RESUMO
Artemis (SNM1C/DCLRE1C) is an endonuclease that plays a key role in development of B- and T-lymphocytes and in dsDNA break repair by non-homologous end-joining (NHEJ). Artemis is phosphorylated by DNA-PKcs and acts to open DNA hairpin intermediates generated during V(D)J and class-switch recombination. Artemis deficiency leads to congenital radiosensitive severe acquired immune deficiency (RS-SCID). Artemis belongs to a superfamily of nucleases containing metallo-ß-lactamase (MBL) and ß-CASP (CPSF-Artemis-SNM1-Pso2) domains. We present crystal structures of the catalytic domain of wildtype and variant forms of Artemis, including one causing RS-SCID Omenn syndrome. The catalytic domain of the Artemis has similar endonuclease activity to the phosphorylated full-length protein. Our structures help explain the predominantly endonucleolytic activity of Artemis, which contrasts with the predominantly exonuclease activity of the closely related SNM1A and SNM1B MBL fold nucleases. The structures reveal a second metal binding site in its ß-CASP domain unique to Artemis, which is amenable to inhibition by compounds including ebselen. By combining our structural data with that from a recently reported Artemis structure, we were able model the interaction of Artemis with DNA substrates. The structures, including one of Artemis with the cephalosporin ceftriaxone, will help enable the rational development of selective SNM1 nuclease inhibitors.
Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Endonucleases/ultraestrutura , Exodesoxirribonucleases/ultraestrutura , Imunodeficiência Combinada Severa/genética , Linfócitos B/enzimologia , Domínio Catalítico/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Cristalografia por Raios X , Reparo do DNA por Junção de Extremidades/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/antagonistas & inibidores , Endonucleases/química , Endonucleases/genética , Inibidores Enzimáticos/química , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Humanos , Fosforilação/genética , Dobramento de Proteína , Imunodeficiência Combinada Severa/enzimologia , Imunodeficiência Combinada Severa/patologia , Linfócitos T/enzimologiaRESUMO
Circadian rhythms are 24-h cycles in physiology and behavior that occur in virtually all organisms. These processes are not simply driven by changes in the external environment as they persist under constant conditions, providing evidence for an internal biological clock. In mammals, this clock is located in the hypothalamic suprachiasmatic nuclei (SCN) and is based upon an intracellular mechanism composed of a transcriptional-translational feedback loop composed of a number of core clock genes. However, a clock is of no use unless it can be set to the correct time. The primary time cue for the molecular clock in the SCN is light detected by the eye. The photoreceptors involved in this process include the rods and cones that mediate vision, as well as the recently identified melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). Light information is conveyed to the SCN via the retinohypothalamic tract, resulting in an intracellular signaling cascade which converges on cAMP-response elements in the promoters of several key clock genes. Over the last two decades a number of studies have investigated the transcriptional response of the SCN to light stimuli with the aim of further understanding these molecular signaling pathways. Here we provide an overview of these studies and provide protocols for studying the molecular responses to light in the SCN clock.