Affinity Modification of the Restriction Endonuclease SsoII by 2´-Aldehyde-Containing Double Stranded DNAs

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A. E. Sud'ina¹, T. S. Zatsepin², V. Pingoud³, A. Pingoud³, T. S. Oretskaya^1,2, and E. A. Kubareva^1*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181; E-mail: kubareva@belozersky.msu.ru

²Faculty of Chemistry, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-3181

³Institut für Biochemie, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany; fax: +49 (641) 99-35409

^* To whom correspondence should be addressed.

Received February 7, 2005; Revision received March 25, 2005

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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 cross-linked 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.

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KEY WORDS: restriction endonuclease SsoII, DNA-protein interactions, modified double stranded DNAs, affinity modification

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