40, phi X174 RF I, M13mp 18 RF I or pBR322 DNAs. pBR328 DNA was cleaved at 1 site by the purified enzyme. The purified enzyme worked best at 37 degrees C and pH 7.5 in a reaction mixture (50 microliters) containing 1.0 micrograms lambda DNA, 10 mM Tris-HCl, 7 mM 2-mercaptoethanol, 7 mM MgCl2 and 50 mM NaCl. The purified enzyme did not require monovalent cations necessarily for the enzyme reaction. The enzyme recognized the palindromic hexanucleotide DNA sequence 5'-ACCGGT-3' and cut between A and C, producing a 5'-cohesive tetranucleotide extension.
Department of Agricultural Chemistry, Shizuoka University, Japan.
H
H
Mizuno
T
T
Suzuki
M
M
Akagawa
K
K
Yamasato
Y
Y
Yamada
D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D004262:DNA Restriction Enzymes / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012231:Rhizobium / Q000201:enzymology*
D002413:Cations, Divalent; D004279:DNA, Viral; D004262:DNA Restriction Enzymes