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PMID: 2843805 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Restriction endonuclease RsrI from Rhodobacter sphaeroides, an isoschizomer of EcoRI: purification and properties.

Nucleic acids research ·Vol. 16 ·No. 16 ·1988-08-25 ·Pages 7901-16

Aiken C, Gumport RI

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 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.

MeSH Terms
Amino Acid Sequence Catalysis Cellulose/analogs & derivatives Chromatography, DEAE-Cellulose DNA/analogs & derivatives DNA Restriction Enzymes/isolation & purification,metabolism Deoxyribonuclease EcoRI Durapatite Enzyme Activation Hydroxyapatites Isoelectric Point Kinetics Molecular Sequence Data Molecular Weight Polyethyleneimine Rhodobacter sphaeroides/enzymology
Chemicals
DNA-cellulose Hydroxyapatites Polyethyleneimine Cellulose DNA phosphocellulose Durapatite DNA Restriction Enzymes Deoxyribonuclease EcoRI
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Aiken C
Department of Biochemistry, College of Medicine, University of Illinois, Urbana 61801.
Gumport R I
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35 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/16.16.7901
Published
1988-08-25
Pages
7901-16
Language
English
Region
England
NLM ID
0411011
PMCID
PMC338499
Subset
IM
Grants
NIGMS NIH HHS · GM07283-11 · United States
NIGMS NIH HHS · GM25621 · United States
External Links
PubMed source DOI: 10.1093/nar/16.16.7901 PMC Full Text View journal details
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