DNA-methyltransferase SsoII as a bifunctional protein: Features of the interaction with the promoter region of SsoII restriction-modification genes

Abstract

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.

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase

“…After formation of the M.SsoII complex with the sets of DNA fragments containing the regulatory site ( 162reg-1 and 162reg-2 ), two types of DNA–protein complexes formed ( С1 and С2 , see above). There was much more complex C2 than complex C1 , just as in Figure 3 , pointing to high cooperativity of M.SsoII binding to the regulatory site in accordance with our earlier data when M.SsoII was cross-linked to DNA duplexes containing a reactive group at the regulatory site [ 67 ]. Complexes C2 showed significant differences in the electrophoretic mobility depending on the position of the regulatory site ( Figure 4 A).…”

“…Because M.SsoII has two different DNA-binding sites located in two different domains, one can imagine a complex where an M.SsoII molecule would be bound simultaneously to the methylation site and to the regulatory site. Although such a complex was fixed in our previous work [ 67 ], where two synthetic DNA duplexes with reactive groups were covalently cross-linked to the protein, we have never detected this complex experimentally under physiological conditions. Moreover, we found that the presence of 60reg-1 in a reaction mixture significantly decreased the binding of M.SsoII to 60met in the presence of cofactor analogue AdoHcy (data not shown).…”

“…The DNA duplex with the methylation site was relatively long (60 bp) for the purpose of comparison with other 60-bp duplexes, although typically, short DNA duplexes (12–25 bp) are used in studies on methylation. One disadvantage of the long DNA was the inability to detect target cytosine flipping and covalent-bond formation directly by UV absorbance at 280 nm (as in [ 67 ]). Therefore, we used duplexes with TAMRA placed near the methylation site in order to detect DNA–protein complex formation and its further conformational changes and duplexes with a 2-aminopurine (2-aPu) residue inside the methylation site in order to study the target base flipping.…”

Thymidine Glycol: The Effect on DNA Structure and DNA Binding by Site-Specific Proteins

“…However, the protein dimer can assemble the proper complex only when the DNA is long enough (60 bp and more) [39]. The 31-bp duplexes used in the present work allow to detect only the intermediate complex where one M.SsoII molecule binds to one DNA duplex -either to the "left" half or to the "right" half of the regulatory site [34] [70] [71]. These two complexes have equal molecular mass and therefore cannot be distinguished by EMSA.…”