Bending and flexibility of methylated and unmethylated Eco RI DNA

The first B-DNA crystal structure that spanned more than a full helical repeat was that of the dodecamer d(CGCGAATTCGCG)2. This sequence still serves as a bench mark for both experimental and theoretical DNA structural studies. The molecule contains in its core the Eco RI endonuclease restriction site GAATTC. We used cyclization kinetics experiments and Monte Carlo simulations to determine a structural model for a DNA decamer containing the Eco RI restriction site. Our findings agree well with recent crystal and NMR structures of the Eco RI dodecamer, where an overall bend of 7 degrees is distributed symmetrically over the molecule. Monte Carlo simulations indicate that the sequence has a higher flexibility, assumed to be isotropic, compared to that of a "generic" DNA sequence. This model was used as a starting point for the investigation of the effect of cytosine methylation on DNA bending and flexibility. While methylation did not affect bend magnitude or position, it resulted in a reduction in bending flexibility and under-winding of the methylated nucleotides.;The effect of methylation on nucleosome stability has not been fully characterized. Competitive reconstitution analysis with chemically synthesized DNA sequences containing the Eco RI decamer addressed the question of the effect of DNA methylation on nucleosome formation. Our initial results show that for the DNA sequences in question, methylation did not affect nucleosome affinity significantly. Affinity was dominated by the position of the CpG triplet and AATT tract with respect to the underlying histone core rather than by methylation.