A structural snapshot of base-pair opening in DNA

Abstract

The response of double-helical DNA to torsional stress may be a driving force for many processes acting on DNA. The 1.55-Å crystal structure of a duplex DNA oligonucleotide d(CCAGGCCTGG)2 with an engineered crosslink in the minor groove between the central guanine bases depicts how the duplex can accommodate such torsional stress. We have captured in the same crystal two rather different conformational states. One duplex contains a strained crosslink that is stabilized by calcium ion binding in the major groove, directly opposite the crosslink. For the other duplex, the strain in the crosslink is relieved through partial rupture of a base pair and partial extrusion of a cytosine accompanied by helix bending. The sequence used is the target sequence for the HaeIII methylase, and this partially flipped cytosine is the same nucleotide targeted for extrusion by the enzyme. Molecular dynamics simulations of these structures show an increased mobility for the partially flipped-out cytosine.T he cellular genome is subjected to strain forces that can drive the DNA double helix to adopt a variety of unusual structures. Many essential operations on the genome such as transcription, replication, recombination, repair, and methylation proceed through transient intermediates in which DNA structure deviates sometimes radically from the canonical B form duplex. Because the cellular context often provides the conditions necessary to drive the formation of such torsionally stressed structures, or because of their fleeting nature, few have been captured in a form that has enabled high-resolution structural studies. As a consequence, little detailed information is available regarding how naked DNA responds to torsional stress.Perhaps the most visually striking distortion brought on DNA by the binding of a protein is so-called ''base flipping,'' wherein an entire nucleoside unit is swiveled out of the DNA helix and inserted into a recognition pocket on the protein. Extrahelical, or flipped-out, bases were initially observed in duplex DNA in a crystal structure (1) and several NMR studies of B form DNA containing unpaired bases (2-4). The first flipped bases in protein complexes were observed for the substrate cytosine residue of the DNA (cytosine-5)-methyltransferases (m5C-MTases) M.HhaI (5) and M.HaeIII (6). Nucleoside extrusion and extrahelical recognition have since been directly implicated in the catalytic mechanism of diverse DNA-processing enzymes. These are exemplified by crystal structures of 3-methyladenine DNA glycosylase (Aag) (7), T4 endonuclease V (8), uracil-DNA glycosylases (9, 10), and G:T͞U mismatch-specific DNA glycosylase (11), all complexed to DNA, and are suggested to occur in numerous other cases (12). These findings have aroused interest in understanding processes through which torsional stress imposed on DNA can induce the opening of base pairs.Verdine and coworkers have developed a strategy for trapping torsionally stressed structures in oligonucleotides. In this system, disulfide crosslinks are engineered ...

The Triplex-Hairpin Transition in Cytosine-Rich DNA

“…The increased stability of the central GC 1 basepairs also results from positional constraints imposed by stacking interactions involving the two neighboring basepairs. This basepair opening has also been observed in MD simulations (Cieplak et al, 1997;van Aalten et al, 1999).…”

Site-Specific Disulfide Crosslinked Nucleosomes with Enhanced Stability

“…Both cysteine point mutations were separately introduced into the H3 tail by site-directed mutagenesis. A thiol group was introduced into the DNA at either position T69 or T70 on both the I- and J-strands through oligonucleotides incorporating a convertible nucleotide [33] , [34] , [35] , [36] . 2-Fluoro-deoxyinosine (2-F-dI) was used in oligonucleotide synthesis and converted post-synthetically to contain a cystamine moiety at the purine N 2 position which projects into the DNA minor groove (dG*).…”

DNA-Directed Base Pair Opening

“…A clear demonstration of the direct opening of a GC base pair induced by a phosphate group of a symmetry related molecule is provided by the high resolution structure of the crosslinked decamer duplex d(CCAG G CTGG) 2 , ( G denotes the ethyldisulfide crosslinked guanines) [ 50 ]. This decamer crystallizes with two distinct molecules in the asymmetric unit, one having a canonical duplex whereas the other displays a distorted DNA structure and an open GC base pair.…”