Use of Oligodeoxyribonucleotides with Conformationally Constrained Abasic Sugar Targets To Probe the Mechanism of Base Flipping by HhaI DNA (Cytosine C5)-methyltransferase

Abstract

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.

5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy

“…Electrophoretic mobility on native gels demonstrated that M.HhaI complexes with ODNs containing DZCyt as a replacement for the Cyt target of M.HhaI had the same 'closed' mobility in the presence of AdoMet or AdoHcy or in the absence of cofactor (Sheikhnejad et al, 1999). Identical behavior was noted for ODNs with abasic carbocyclic and furanose sugars in place of Cyt (Wang et al, 2000). An improved method for the automated synthesis of ODNs containing ZCyt (Garcı´a et al, 2001) allowed us to examine the formation of complexes between M.HhaI and ZCyt-ODN and to determine that formation of 'closed' complexes with ZCyt-ODN is also independent of cofactor (Brank et al, in preparation).…”

“…While DZCyd has the advantage of lower toxicity and mutagenic potential, it has not proven to be particularly effective at inducing a therapeutic response. With a better understanding of how mechanism based inhibitors interact with DNA methyltransferases, it should be possible to design small 'decoy' DNAs containing these inhibitors for use as therapeutics (Brank et al, in preparation;Garcı´a et al, 2001;Sheikhnejad et al, 1999;Wang et al, 2000). Dnmt1, the most abundant of the three catalytically active DNA MTase in mammalian cells (Figure 9) and the best studied, is assumed to be the major target for inhibition by Cyt analogs in DNA.…”

Observing an Induced-fit Mechanism during Sequence-specific DNA Methylation

“…These substrates most likely have severe backbone distortions due to steric clashes and incorrect hydrogen donor/acceptor alignment (51-53). The abasic DNA sub-strate, which most likely has the DNA backbone largely in the extrahelical conformer (54), shows the greatest biphasic behavior for loop motion. This suggests that any perturbation in DNA structure that alters the extrahelical nature of the target base will contribute to the biphasic nature of loop motion.…”

The Coupling of Tight DNA Binding and Base Flipping

“…Many M.HhaI crystal structures provide structural insights into the mechanisms of DNA methylation and base flipping (8). Functional analysis of the WT M.HhaI has been extensive (9 -12), including K D DNA determination for a variety of DNA substrates (13,14). Many structural components of the M.HhaI mechanism have been examined by mutagenesis including Gln 237 , which positions itself into the DNA helix and interacts with the lone guanine (15), and Cys 81 , which forms a covalent bond to the target cytosine (16).…”