XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining

International audienceThe Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 alpha/beta domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 alpha/beta domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ

Assessment of impact damage in Kevlar{reg_sign}-epoxy, filament-wound spherical test specimens by acoustic emission techniques

The results of a study of the acoustic emission (AE) behavior of impact-damaged, spherical, composite test specimens subjected to thermal cycling and biaxial mechanical loading are presented. Seven Kevlar{reg_sign}-epoxy, filament-wound, spherical composite test specimens were subjected to different levels of impact damage. The seven specimens were a subset of a group of 77 specimens made with simulated fabrication-induced flaws. The specimens were subjected to two or three cycles of elevated temperature and then hydraulically pressurized to failure. The pressurization regime consisted of two cycles to different intermediate levels with a hold at each peak pressure level; a final pressurization to failure followed. The thermal and pressurization cycles were carefully designed to stimulate AE production under defined conditions. Both impacted and nonimpacted specimens produced thermo-AE (the term given to emission stimulated by thermal loading), but impacted specimens produced significantly more. Thermo-AE was produced primarily by damaged composite material. Damaged material produced emission as a function of both rising and falling temperature, but the effect was not repeatable. More seriously damaged specimens produced very large quantities of emission. Emission recorded during the static portion of the hydraulic loading cycles varied with load, time, and degree of damage. Static load AE behavior was quantified using a newly developed concept, the event-rate moment, and various correlations with residual strength were attempted. Correlations between residual strength, long-duration events, and even-rate moments were developed with varying degrees of success

Integrative Structural Biology: Using X-ray Crystallography, Small-Angle X-ray Scattering, and Cryogenic Electron Microscopy to Determine Protein Structures

The field of structural biology focuses on determining and studying the structures of macromolecules in order to understand how three-dimensional shape dictates function at the molecular level. There are a variety of experimental tools that can be used to determine protein structures, and each technique has its strengths and weaknesses. This chapter focuses on three of these techniques: X-ray crystallography, small-angle X-ray scattering (SAXS), and cryogenic electron microscopy (cryo-EM). Each technique is introduced, and its strengths and weaknesses as a tool for protein structure determination are discussed. The emphasis of this chapter is that while these techniques on their own can provide a wealth of information regarding protein structure, when combined they complement each other to paint a more complete picture of the three-dimensional architecture of proteins. Two examples from the literature are provided where all three techniques were utilized to learn the fine details of protein structure. The first example reveals the structural details of how multiple proteins assemble to replicate DNA, while the second shows how multiple structures of a single enzyme with and without substrate bound can provide the molecular details of a catalytic cycle