Abstract
AbstractFlap 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 protein’s 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.
Publisher
Cold Spring Harbor Laboratory
Reference97 articles.
1. . Okazaki , R., Okazaki , T., Sakabe , K., Sugimoto , K., & Sugino , A. (1968). Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proceedings of the National Academy of Sciences of the United States of America, 59(2), 598–605.
2. DNA Polymerase I: Essential Replication Enzyme
3. Watson, J. D. , Baker, T. A. , Bell, S. P. , Gann, A. , Levine, M. , Losick, R . (2013). Molecular Biology of the Gene. Pearson Education.
4. Flap Endonuclease 1: A Central Component of DNA Metabolism
5. Flap Endonuclease 1
Cited by
1 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献