Modifying the helical structure of DNA by design: recruitment of an architecture-specific protein to an enforced DNA bend.
BACKGROUND
Proteins can force DNA to adopt distorted helical structures that are rarely if ever observed in naked DNA. The ability to synthesize DNA that contains defined helical aberrations would offer a new avenue for exploring the structural and energetic plasticity of DNA. Here we report a strategy for the enforcement of non-canonical helical structures through disulfide cross-linking; this approach is exemplified by the design and synthesis of an oligonucleotide containing a pronounced bend.


RESULTS
A localized bend was site-specifically introduced into DNA by the formation of a disulfide cross-link between the 5' adenines of a 5'-AATT-3' region in complementary strands of DNA. The DNA bend was characterized by high-resolution NMR structure determination of a cross-linked dodecamer and electrophoretic mobility assays on phased multimers, which together indicate that the cross-linked tetranucleotide induces a helical bend of approximately 30 degrees and a modest degree of unwinding. The enforced bend was found to stimulate dramatically the binding of an architecture-specific protein, HMG-D, to the DNA. DNase I foot-printing analysis revealed that the protein is recruited to the section of DNA that is bent.


CONCLUSIONS
The present study reports a novel approach for the investigation of non-canonical DNA structures and their recognition by architecture-specific proteins. The mode of DNA bending induced by disulfide cross-linking resembles that observed in structures of protein-DNA complexes. The results reveal common elements in the DNA-binding mode employed by sequence-specific and architecture-specific HMG proteins.
Wolfe
S A
SA
Department of Chemistry, Harvard University, Cambridge, MA 02138, USA.
Ferentz
A E
AE
Grantcharova
V
V
Churchill
M E
ME
Verdine
G L
GL
D003432:Cross-Linking Reagents; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry*; D018983:DNA Footprinting; D003850:Deoxyribonuclease I / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006609:High Mobility Group Proteins / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry
D003432:Cross-Linking Reagents; D004220:Disulfides; D006609:High Mobility Group Proteins; D009841:Oligonucleotides; D011506:Proteins; D004247:DNA; D003850:Deoxyribonuclease I