Probing of unusual DNA structures in topologically constrained form V DNA: use of restriction enzymes as structural probe

Abstract

The ability of DNA sequences to adopt unusual structures under the superhelical torsional stress has been studied. Sequences that are forced to adopt unusual conformation in topologically constrained pBR322 form V DNA (Lk = 0) were mapped using restriction enzymes as probes. Restriction enzymes such as BamHl, Pstl, Aval and HindlIl could not cleave their recognition sequences. The removal of topological constraint relieved this inhibition. The influence of neighbouring sequences on the ability of a given sequence to adopt unusual DNA structure, presumably left handed Z conformation, was studied through single hit analysis. Using multiple cut restriction enzymes such as Narl and FspI, it could be shown that under identical topological strain, the extent of structural alteration is greatly influenced by the neighbouring sequences. In the light of the variety of sequences and locations that could be mapped to adopt non-B conformation in pBR322 form V DNA, restriction enzymes appear as potential structural probes for natural DNA sequences. INTRODUCTIONOver the last decade our understanding of DNA structure as a rigid uniform double helix has significantly changed to that of a sequence-dependent dynamic conformation (1,2). Although several crystal structures of oligonucleotides have shown that the DNA conformation is indeed sequence-dependent (3), paucity of data does not enable us to arrive at a generalised rule. However, the influence of ionic environment and topological strain (resulting from supercoiling) in altering DNA secondary structure has provided valuable information with regard to structural transition from B to Z form (4-8). Enzymes such as DNase I and SI nuclease have been extensively used to probe DNA conformation in solution (2,9-11). Although restriction endonucleases are believed to be only sequence-specific and not structure sensitive, recent studies have shown that some restriction enzymes fail to recognize and cleave DNA in left handed Z conformation (12)(13)(14).

CyDNA: Synthesis and Replication of Highly Cy-Dye Substituted DNA by an Evolved Polymerase

“…To probe CyDNA structure we initially used restriction endonucleases, which are sensitive probes of noncanonical DNA conformations such as those which occur under torsional strain. (47) Indeed, some like DdeI (C’TNAG) failed to cut CyDNA, presumably due to steric hindrance by bulky Cyanine dyes, while others, notably MseI (T’TAA), cut CyDNA efficiently (Figure 4b). This indicates that at least the local regions of AT-sequence in Cy-DNA adopt a canonical B-form conformation.…”

“…We therefore wondered if CyDNA would still adopt a canonical double helical structure or if the presence of the heterocyclic dyes had promoted a structural transition. To probe CyDNA structure we initially used restriction endonucleases, which are sensitive probes of noncanonical DNA conformations such as those which occur under torsional strain . Indeed, some like DdeI (C’TNAG) failed to cut CyDNA, presumably due to steric hindrance by bulky Cyanine dyes, while others, notably MseI (T’TAA), cut CyDNA efficiently (Figure b).…”

Synergetic enhancing effects between platinum nano-sensitizers and clinically approved stabilizing ligands in proton therapy, causing high-yield double-strand breaks of plasmid DNA at relevant dose

“…[69] To unmistakably associate the bands in the gels with different conformations of pBR322, we initially spiked the intact DNA with the restriction enzyme Eco R1, which breaks the plasmid at exactly one point, selectively creating a DSB. [70] The usage of restriction enzymes to selectively induce a DSB is commonly known and plays an important part in gene analysis and cloning work. [71] The control sample containing Eco R1 was then run on the gel next to the irradiated sample and the bands were at the same level (Figure S4).…”

Restriction Enzyme Analysis of Double-Stranded DNA on Pristine Single-Walled Carbon Nanotubes

“…Importantly, these enzymes are mostly specific for dsDNA in the native B-DNA conformation. 31,32 We take advantage of this conformational specificity to understand the structure and stability of DNA duplexes in the vicinity of the SWCNT surface. Additional Forster resonance electron transfer (FRET) measurements and MD simulation results show that short dsDNA strands can retain their native conformation in the proximity of a SWCNT surface.…”

“…Type-II RE-mediated DNA cleavage is widely used in different techniques, such as restriction fragment length polymorphism, single nucleotide polymorphism, and post-modification analysis as the cleavage activity can be easily investigated by gel electrophoresis. Importantly, these enzymes are mostly specific for dsDNA in the native B-DNA conformation. , We take advantage of this conformational specificity to understand the structure and stability of DNA duplexes in the vicinity of the SWCNT surface. Additional Förster resonance electron transfer (FRET) measurements and MD simulation results show that short dsDNA strands can retain their native conformation in the proximity of a SWCNT surface.…”