RESUMO
TLS (also known as FUS) is an RNA-binding protein that contributes the N-terminal half of fusion oncoproteins implicated in the development of human liposarcomas and leukemias. Here we report that male mice homozygous for an induced mutation in TLS are sterile with a marked increase in the number of unpaired and mispaired chromosomal axes in pre-meiotic spermatocytes. Nuclear extracts from TLS(-/-) testes lack an activity capable of promoting pairing between homologous DNA sequences in vitro, and TLS(-/-) mice and embryonic fibroblasts exhibit increased sensitivity to ionizing irradiation. These results are consistent with a role for TLS in homologous DNA pairing and recombination.
Assuntos
Infertilidade/genética , Tolerância a Radiação/genética , Ribonucleoproteínas/genética , Fatores Associados à Proteína de Ligação a TATA , Animais , Aberrações Cromossômicas , Transtornos Cromossômicos , Embrião de Mamíferos/efeitos da radiação , Feminino , Fibroblastos , Genitália/patologia , Genitália/efeitos da radiação , Ribonucleoproteínas Nucleares Heterogêneas , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , RNA/metabolismo , RNA Mensageiro/efeitos dos fármacos , Proteína FUS de Ligação a RNA , Proteínas de Ligação a RNA/genética , Radiação Ionizante , Recombinação Genética , Ribonucleoproteínas/metabolismo , Espermatozoides/patologia , Espermatozoides/efeitos da radiação , Complexo Sinaptonêmico/genética , Testículo/efeitos da radiação , Fatores de Transcrição/metabolismo , Raios UltravioletaRESUMO
Homologous recombination plays a fundamental role in DNA double-strand break repair. Previously, we detected two mammalian nuclear proteins of 100 and 75 kDa (POMp100 and POMp75, respectively) that are able to promote homologous DNA pairing, a key step in homologous recombination. Here we describe the identification of human (h) POMp75 as the pro-oncoprotein TLS/FUS. hPOMp75/TLS binds both single- and double-stranded DNAs and mediates annealing of complementary DNA strands. More important, it promotes the uptake of a single-stranded oligonucleotide into a homologous superhelical DNA to form a D-loop. The formation of a D-loop is an essential step in DNA double-strand break repair through recombination. DNA annealing and D-loop formation catalyzed by hPOMp75/TLS require Mg(2+) and are ATP-independent. Interestingly, the oncogenic fusion form TLS-CHOP is not able to promote DNA pairing. These data suggest a possible role for hPOMp75/TLS in maintenance of genomic integrity.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , DNA/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Pareamento de Bases , Ligação Competitiva , DNA/química , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas , Humanos , Peso Molecular , Proteínas Nucleares/química , Proteínas Nucleares/isolamento & purificação , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/metabolismo , Proteína FUS de Ligação a RNA , Fator de Transcrição CHOPRESUMO
Recombination protein complex RC-1, purified from calf thymus nuclear extracts, catalyzes cell-free DNA strand transfer and repair of gaps and deletions through DNA recombination. DNA polymerase E, DNA ligase III and a DNA structure-specific endonuclease co-purify with the five polypeptide complex. Here we describe the identification of two hitherto unknown subunits of RC-1. N-terminal amino acid sequences of the 160 and 130 kDa polypeptides display up to 100% identity to proteins of the structural maintenance of chromosomes (SMC) subfamilies 1 and 2. SMC proteins are involved in mitotic chromosome segregation and condensation, as well as in certain DNA repair pathways in fission (rad18 gene) and budding (RHC18 gene) yeast. The assignment was substantiated by immuno-cross-reactivity of the RC-1 subunits with polyclonal antibodies specific for Xenopus laevis SMC proteins. These antibodies, and polyclonal antibodies directed against the bovine 160 and 130 kDa polypeptides, named BSMC1 and BSMC2 (bovine SMC), inhibited RC-1-mediated DNA transfer, indicating that the SMC proteins are necessary components of the reaction. Two independent assays revealed DNA reannealing activity of RC-1, which resides in its BSMC subunits, thereby demonstrating a novel function of these proteins. To our knowledge, this is the first evidence for the association of mammalian SMC proteins with a multiprotein complex harboring, among others, DNA recombination, DNA ligase and DNA polymerase activities.