S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes

Pradeep Bist, Srivani Sistla, Vinita Krishnamurthy, Asha Acharya, Basavannachar Chandrakala, Desirazu N. Rao

Research output: Contribution to journalArticle

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Abstract

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 rood allele with a wild-type rood 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.

Original languageEnglish (US)
Pages (from-to)93-109
Number of pages17
JournalJournal of Molecular Biology
Volume310
Issue number1
DOIs
StatePublished - Jun 29 2001

Fingerprint

Type III Site Specific Deoxyribonucleases
S-Adenosylmethionine
DNA Cleavage
Methionine
Enzymes
Apoenzymes
Gel Chromatography
Alleles
Native Polyacrylamide Gel Electrophoresis
Holoenzymes
DNA
Circular Dichroism

Keywords

  • AdoMet
  • Apoenzyme
  • Clear-plaque mutants
  • DNA cleavage
  • Type III restriction-modification enzymes

ASJC Scopus subject areas

  • Virology

Cite this

Bist, P., Sistla, S., Krishnamurthy, V., Acharya, A., Chandrakala, B., & Rao, D. N. (2001). S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes. Journal of Molecular Biology, 310(1), 93-109. https://doi.org/10.1006/jmbi.2001.4744

S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes. / Bist, Pradeep; Sistla, Srivani; Krishnamurthy, Vinita; Acharya, Asha; Chandrakala, Basavannachar; Rao, Desirazu N.

In: Journal of Molecular Biology, Vol. 310, No. 1, 29.06.2001, p. 93-109.

Research output: Contribution to journalArticle

Bist, P, Sistla, S, Krishnamurthy, V, Acharya, A, Chandrakala, B & Rao, DN 2001, 'S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes', Journal of Molecular Biology, vol. 310, no. 1, pp. 93-109. https://doi.org/10.1006/jmbi.2001.4744
Bist P, Sistla S, Krishnamurthy V, Acharya A, Chandrakala B, Rao DN. S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes. Journal of Molecular Biology. 2001 Jun 29;310(1):93-109. https://doi.org/10.1006/jmbi.2001.4744
Bist, Pradeep ; Sistla, Srivani ; Krishnamurthy, Vinita ; Acharya, Asha ; Chandrakala, Basavannachar ; Rao, Desirazu N. / S-adenosyl-L-methionine is required for DNA cleavage by type III restriction enzymes. In: Journal of Molecular Biology. 2001 ; Vol. 310, No. 1. pp. 93-109.
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