Structural Recognition between a Four-way DNA Junction and a Resolving Enzyme

• Published in: Journal of molecular biology Vol. 359; no. 5; pp. 1261 - 1276
• Main Authors: Déclais, Anne-Cécile, Liu, Jia, Freeman, Alasdair D.J., Lilley, David M.J.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 23-06-2006
• Subjects: Bacteriophage T7 - enzymology; Base Sequence; Deoxyribonuclease I - chemistry; Deoxyribonuclease I - metabolism; DNA Footprinting; DNA, Circular - chemistry; DNA, Cruciform - chemistry; DNA, Cruciform - genetics; DNA, Cruciform - metabolism; Holliday junction; Holliday Junction Resolvases - chemistry; Holliday Junction Resolvases - metabolism; Models, Molecular; Molecular Sequence Data; nucleases; Nucleic Acid Conformation; Organophosphorus Compounds - metabolism; Protein Binding; recombination; structure-selective DNA-protein interaction; Substrate Specificity; Holliday junction; structure-selective DNA-protein interaction; recombination; nucleases
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2006.04.037

Resolving enzymes bind highly selectively to four-way DNA junctions, but the mechanism of this structural specificity is poorly understood. In this study, we have explored the role of interactions between the dimeric enzyme and the helical arms of the junction, using junctions with either shortened arms, or circular permutation of arms. We find that DNA–protein contacts in the arms containing the 5′ ends of the continuous strands are very important, conferring a significant level of sequence discrimination upon both the choice of conformer and the order of strand cleavage. We have exploited these properties to obtain hydroxyl radical footprinting data on endonuclease I–junction complexes that are not complicated by the presence of alternative conformers, with results that are in good agreement with the arm permutation and shortening experiments. Substitution of phosphate groups at the center of the junction reveals the importance of electrostatic interactions at the point of strand exchange in the complex. Our data show that the form of the complex between endonuclease I and a DNA junction depends on the core of the junction and on interactions with the first six base-pairs of the arms containing the 5′ ends of the continuous strands.