Analysis of the Intrinsically Disordered N-Terminusof the DNA Junction-Resolving Enzyme T7 Endonuclease I: Identificationof Structure Formed upon DNA Binding

摘要

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 distributedin the free protein but are significantly shortened and become moredefined on binding to DNA. These results suggest that the N-terminalpeptides become more organized on binding to the DNA junction andnestle into the minor grooves at the branchpoint, consistent withthe biochemical data indicating an important role in the resolutionprocess. This study demonstrates the presence of structure withina protein region that cannot be viewed by crystallography.