Affinity modification of the restriction endonuclease SsoII by 2'-aldehyde-containing double stranded DNAs

A. E. Sud'ina, T. S. Zatsepin, V. Pingoud, A. Pingoud, T. S. Oretskaya, E. A. Kubareva

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1 Citation (Scopus)

Abstract

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully crosslinked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.

Original languageEnglish
Pages (from-to)1137-1144
Number of pages8
JournalBiokhimiya
Volume70
Issue number8
Publication statusPublished - 2005
Externally publishedYes

Keywords

  • Affinity modification
  • DNA-protein interactions
  • Modified double stranded DNAs
  • Restriction endonuclease SsoII

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