ABSTRACT
The 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 α/ß domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 α/ß 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.
Subject(s)
DNA End-Joining Repair , DNA Repair Enzymes/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-Binding Proteins/chemistry , Ku Autoantigen/chemistry , Poly-ADP-Ribose Binding Proteins/chemistry , Conserved Sequence , Crystallography, X-Ray , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/physiology , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/physiology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/physiology , Humans , Ku Autoantigen/metabolism , Ku Autoantigen/physiology , Models, Molecular , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/physiology , Protein DomainsABSTRACT
Skeletal dysplasias are a clinically and genetically heterogeneous group of bone and cartilage disorders. A total of 436 skeletal dysplasias are listed in the 2015 revised version of the nosology and classification of genetic skeletal disorders, of which nearly 20% are still genetically and molecularly uncharacterized. We report the clinical and molecular characterization of a lethal skeletal dysplasia of the short-rib group caused by mutation of the mouse Fop gene. Fop encodes a centrosomal and centriolar satellite (CS) protein. We show that Fop mutation perturbs ciliogenesis in vivo and that this leads to the alteration of the Hedgehog signaling pathway. Fop mutation reduces CSs movements and affects pericentriolar material composition, which probably participates to the ciliogenesis defect. This study highlights the role of a centrosome and CSs protein producing phenotypes in mice that recapitulate a short rib-polydactyly syndrome when mutated.
Subject(s)
Ciliopathies/genetics , Proto-Oncogene Proteins/genetics , Short Rib-Polydactyly Syndrome/genetics , Transcription Factors/genetics , Animals , Centrioles/genetics , Centrosome/metabolism , Centrosome/pathology , Cilia/genetics , Cilia/pathology , Ciliopathies/physiopathology , Humans , Mice , Mutation , Short Rib-Polydactyly Syndrome/physiopathologyABSTRACT
Brain lipid homeostasis is important for maintenance of brain cell function and synaptic communications, and is intimately linked to age-related cognitive decline. Because of the blood-brain barrier's limiting nature, this tissue relies on a complex system for the synthesis and receptor-mediated uptake of lipids between the different networks of neurons and glial cells. Using immunofluorescence, we describe the region-specific expression of the lipolysis-stimulated lipoprotein receptor (LSR), in the mouse hippocampus, cerebellum Purkinje cells, the ependymal cell interface between brain parenchyma and cerebrospinal fluid, and the choroid plexus. Colocalization with cell-specific markers revealed that LSR was expressed in neurons, but not astrocytes. Latency in arms of the Y-maze exhibited by young heterozygote LSR(+/-) mice was significantly different as compared to control LSR(+/+), and increased in older LSR(+/-) mice. Filipin and Nile red staining revealed membrane cholesterol content accumulation accompanied by significantly altered distribution of LSR in the membrane, and decreased intracellular lipid droplets in the cerebellum and hippocampus of old LSR(+/-) mice, as compared to control littermates as well as young LSR(+/-) animals. These data therefore suggest a potential role of LSR in brain cholesterol distribution, which is particularly important in preserving neuronal integrity and thereby cognitive functions during aging.