Intercalation of small molecules into DNA in chromatin is primarily controlled by superhelical constraint

The restricted access of regulatory factors to their binding sites on DNA wrapped around the nucleosomes is generally interpreted in terms of molecular shielding exerted by nucleosomal structure and internucleosomal interactions. Binding of proteins to DNA often includes intercalation of hydrophobic amino acids into the DNA. To assess the role of constrained superhelicity in limiting these interactions, we studied the binding of small molecule intercalators to chromatin in close to native conditions by laser scanning cytometry. We demonstrate that the nucleosome-constrained superhelical configuration of DNA is the main barrier to intercalation. As a result, intercalating compounds are virtually excluded from the nucleosome-occupied regions of the chromatin. Binding of intercalators to extranucleosomal regions is limited to a smaller degree, in line with the existence of net supercoiling in the regions comprising linker and nucleosome free DNA. Its relaxation by inducing as few as a single nick per ~50 kb increases intercalation in the entire chromatin loop, demonstrating the possibility for long-distance effects of regulatory potential.调控因子（regulatory factors）结合于缠绕核小体（nucleosome）的DNA上的位点存在受限现象，该现象通常被归因于核小体结构与核小体间相互作用（internucleosomal interactions）所施加的分子屏蔽（molecular shielding）。蛋白质与DNA的结合过程常涉及疏水氨基酸（hydrophobic amino acids）嵌入DNA双螺旋结构。为评估受限超螺旋（constrained superhelicity）在限制此类相互作用中的作用，我们采用激光扫描细胞术（laser scanning cytometry），在接近天然的实验条件下，研究了小分子嵌入剂与染色质（chromatin）的结合行为。我们证实，DNA受核小体约束的超螺旋构型是嵌入反应的主要阻碍。据此，嵌入化合物几乎无法进入染色质中被核小体占据的区域。核小体外（extranucleosomal）区域的嵌入剂结合则受限于较小程度，这与连接DNA（linker DNA）及无核小体DNA（nucleosome free DNA）区域存在净超螺旋的现象相符。通过在每~50 kb的DNA片段上引入仅一个单链切口（single nick）即可使其松弛，该操作可提升整个染色质环（chromatin loop）内的嵌入反应效率，证明了调控潜能（regulatory potential）可产生长距离效应的可能性。