Nucleic Acids Research, Vol 25, Issue 14 2773-2783, Copyright © 1997 by Oxford University Press
S Kumar, JR Horton, GD Jones, RT Walker, RJ Roberts and X Cheng
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.
ARTICLES
DNA containing 4'-thio-2'-deoxycytidine inhibits methylation by HhaI methyltransferase
New England Biolabs, 32 Tozer Road, Beverly, MA 01915, USA. kumar@neb.com
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