S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes.
The requirement of S-adenosyl-L-methionine (AdoMet) in the cleavage reaction carried out by type III restriction-modification enzymes has been investigated. We show that DNA restriction by EcoPI restriction enzyme does not take place in the absence of exogenously added AdoMet. Interestingly, the closely related EcoP15I enzyme has endogenously bound AdoMet and therefore does not require the addition of the cofactor for DNA cleavage. By employing a variety of AdoMet analogs, which differ structurally from AdoMet, this study demonstrates that the carboxyl group and any substitution at the epsilon carbon of methionine is absolutely essential for DNA cleavage. Such analogs could bring about the necessary conformational change(s) in the enzyme, which make the enzyme proficient in DNA cleavage. Our studies, which include native polyacrylamide gel electrophoresis, molecular size exclusion chromatography, UV, fluorescence and circular dichroism spectroscopy, clearly demonstrate that the holoenzyme and apoenzyme forms of EcoP15I restriction enzyme have different conformations. Furthermore, the Res and Mod subunits of the EcoP15I restriction enzyme can be separated by gel filtration chromatography in the presence of 2 M NaCl. Reconstitution experiments, which involve mixing of the isolated subunits, result in an apoenzyme form, which is restriction proficient in the presence of AdoMet. However, mixing the Res subunit with Mod subunit deficient in AdoMet binding does not result in a functional restriction enzyme. These observations are consistent with the fact that AdoMet is required for DNA cleavage. In vivo complementation of the defective mod allele with a wild-type mod allele showed that an active restriction enzyme could be formed. Furthermore, we show that while the purified c2-134 mutant restriction enzyme is unable to cleave DNA, the c2-440 mutant enzyme is able to cleave DNA albeit poorly. Taken together, these results suggest that AdoMet binding causes conformational changes in the restriction enzyme and is necessary to bring about DNA cleavage.
Bist
P
P
Department of Biochemistry, Indian Institute of Science, Bangalore, 560 012, India.
Sistla
S
S
Krishnamurthy
V
V
Acharya
A
A
Chandrakala
B
B
Rao
D N
DN
D000483:Alleles; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015263:Deoxyribonucleases, Type III Site-Specific / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D010777:Photochemistry; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis
D001051:Apoenzymes; D003067:Coenzymes; D020035:Holoenzymes; D021122:Protein Subunits; D012436:S-Adenosylmethionine; D004247:DNA; D008780:Methyltransferases; C030432:endodeoxyribonuclease EcoP1; C023451:endodeoxyribonuclease EcoP15I; D015263:Deoxyribonucleases, Type III Site-Specific