RESUMO
The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.
Assuntos
Bacteriófagos/fisiologia , Empacotamento do DNA , Proteínas Virais/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Bacteriófagos/ultraestrutura , Domínio Catalítico , Dicroísmo Circular , Endodesoxirribonucleases/metabolismo , Escherichia coli/virologia , Histidina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Subunidades Proteicas/metabolismo , Proteínas Virais/químicaRESUMO
BACKGROUND: The CTCF insulator protein is a highly conserved zinc finger protein that has been implicated in many aspects of gene regulation and nuclear organization. The protein has been hypothesized to organize the human genome by forming DNA loops. RESULTS: In this paper, we report biochemical evidence to support the role for CTCF in forming DNA loops. We have measured DNA bending by CTCF at the chicken HS4 ß-globin FII insulator element in vitro and have observed a unique DNA structure with aberrant electrophoretic mobility which we believe to be a DNA loop. CTCF is able to form this unusual DNA structure at two other binding sites: the c-myc P2 promoter and the chicken F1 lysozyme gene silencer. We also demonstrate that the length though not the sequence of the DNA downstream of the binding site is important for the ability of CTCF to form this unusual DNA structure. We hypothesize that a single CTCF protein molecule is able to act as a "looper" possibly through the use of several of its zinc fingers. CONCLUSIONS: CTCF is able to form an unusual DNA structure through the zinc finger domain of the protein. This unusual DNA structure is formed in a directional manner by the CTCF protein. The findings described in this paper suggest mechanisms by which CTCF is able to form DNA loops, organize the mammalian genome and function as an insulator protein.
Assuntos
DNA/metabolismo , Proteínas Repressoras/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Fator de Ligação a CCCTC , Galinhas , DNA/química , Camundongos , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Repressoras/química , Dedos de ZincoRESUMO
The final step in the morphogenesis of long-tailed double-stranded DNA bacteriophages is the joining of the DNA-filled head to the tail. The connector is a specialized structure of the head that serves as the interface for tail attachment and the point of egress for DNA from the head during infection. Here, we report the determination of a 2.1 A crystal structure of gp6 of bacteriophage HK97. Through structural comparisons, functional studies, and bioinformatic analysis, gp6 has been determined to be a component of the connector of phage HK97 that is evolutionarily related to gp15, a well-characterized connector component of bacteriophage SPP1. Whereas the structure of gp15 was solved in a monomeric form, gp6 crystallized as an oligomeric ring with the dimensions expected for a connector protein. Although this ring is composed of 13 subunits, which does not match the symmetry of the connector within the phage, sequence conservation and modeling of this structure into the cryo-electron microscopy density of the SPP1 connector indicate that this oligomeric structure represents the arrangement of gp6 subunits within the mature phage particle. Through sequence searches and genomic position analysis, we determined that gp6 is a member of a large family of connector proteins that are present in long-tailed phages. We have also identified gp7 of HK97 as a homologue of gp16 of phage SPP1, which is the second component of the connector of this phage. These proteins are members of another large protein family involved in connector assembly.
Assuntos
Siphoviridae/química , Proteínas Virais/química , Sequência de Aminoácidos , Cristalografia por Raios X , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestrutura , Homologia de Sequência de Aminoácidos , Siphoviridae/genética , Siphoviridae/fisiologia , Siphoviridae/ultraestrutura , Eletricidade Estática , Homologia Estrutural de Proteína , Proteínas Virais/genética , Proteínas Virais/fisiologia , Proteínas Virais/ultraestrutura , Montagem de VírusRESUMO
The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sequência de Aminoácidos , Animais , Fator de Ligação a CCCTC , Estruturas do Núcleo Celular/metabolismo , Cisteína Endopeptidases , DNA/metabolismo , Proteínas de Ligação a DNA/química , Endopeptidases/metabolismo , Células HeLa , Humanos , Isoenzimas/metabolismo , Ligases , Camundongos , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Proteínas do Grupo Polycomb , Regiões Promotoras Genéticas/genética , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Repressoras/química , Proteína SUMO-1/metabolismo , Ubiquitina-Proteína Ligases , Ubiquitinas/metabolismoRESUMO
The paternal repression of several genes in human chromosome 11p15.5 (mouse chromosome 7) is associated with paternal expression of a transcript called KCNQ1OT1 (also known as LIT1). This long transcript originates from a promoter that resides in a CpG island in intron 10 of the KCNQ1 gene and runs in an antisense orientation to the direction of the coding KCNQ1 transcript. The CpG island is maternally methylated but paternally nonmethylated. The CpG island loses its maternal methylation in over 50% of cases of Beckwith-Wiedemann syndrome who lack uniparental disomy. This loss is usually accompanied by biallelic expression of the KCNQ1OT1 transcript. We have examined the methylation status of this CpG island in somatic cell hybrids and diploid lymphoblasts using Southern hybridization and bisulfite sequencing techniques. We find that the maternal copy of the CpG island is methylated at all CpGs examined within the CpG island and uniformly paternally unmethylated. In addition, in BWS patients who have lost methylation of the CpG island, this loss occurs throughout the CpG island. Finally, we find that there is a switch in methylation patterns outside the CpG island from maternal methylation within the island to predominantly paternal methylation at sites flanking the CpG island.
Assuntos
Cromossomos Humanos Par 11/genética , Ilhas de CpG/genética , Metilação de DNA , Impressão Genômica/genética , Proteínas de Membrana/genética , Animais , Síndrome de Beckwith-Wiedemann/genética , Humanos , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Antissenso/genética , Análise de Sequência de DNA/métodos , Transcrição GênicaAssuntos
Recombinases/metabolismo , Tirosina/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/química , DNA/metabolismo , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/metabolismo , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Integrases/química , Integrases/metabolismo , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Recombinases/química , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The human 11p15.5 region contains several maternally and paternally imprinted genes. Dysregulation of imprinting of some of these genes occurs in the Beckwith-Wiedemann syndrome and several tumors. Imprinting in this region is controlled by two imprinting control regions (ICR). ICR1 acts as an insulator that regulates the reciprocal imprinting of the IGF2 and H19 genes. A differentially methylated region in ICR2 regulates the expression of a long transcript called KCNQ1OT1. This paternally expressed transcript negatively regulates several paternally imprinted genes around ICR2. Biallelic expression of the KCNQ1OT1 transcript is the primary molecular defect in over 50% of cases of Beckwith-Wiedemann syndrome. To understand the role of KCNQ1OT1 in regulating ICR2 we characterized its promoter. The critical promoter is approximately 300 bp and it is surrounded by inhibitory elements within the CpG island. The promoter activity is strongly inhibited by cytosine methylation in keeping with the finding that the inactive maternal promoter is methylated in vivo. We have identified the transcription start sites and four CCAAT boxes upstream of the 5'-most start site. Mutation of the CCAAT boxes produced impairment of promoter activity. Transfection and gel mobility shift experiments suggest that binding of the factor NF-Y to the CCAAT boxes is important for promoter activity.
Assuntos
Cromossomos Humanos Par 11/genética , Impressão Genômica/genética , Proteínas de Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Regiões Promotoras Genéticas/genética , Sequência de Bases , Fator de Ligação a CCAAT/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Metilação de DNA , Genes Reporter/genética , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Mutação/genética , Ligação Proteica , Elementos de Resposta/genética , Sítio de Iniciação de TranscriçãoRESUMO
Wiedemann-Beckwith syndrome (WBS) is a human overgrowth disorder that is accompanied by an increased risk of embryonal tumors and is associated with dsyregulation of the imprinting of genes in chromosome 11p15.5. Maternally inherited mutations in the imprinted CDKN1C gene are known to be associated with WBS. We have identified a novel mutation in several members of a large family affected by WBS. The mutation is a G --> T change in a run of seven G's near the 5' splice site of intron 3. All obligate carriers and affected individuals carry the mutation, and in each affected case, the allele was inherited maternally, strongly suggesting a role in causing WBS. The mutation is located in a poly-G tract in the intron; intronic G-rich sequences in other genes have been shown to have a role in promoting splicing. In transfected 293HEK cells, we found that the G --> T mutation reduced splicing efficiency. Mutation of all seven G's in the poly-G tract further reduced splicing efficiency, supporting a role for the G-tract as a splicing enhancer. The fibroblasts of one affected patient showed a similar reduction in splicing efficiency. Maternal monoallelic expression of CDKN1C was verified in this patient cell line. However, the total amount of spliced message was not reduced by the mutation in spite of the reduced efficiency of splicing. We discuss the possible role of the splicing defect in the pathogenesis of WBS in this pedigree.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Proteínas Nucleares/genética , Splicing de RNA , Sequência de Bases , Linhagem Celular Transformada , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p57 , DNA , Primers do DNA , Humanos , Íntrons , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The Cre protein of bacteriophage P1 is a tyrosine recombinase and catalyzes recombination via formation of a covalent protein-DNA complex and a Holliday junction intermediate. Several co-crystal structures of Cre bound to its target lox site have provided novel insights into its biochemical activities. We have used these structures to guide the mutagenesis of several Cre residues that contact the lox spacer region and/or are involved in intersubunit protein-protein interactions. None of the mutant proteins had significant defects in DNA binding, DNA bending, or strand-specific initiation of recombination. We have identified novel functions of several amino acids that are involved in three aspects of the Cre reaction. 1) Single mutation of several NH2-terminal basic residues that contact the spacer region of loxP caused the accumulation of Holliday junction (HJ) intermediates but only a modest impairment of recombination. These residues may be involved in the isomerization of the Holliday intermediate. 2) We identified three new residues (Arg-118, Lys-122, and Glu-129) that are involved in synapsis. Cre R118A, K122A, and E129Q were catalytically competent. 3) Mutations E129R, Q133H, and K201A inactivated catalysis by the protein. The function of these Cre residues in recombination is discussed.
Assuntos
Bacteriófago P1/metabolismo , Integrases/química , Integrases/fisiologia , Proteínas Virais/química , Proteínas Virais/fisiologia , Sequência de Bases , Catálise , Cristalografia por Raios X , DNA/metabolismo , Eletroforese em Gel de Poliacrilamida , Integrases/genética , Modelos Biológicos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Recombinação Genética , Temperatura , Fatores de Tempo , Proteínas Virais/genéticaRESUMO
BACKGROUND: While conventional cloning methods using restriction enzymes and polynucleotide ligase are adequate for most DNAs, fragments made by the polymerase chain reaction are difficult to clone because the amplifying DNA polymerase tends to add untemplated nucleotides to the 3'-termini of the amplified strands. Conservative site-specific recombinases offer an efficient alternative to conventional cloning methods. RESULTS: In this paper I describe the use of the Flp site-specific recombinase for cloning PCR-amplified fragments. A DNA fragment is amplified with primers that contain at their ends inverted target sequences for Flp. Flp readily recombines these fragments in vitro into a vector that also contains two inverted Flp target sequences surrounding the alpha-complementing region of the lacZ gene of E. coli. The recombinants are conveniently detected as white colonies by the familiar blue/white screening test for lacZ activity. A useful feature of the system is that both orientations of the inserted DNA are usually obtained. If the recipient vector is cut between the two inverted Flp targets, Flp "heals" the double-strand break by inserting a linear fragment flanked by Flp targets. CONCLUSION: This system ("The Flp Double Cross System") should be useful for cloning multiple PCR fragments into many sites in several vectors. It has certain advantages over other available recombinase-based cloning procedures.
Assuntos
Clonagem Molecular/métodos , DNA Nucleotidiltransferases/genética , DNA Bacteriano/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Proteínas da Membrana Bacteriana Externa/genética , Cromossomos Humanos Par 11/genética , DNA Recombinante/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Humanos , Íntrons/genética , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Canais de Potássio/genética , beta-Galactosidase/genéticaRESUMO
The imprinting of the genes on human chromosome 11p15.5 is thought to be controlled by two imprinting control regions located in two differentially methylated CpG islands upstream of the H19 gene (H19 DMR) and in intron 10 of the KCNQ1 gene (KvDMR). We have examined sequences in the human 11p15.5 genomic imprinted region for the presence of insulators and silencers using a position- and enhancer-dependent stable transfection assay. We have confirmed the existence of insulators in H19 DMR and discovered two novel insulators in the IGF2 gene. We have also found two novel silencer sequences; one is located in KvDMR, a region that is thought to contain the promoter for the KCNQ1OT1 transcript, and another is in the CDKN1C gene. We have demonstrated binding of CTCF protein in vitro to all the insulator and silencer sequences that we have detected. We discuss the differences in the regulation of imprinting controlled by the two imprinting control regions in chromosome 11p.
Assuntos
Cromossomos Humanos Par 11/genética , Impressão Genômica/genética , Elementos Isolantes/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Elementos Silenciadores Transcricionais/genética , Fator de Ligação a CCCTC , Mapeamento Cromossômico , Inibidor de Quinase Dependente de Ciclina p57 , Primers do DNA , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Células K562 , Canais de Potássio KCNQ , Canal de Potássio KCNQ1 , Modelos Genéticos , Proteínas Nucleares/genética , Plasmídeos , Canais de Potássio/genética , RNA Longo não Codificante , RNA não Traduzido/genética , Proteínas Repressoras/metabolismoRESUMO
Cre initiates recombination by preferentially exchanging the bottom strands of the loxP site to form a Holliday intermediate, which is then resolved on the top strands. We previously found that the scissile AT and GC base pairs immediately 5' to the scissile phosphodiester bonds are critical in determining this order of strand exchange. We report here that the scissile base pairs also influence the Cre-induced DNA bends, the position of which correlates with the initial site of strand exchange. The binding of one Cre molecule to a loxP site induces a approximately 35 degrees asymmetric bend adjacent to the scissile GC base pair. The binding of two Cre molecules to a loxP site induces a approximately 55 degrees asymmetric bend near the center of the spacer region with a slight bias toward the scissile A. Lys-86, which contacts the scissile nucleotides, is important for establishing the bend near the scissile GC base pair when one Cre molecule is bound but has little role in positioning the bend when two Cre molecules are bound to a loxP site. We present a model relating the position of the Cre-induced bends to the order of strand exchange in the Cre-catalyzed recombination reaction.
Assuntos
DNA/química , Integrases/metabolismo , Recombinação Genética , Proteínas Virais/metabolismo , Substituição de Aminoácidos , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , DNA/metabolismo , Metilação de DNA , Integrases/química , Integrases/genética , Cinética , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Especificidade por Substrato , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Conservative site-specific recombinases of the integrase family carry out recombination via a Holliday intermediate. The Cre recombinase, a member of the integrase family, was previously shown to initiate recombination by cleaving and exchanging preferentially on the bottom strand of its loxP target sequence. We have confirmed this strand bias for an intermolecular recombination reaction that used wild-type loxP sites and Cre protein. We have examined the sequence determinants for this strand preference by selectively mutating the two asymmetric scissile base-pairs in the lox site (those immediately adjacent to the sites of cleavage by Cre). We found that the initial strand exchange occurs preferentially next to the scissile G residue. Resolution of the Holliday intermediate thus formed takes place preferentially next to the scissile A residue. Lys86, which contacts the scissile nucleotides in the Cre-lox crystal structures, was important for establishing the strand preference in the resolution of the loxP-Holliday intermediate, but not for the initiation of recombination between loxP sites.