Flap endonuclease Substrate Interactions studied using Dynamic Single-Molecule Atomic Force Microscopy
Verma, V.; Brudenell, E. L.; Cussons, S.; Phipps, J.; Kong, S. C.; Sayers, J. R.; Hobbs, J. K.
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Flap endonucleases (FENs) recognise and cleave DNA substrates containing a 5-single-strand (ss) of nucleic acid branching off a double-stranded (ds) DNA to yield a nicked duplex during DNA replication. Dynamic Atomic Force Microscopy of an inactive FEN mutant complexed with branched DNA revealed mobilisation of immobilised DNA, indicating that protein interaction affected substrate conformation and disrupted the forces that anchored it to the poly-L-ornithine -treated mica surface. Enzymatically-active FEN was seen intermittently binding DNA, altering its conformation and cleaving the ssDNA branch. We developed a method using motion tracking for quantifying the movement of DNA sections, by visually segmenting DNA and tracking each segment to recognise the DNA sections most affected by the protein. It was found that whilst bound, FEN caused localised DNA bending, and changes in DNA shape were witnessed in the short time span of the proteins appearance close to the nucleic acid, followed by protein adsorption on the mica surface. The results provide the first dynamic observations of FEN-DNA interaction. FEN initially binds to the dsDNA, slides to find the ds/ssDNA junction, and the 5 ssDNA likely threads through a hole in the enzyme which leads to enzymatic hydrolysis of the branched substrate.
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