2000/12/01 by Peiyuan Wang, Adam S. Brank, Nilesh K. Banavali +4 · 1 voice · 1 citation
Biochemistry, Genetics and Molecular Biology · #CRISPR and Genetic Engineering #Epigenetics and DNA Methylation #RNA modifications and cancer
paper · doi:10.1021/ja001989s
openalex publication_date 2000/12/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/22
X-ray crystallographic studies of Hha I DNA (cytosine-C5)-methyltransferase (M. Hha I) 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. Hha I 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. Hha I recognition sequence. Biochemical studies demonstrate that binding affinity of M. Hha I 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. Hha I 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. Hha I 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. Hha I 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. Hha I 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. Hha I catalytic cycle are discussed.