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Analysis of the intrinsically disordered N-terminus of the DNA junction-resolving enzyme T7 endonuclease I : identification of structure formed upon DNA binding

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Mkami_2016_BioChem_Analysis_CCBY.pdf (2.468Mb)
Date
02/08/2016
Author
Freeman, Alasdair D J
Stevens, Michael
Declais, Anne Cecile
Leahy, Adam
Mackay, Katherine
El Mkami, Hassane
Lilley, David M. J.
Norman, David G.
Keywords
QD Chemistry
Biochemistry
NDAS
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Abstract
The four-way (Holliday) DNA junction of homologous recombination is processed by the symmetrical cleavage of two strands by a nuclease. These junction-resolving enzymes bind to four-way junctions in dimeric form, distorting the structure of the junction in the process. Crystal structures of T7 endonuclease I have been determined as free protein, and the complex with a DNA junction. In neither crystal structure was the N-terminal 16-amino acid peptide visible, yet deletion of this peptide has a marked effect on the resolution process. Here we have investigated the N-terminal peptide by inclusion of spin-label probes at unique sites within this region, studied by electron paramagnetic resonance. Continuous wave experiments show that these labels are mobile in the free protein but become constrained on binding a DNA junction, with the main interaction occurring for residues 7-10 and 12. Distance measurements between equivalent positions within the two peptides of a dimer using PELDOR showed that the intermonomeric distances for residues 2-12 are long and broadly distributed in the free protein but are significantly shortened and become more defined on binding to DNA. These results suggest that the N-terminal peptides become more organized on binding to the DNA junction and nestle into the minor grooves at the branchpoint, consistent with the biochemical data indicating an important role in the resolution process. This study demonstrates the presence of structure within a protein region that cannot be viewed by crystallography.
Citation
Freeman , A D J , Stevens , M , Declais , A C , Leahy , A , Mackay , K , El Mkami , H , Lilley , D M J & Norman , D G 2016 , ' Analysis of the intrinsically disordered N-terminus of the DNA junction-resolving enzyme T7 endonuclease I : identification of structure formed upon DNA binding ' , Biochemistry , vol. 55 , no. 30 , pp. 4166-4172 . https://doi.org/10.1021/acs.biochem.6b00242
Publication
Biochemistry
Status
Peer reviewed
DOI
https://doi.org/10.1021/acs.biochem.6b00242
ISSN
0006-2960
Type
Journal article
Rights
Copyright © 2016 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
Description
This work was supported by grants from The Engineering and Physical Sciences Research Council (EPSRC), Basic Technology EP/F039034/1, The Wellcome Trust, 099149/Z/12/Z, and Cancer Research UK (CRUK), C28/A18604.
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  • University of St Andrews Research
URL
http://pubs.acs.org/doi/suppl/10.1021/acs.biochem.6b00242
URI
http://hdl.handle.net/10023/9323

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