4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite 
compatible with solid phase DNA synthesis. When incorporated as the target 
cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. 
HhaI, methyl transfer was strongly inhibited. In contrast, these same 
oligonucleotides were normal substrates for the cognate restriction endonuclease 
R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 
4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium 
conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the 
half-life of the complex by >10-fold. The crystal structure of a ternary complex 
of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 
2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. 
The structure is not grossly different from previously solved ternary complexes 
containing M. HhaI, DNA and AdoHcy. The difference electron density suggests 
partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The 
inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to 
perturbation of a step in the methylation reaction after DNA binding but prior 
to methyl transfer. This inhibitory effect can be partially overcome after a 
considerably long time in the crystal environment where the packing prevents 
complex dissociation and the target is accurately positioned within the active 
site.