Structural Characterization of the C-terminal Domain of Human DNA Ligase IV Bound to Xrcc4

Abstract

<p> Non-homologous end joining (NHEJ) is the predominant mode of DNA double strand break (DSB) repair pathway in mammalian cells. At the heart of this repair pathway is Xrcc4-DNA ligase IV complex, which mediates ligation of the broken DNA strands. The C-terminal tandem BRCT repeats of human DNA ligase IV spanning residues 654-911 in complex with the functional fragment of Xrcc4 comprised of residues 1-203 were crystallized by the hanging drop vapour diffusion method at 20°C. Generation of single, well-packed, diffraction quality crystals suitable for structure determination involved usage of an Xrcc4 point mutant (A60E). Arriving at the crystallization condition included optimization of pH, variation of the precipitant concentration, investigation of the effects of small molecules, and alteration of the amount of crystal seed used as initial nuclei. A Crystal of selenomethionine-derived protein complex was grown using the above optimization steps and diffracted to 2.4 A resolution. Data processing revealed that the crystal belonged to space group P1 with unit cell dimensions a= 67.33 b = 86.00 c = 111.52; a= 67.37 ~ = 83.00 y = 74.56. The crystal structure of Xrcc4-DNA ligase IV complex was solved by single-wavelength anomalous diffraction using data collected at a wavelength of 0.9785A corresponding to peak energy. </p> <p> The structure maintains a 2:1 stoichiometry of Xrcc4 to the C-terminal domain of DNA ligase IV. The structure of the complex not only confirms the overall novel mode of interaction first observed in the 3.9 A structure of the yeast ortholog liflp-lig4p complex, but it also discloses additional key features such as the DNA binding surface of the complex and the striking conformational changes occurring within Xrcc4 upon interaction with DNA ligase IV. Together, the structural information procured forms an important basis for a better understanding of the mechanism involved in the NHEJ repair pathway. </p>