The type I restriction endonuclease R.EcoR124I: over-production and biochemical properties.
In this paper we describe a two-plasmid system which allows over-production of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine (SAM), which has been described as a cofactor of type I restriction enzymes, is not required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage. This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM, as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes, namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1. Supercoiled DNA with one or two SR124I recognition sites is cleaved by the same mechanism inferring co-operation between specifically bound and excess enzymes. Nicked-circle DNA is an intermediate of cleavage reaction. Cleavage of DNA was inhibited by an increased degree of negative supercoiling, which may reflect an increased difficulty for the enzyme to translocate the DNA. Hemi-methylated DNA was the preferred substrate for methylation.
Janscak
P
P
Biophysics Laboratories School of Biological Sciences, University of Portsmouth, UK.
Abadjieva
A
A
Firman
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D015253:Deoxyribonucleases, Type I Site-Specific / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012435:S-Adenosylhomocysteine; D012436:S-Adenosylmethionine / Q000494:pharmacology
D004270:DNA, Circular; D004278:DNA, Superhelical; D012436:S-Adenosylmethionine; D000255:Adenosine Triphosphate; D004247:DNA; D012435:S-Adenosylhomocysteine; C048242:endodeoxyribonuclease EcoR124I; D015253:Deoxyribonucleases, Type I Site-Specific; D000251:Adenosine Triphosphatases; D008274:Magnesium