The Structure of Xis reveals the basis for Filament Formation and insight into DNA bending within a mycobacteriophage Intasome Shweta Singh 1 , Joseph G. Plaks 1 , Nicholas J. Homa 1,4 , Christopher G. Amrich 1 , Annie Heroux 2 , Graham F. Hatfull 1 , and Andrew P. VanDemark 1,3 1 Department of Biological Sciences, University of Pittsburgh, Pittsburgh PA 15260, USA 2 Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA Abstract The Recombination Directionality Factor, Xis, is a DNA bending protein that determines the outcome of integrase-mediated site-specific recombination by redesign of higher-order protein- DNA architectures. Although the attachment site DNA of Mycobacteriophage Pukovnik is likely to contain four sites for Xis binding, Xis crystals contain five subunits in the asymmetric unit, four of which align into a Xis filament, and a fifth that is generated by an unusual domain swap. Extensive intersubunit contacts stabilize a bent filament-like arrangement with Xis monomers aligned head-to-tail. The structure implies a DNA bend of ~120°, which is in agreement with DNA bending measured in vitro. Formation of attR-containing intasomes requires only Int and Xis, distinguishing Pukovnik from lambda. Therefore, we conclude that in Pukovnik, Xis-induced DNA bending is sufficient to promote intramolecular Int-mediated bridges during intasome formation. Keywords DNA recombination; mycobacteriophage Pukovnik; Xis; DNA bending; filament; structure Introduction Prophage establishment by temperate bacteriophages typically involves integrase-mediated site-specific recombination between the phage attP and chromosomal attB site 1 . Of the large family of phage-encoded integrases of the tyrosine recombinase type that mediate these events, phage lambda represents a well-studied prototype 1; 2; 3 . In lambda, integration requires integrase (Int), the host integration factor (IHF), a large (250 bp) attP site that contains both core-type and arm-type integrase binding sites, and a smaller attB site (25 bp). Strand exchange occurs within the shared common core sequence and proceeds through a © 2013 Elsevier Ltd. All rights reserved. 3 Corresponding Authors Contact Information 4249 Fifth Ave, 360A Langley Hall, University of Pittsburgh, Pittsburgh PA 15260 andyv@pitt.edu, phone: (412) 648-0110, fax: (412) 624-4759. 4 Present address: 426 CARL building, Duke University, Durham, NC, 27710, njh16@duke Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Accession Codes. Coordinates and structure factors for Pukovnik Xis have been deposited at the Protein Data Band under accession number 4J2N. NIH Public Access Author Manuscript J Mol Biol. Author manuscript; available in PMC 2015 January 23. Published in final edited form as: J Mol Biol. 2014 January 23; 426(2): 412–422. doi:10.1016/j.jmb.2013.10.002. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript