The dynamics of the monomeric restriction endonuclease BcnI during its interaction with DNA

作者：Kostiuk Georgij; Dikic Jasmina; Schwarz Friedrich W; Sasnauskas Giedrius; Seidel Ralf^*; Siksnys Virginijus^*

来源：Nucleic Acids Research, 2017, 45(10): 5968-5979.

DOI：10.1093/nar/gkx294

Endonucleases that generate DNA double strand breaks often employ two independent subunits such that the active site from each subunit cuts either DNA strand. Restriction enzyme BcnI is a remarkable exception. It binds to the 5'-CC/SGG-3' (where S = C or G, '/' designates the cleavage position) target as a monomer forming an asymmetric complex, where a single catalytic center approaches the scissile phosphodiester bond in one of DNA strands. Bulk kinetic measurements have previously shown that the same BcnI molecule cuts both DNA strands at the target site without dissociation from the DNA. Here, we analyse the BcnI DNA binding and target recognition steps at the single molecule level. We find, using FRET, that BcnI adopts either 'open' or 'closed' conformation in solution. Next, we directly demonstrate that BcnI slides over long distances on DNA using 1D diffusion and show that sliding is accompanied by occasional jumping events, where the enzyme leaves the DNA and rebinds immediately at a distant site. Furthermore, we quantify the dynamics of the BcnI interactions with cognate and non-cognate DNA, and determine the preferred binding orientation of BcnI to the target site. These results provide new insights into the intricate dynamics of BcnI-DNA interactions.

• 出版日期2017-6-2