Nucleoprotein architectures regulating the directionality of viral integration and excision

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-08, Vol.111 (34), p.12372-12377
• Main Authors: Seah, Nicole E., Warren, David, Tong, Wenjun, Laxmikanthan, Gurunathan, Van Duyne, Gregory D., Landy, Arthur
• Format: Article
• Language: English
• Subjects: Architectural models; Architecture; Bacteriophage lambda; Bacteriophage lambda - genetics; Bacteriophage lambda - physiology; Bacteriophages; Bending; Binding Sites; Biological Sciences; Computer Simulation; Crystal structure; Deoxyribonucleic acid; DNA; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; DNA, Cruciform - chemistry; DNA, Cruciform - genetics; DNA, Cruciform - metabolism; Dyes; Escherichia coli - genetics; Escherichia coli - metabolism; Escherichia coli - virology; Fluorescence Resonance Energy Transfer; Gene expression regulation; Integrases - chemistry; Integrases - genetics; Integrases - metabolism; Integration; Lysogeny - genetics; Models, Molecular; Multiprotein Complexes - chemistry; Multiprotein Complexes - genetics; Multiprotein Complexes - metabolism; Nucleic Acid Conformation; Nucleoproteins - chemistry; Nucleoproteins - genetics; Nucleoproteins - metabolism; Protein Binding; Protein Interaction Domains and Motifs; Proteins; Recombination, Genetic; Topology; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Activation - genetics
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1413019111

The virally encoded site-specific recombinase Int collaborates with its accessory DNA bending proteins IHF, Xis, and Fis to assemble two distinct, very large, nucleoprotein complexes that carry out either integrative or excisive recombination along regulated and essentially unidirectional pathways. The core of each complex consists of a tetramer of Integrase protein (Int), which is a heterobivalent DNA binding protein that binds and bridges a core-type DNA site (where strand cleavage and ligation are executed), and a distal arm-type site, that is brought within range by one or more DNA bending proteins. The recent determination of the patterns of these Int bridges has made it possible to think realistically about the global architecture of the recombinogenic complexes. Here, we combined the previously determined Int bridging patterns with in-gel FRET experiments and in silico modeling to characterize and differentiate the two 400-kDa multiprotein Holiday junction recombination intermediates formed during λ integration and excision. The results lead to architectural models that explain how integration and excision are regulated in λ site-specific recombination. Our confidence in the basic features of these architectures is based on the redundancy and self-consistency of the underlying data from two very different experimental approaches to establish bridging interactions, a set of strategic intracomplex distances from FRET experiments, and the model’s ability to explain key aspects of the integrative and excisive recombination pathways, such as topological changes, the mechanism of capturing att B, and the features of asymmetry and flexibility within the complexes.