RESUMO
Homing endonucleases are highly specific catalysts of DNA strand breaks, leading to the transfer of mobile intervening sequences containing the endonuclease ORF. We have determined the structure and DNA recognition behavior of I-CeuI, a homodimeric LAGLIDADG endonuclease from Chlamydomonas eugametos. This symmetric endonuclease displays unique structural elaborations on its core enzyme fold, and it preferentially cleaves a highly asymmetric target site. This latter property represents an early step, prior to gene fusion, in the generation of asymmetric DNA binding platforms from homodimeric ancestors. The divergence of the sequence, structure, and target recognition behavior of homing endonucleases, as illustrated by this study, leads to the invasion of novel genomic sites by mobile introns during evolution.
Assuntos
Chlamydomonas/enzimologia , DNA/química , Endodesoxirribonucleases/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Endodesoxirribonucleases/classificação , Dados de Sequência Molecular , Filogenia , Conformação Proteica , Dobramento de Proteína , Especificidade por SubstratoRESUMO
The homing endonuclease I-CreI recognizes and cleaves a particular 22 bp DNA sequence. The crystal structure of I-CreI bound to homing site DNA has previously been determined, leading to a number of predictions about specific protein-DNA contacts. We test these predictions by analyzing a set of endonuclease mutants and a complementary set of homing site mutants. We find evidence that all structurally predicted I-CreI/DNA contacts contribute to DNA recognition and show that these contacts differ greatly in terms of their relative importance. We also describe the isolation of a collection of altered specificity I-CreI derivatives. The in vitro DNA-binding and cleavage properties of two such endonucleases demonstrate that our genetic approach is effective in identifying homing endonucleases that recognize and cleave novel target sequences.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , DNA/genética , Enzimas de Restrição do DNA/genética , Escherichia coli/genética , Cinética , Mutação , Plasmídeos/genética , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA/genética , Endodesoxirribonucleases/metabolismo , Mutagênese , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalização , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Enzimas de Restrição do DNA/genética , Endodesoxirribonucleases/genética , Escherichia coli/genética , Íntrons/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/genética , Conformação Proteica , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade por SubstratoRESUMO
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco/fisiologia , Animais , Proteínas de Transporte/genética , Células Cultivadas , Perfilação da Expressão Gênica , Humanos , Complexos Multiproteicos , Proteínas de Neoplasias , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 2 , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Transdução de Sinais/fisiologia , Células-Tronco/citologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The mixed-lineage leukemia (MLL1/ALL-1/HRX) histone methyltransferase is involved in the epigenetic maintenance of transcriptional memory and the pathogenesis of human leukemias. To understand its role in cell type specification, we determined the human genomic binding sites of MLL1. We found that MLL1 functions as a human equivalent of yeast Set1. Like Set1, MLL1 localizes with RNA polymerase II (Pol II) to the 5' end of actively transcribed genes, where histone H3 lysine 4 trimethylation occurs. Consistent with this global role in transcription, MLL1 also localizes to microRNA (miRNA) loci that are involved in leukemia and hematopoiesis. In contrast to the 5' proximal binding behavior at most protein-coding genes, MLL1 occupies an extensive domain within a transcriptionally active region of the HoxA cluster. The ability of MLL1 to serve as a start site-specific global transcriptional regulator and to participate in larger chromatin domains at the Hox genes reveals dual roles for MLL1 in maintenance of cellular identity.
Assuntos
Cromossomos Humanos Par 7/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Genes Reguladores/genética , Genoma Humano , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 7/genética , Proteínas de Ligação a DNA/genética , Genes Homeobox/genética , Genômica/métodos , Histona Metiltransferases , Humanos , MicroRNAs/metabolismo , Proteína de Leucina Linfoide-Mieloide , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Metiltransferases , Proto-Oncogenes/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genéticaRESUMO
An oil tanker ran aground on the Galapagos island of San Cristóbal on 17 January 2001, spilling roughly three million litres of diesel and bunker oil. The slick started to spread westwards and was dispersed by strong currents, so only a few marine animals were killed immediately as a result. Here we draw on the long-term data sets gathered before the spill to show that a population of marine iguanas (Amblyrhychus cristatus) on Sante Fe island suffered a massive 62% mortality in the year after the accident, due to a small amount of residual oil contamination in the sea. Another population on the more remote island of Genovesa was unaffected.
Assuntos
Poluição Ambiental/efeitos adversos , Eucariotos/efeitos dos fármacos , Cadeia Alimentar , Iguanas/fisiologia , Petróleo/efeitos adversos , Corticosteroides/sangue , Animais , Digestão/efeitos dos fármacos , Desastres , Equador , Eucariotos/fisiologia , Gasolina/efeitos adversos , Iguanas/sangue , Modelos Biológicos , Simbiose/efeitos dos fármacosRESUMO
We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.
Assuntos
Endonucleases/química , Endonucleases/metabolismo , Engenharia de Proteínas/métodos , Algoritmos , Sequência de Bases , Sítios de Ligação , Catálise , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/química , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Desoxirribonucleases de Sítio Específico do Tipo I/metabolismo , Endonucleases/genética , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , TermodinâmicaRESUMO
The LAGLIDADG homing endonucleases include free-standing homodimers, pseudosymmetric monomers, and related enzyme domains embedded within inteins. DNA-bound structures of homodimeric I-CreI and monomeric I-SceI indicate that three catalytic divalent metal ions are distributed across a pair of overlapping active sites, with one shared metal participating in both strand cleavage reactions. These structures differ in the precise position and binding interactions of the metals. We have studied the metal dependence for the I-CreI homodimer using site-directed mutagenesis of active site residues and assays of binding affinity and cleavage activity. We have also reassessed the binding of a nonactivating metal ion (calcium) in the wild-type enzyme-substrate complex, and determined the DNA-bound structure of two inactive enzyme mutants. The conclusion of these studies is that the catalytic mechanism of symmetric LAGLIDADG homing endonucleases, and probably many of their monomeric cousins, involves a canonical two-metal mechanism in each of two active sites, which are chemically and structurally tethered to one another by a shared metal ion. Failure to occupy the shared metal site, as observed in the presence of calcium or when the metal-binding side chain from the LAGLIDADG motif (Asp 20) is mutated to asparagine, prevents cleavage by the enzyme.