X-ray crystallographic studies of HhaI DNA (cytosine-C5)-methyltransferase (M.HhaI) covalently
linked to methylated 5-fluorocytosine in DNA provided the first direct evidence that the cytosine residue
targeted for methylation was “flipped” out of the helix during the transfer reaction. Subsequent studies indicated
that removal of the target cytosine base, i.e., introduction of an abasic site, enhanced binding of M.HhaI to
DNA and that the conformation of the sugar−phosphate backbone at the abasic site in the resultant complexes
was the same as that of the sugar attached to a “flipped” cytosine. In the present study, pseudorotationally
constrained sugar analogues, based on bicyclo[3.1.0]hexane templates, were placed in DNA duplexes as abasic
target sites in the M.HhaI recognition sequence. Biochemical studies demonstrate that binding affinity of M.HhaI
for abasic sites increases when the abasic target sugar analogue is constrained to the south conformation and
decreases when it is constrained to the north conformation. In native gel-shift assays, M.HhaI exhibits a “closed”
conformation when bound to the abasic south or abasic furanose analogues, whereas an “open” conformation
predominates with the abasic north analogue. A structural understanding of these results was obtained via
molecular dynamics simulations of the DNA duplex alone and in ternary complex with M.HhaI and cofactor,
along with quantum mechanical calculations on model compounds representative of the abasic and modified
sugars. Binding affinities are shown to be related to the ability of the abasic sugar analogues to spontaneously
flip out of the DNA duplex. Enhanced binding of the abasic south analogue is suggested to be due to its
increased capacity for sampling the experimentally observed conformation of the DNA target site in the M.HhaI
ternary complex. Decreased binding of the north analogue is due to decreased flexibility of the phosphodiester
backbone associated with a north pseudorotation angle, thereby inhibiting spontaneous flipping of the sugar
moiety out of the DNA duplex. Spontaneous flipping of the sugar moiety out of the DNA duplex is also
suggested to facilitate formation of a “closed” complex between M.HhaI and DNA whereas partial or no
flipping favors the “open” conformation. These results show that introduction of structural constraints into
DNA that induce enhanced sampling of protein-bound conformations facilitate DNA−protein binding.
Implications of the present results with respect to the mechanism of base flipping in the M.HhaI catalytic
cycle are discussed.
The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.
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