2Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, ul. Gamalei 18, 123098 Moscow, Russia
3All-Russian Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Timiryazevskaya ul. 42, 127550 Moscow, Russia
4Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (495) 939-3181; E-mail: email@example.com
* To whom correspondence should be addressed.
Received June 21, 2006; Revision received August 17, 2006
DNA duplexes bearing an aldehyde group at the 2´-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2´-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2´-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.
KEY WORDS: (cytosine-5)-DNA methyltransferases, modified oligonucleotides, affinity modification, 2´-aldehyde group, phosphoryldisulfide group