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1.
Cell Rep ; 43(4): 114001, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38547127

RESUMO

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.


Assuntos
Clivagem do DNA , Paramecium , Paramecium/genética , Paramecium/metabolismo , Quebras de DNA de Cadeia Dupla , Genoma de Protozoário , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Reparo do DNA , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Reparo do DNA por Junção de Extremidades
2.
Mob DNA ; 12(1): 12, 2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33926516

RESUMO

BACKGROUND: Transposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive. RESULTS: We provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state. CONCLUSIONS: The present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

3.
PLoS Genet ; 16(4): e1008723, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298257

RESUMO

Gene duplication and diversification drive the emergence of novel functions during evolution. Because of whole genome duplications, ciliates from the Paramecium aurelia group constitute a remarkable system to study the evolutionary fate of duplicated genes. Paramecium species harbor two types of nuclei: a germline micronucleus (MIC) and a somatic macronucleus (MAC) that forms from the MIC at each sexual cycle. During MAC development, ~45,000 germline Internal Eliminated Sequences (IES) are excised precisely from the genome through a 'cut-and-close' mechanism. Here, we have studied the P. tetraurelia paralogs of KU80, which encode a key DNA double-strand break repair factor involved in non-homologous end joining. The three KU80 genes have different transcription patterns, KU80a and KU80b being constitutively expressed, while KU80c is specifically induced during MAC development. Immunofluorescence microscopy and high-throughput DNA sequencing revealed that Ku80c stably anchors the PiggyMac (Pgm) endonuclease in the developing MAC and is essential for IES excision genome-wide, providing a molecular explanation for the previously reported Ku-dependent licensing of DNA cleavage at IES ends. Expressing Ku80a under KU80c transcription signals failed to complement a depletion of endogenous Ku80c, indicating that the two paralogous proteins have distinct properties. Domain-swap experiments identified the α/ß domain of Ku80c as the major determinant for its specialized function, while its C-terminal part is required for excision of only a small subset of IESs located in IES-dense regions. We conclude that Ku80c has acquired the ability to license Pgm-dependent DNA cleavage, securing precise DNA elimination during programmed rearrangements. The present study thus provides novel evidence for functional diversification of genes issued from a whole-genome duplication.


Assuntos
Genoma de Protozoário , Instabilidade Genômica , Autoantígeno Ku/genética , Proteínas de Protozoários/genética , Duplicação Gênica , Autoantígeno Ku/química , Autoantígeno Ku/metabolismo , Macronúcleo/genética , Macronúcleo/metabolismo , Micronúcleo Germinativo/genética , Micronúcleo Germinativo/metabolismo , Paramecium/genética , Paramecium/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo
4.
Elife ; 72018 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-30223944

RESUMO

The domestication of transposable elements has repeatedly occurred during evolution and domesticated transposases have often been implicated in programmed genome rearrangements, as remarkably illustrated in ciliates. In Paramecium, PiggyMac (Pgm), a domesticated PiggyBac transposase, carries out developmentally programmed DNA elimination, including the precise excision of tens of thousands of gene-interrupting germline Internal Eliminated Sequences (IESs). Here, we report the discovery of five groups of distant Pgm-like proteins (PgmLs), all able to interact with Pgm and essential for its nuclear localization and IES excision genome-wide. Unlike Pgm, PgmLs lack a conserved catalytic site, suggesting that they rather have an architectural function within a multi-component excision complex embedding Pgm. PgmL depletion can increase erroneous targeting of residual Pgm-mediated DNA cleavage, indicating that PgmLs contribute to accurately position the complex on IES ends. DNA rearrangements in Paramecium constitute a rare example of a biological process jointly managed by six distinct domesticated transposases.


Assuntos
DNA de Protozoário/genética , Paramecium/genética , Transposases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Genoma de Protozoário , Funções Verossimilhança , Modelos Biológicos , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Transposases/química , Transposases/genética
5.
Nucleic Acids Res ; 46(5): 2660-2677, 2018 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-29385532

RESUMO

The piggyBac transposase (PB) is distinguished by its activity and utility in genome engineering, especially in humans where it has highly promising therapeutic potential. Little is known, however, about the structure-function relationships of the different domains of PB. Here, we demonstrate in vitro and in vivo that its C-terminal Cysteine-Rich Domain (CRD) is essential for DNA breakage, joining and transposition and that it binds to specific DNA sequences in the left and right transposon ends, and to an additional unexpectedly internal site at the left end. Using NMR, we show that the CRD adopts the specific fold of the cross-brace zinc finger protein family. We determine the interaction interfaces between the CRD and its target, the 5'-TGCGT-3'/3'-ACGCA-5' motifs found in the left, left internal and right transposon ends, and use NMR results to propose docking models for the complex, which are consistent with our site-directed mutagenesis data. Our results provide support for a model of the PB/DNA interactions in the context of the transpososome, which will be useful for the rational design of PB mutants with increased activity.


Assuntos
Proteínas de Ligação a DNA/química , Transposases/química , Sequência de Bases , DNA/química , DNA/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Simulação de Acoplamento Molecular , Mutação , Ligação Proteica , Domínios Proteicos , Transposases/genética , Transposases/metabolismo , Zinco/química , Dedos de Zinco
6.
Nucleic Acids Res ; 45(6): 3204-3216, 2017 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-28104713

RESUMO

During sexual processes, the ciliate Paramecium eliminates 25-30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.


Assuntos
Clivagem do DNA , Transposases/genética , Sequência de Bases , Genoma , Mutação , Paramecium tetraurellia/genética , Domínios Proteicos , Multimerização Proteica , Deleção de Sequência , Transgenes , Transposases/química , Transposases/metabolismo
7.
PLoS Genet ; 10(8): e1004552, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25166013

RESUMO

During somatic differentiation, physiological DNA double-strand breaks (DSB) can drive programmed genome rearrangements (PGR), during which DSB repair pathways are mobilized to safeguard genome integrity. Because of their unique nuclear dimorphism, ciliates are powerful unicellular eukaryotic models to study the mechanisms involved in PGR. At each sexual cycle, the germline nucleus is transmitted to the progeny, but the somatic nucleus, essential for gene expression, is destroyed and a new somatic nucleus differentiates from a copy of the germline nucleus. In Paramecium tetraurelia, the development of the somatic nucleus involves massive PGR, including the precise elimination of at least 45,000 germline sequences (Internal Eliminated Sequences, IES). IES excision proceeds through a cut-and-close mechanism: a domesticated transposase, PiggyMac, is essential for DNA cleavage, and DSB repair at excision sites involves the Ligase IV, a specific component of the non-homologous end-joining (NHEJ) pathway. At the genome-wide level, a huge number of programmed DSBs must be repaired during this process to allow the assembly of functional somatic chromosomes. To understand how DNA cleavage and DSB repair are coordinated during PGR, we have focused on Ku, the earliest actor of NHEJ-mediated repair. Two Ku70 and three Ku80 paralogs are encoded in the genome of P. tetraurelia: Ku70a and Ku80c are produced during sexual processes and localize specifically in the developing new somatic nucleus. Using RNA interference, we show that the development-specific Ku70/Ku80c heterodimer is essential for the recovery of a functional somatic nucleus. Strikingly, at the molecular level, PiggyMac-dependent DNA cleavage is abolished at IES boundaries in cells depleted for Ku80c, resulting in IES retention in the somatic genome. PiggyMac and Ku70a/Ku80c co-purify as a complex when overproduced in a heterologous system. We conclude that Ku has been integrated in the Paramecium DNA cleavage factory, enabling tight coupling between DSB introduction and repair during PGR.


Assuntos
Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Rearranjo Gênico/genética , Instabilidade Genômica , Paramecium tetraurellia/genética , Sequência de Bases/genética , Núcleo Celular/genética , Clivagem do DNA , Reparo do DNA , DNA de Protozoário/genética , Genoma , Células Germinativas , Transposases/metabolismo
8.
EMBO J ; 31(18): 3757-67, 2012 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-22863778

RESUMO

Toxigenic conversion of Vibrio cholerae bacteria results from the integration of a filamentous phage, CTX phage. Integration is driven by the bacterial Xer recombinases, which catalyse the exchange of a single pair of strands between the phage single-stranded DNA and the host double-stranded DNA genomes; replication is thought to convert the resulting pseudo-Holliday junction (HJ) intermediate into the final recombination product. The natural tendency of the Xer recombinases to recycle HJ intermediates back into substrate should thwart this integration strategy, which prompted a search for additional co-factors aiding directionality of the process. Here, we show that Endo III, a ubiquitous base excision repair enzyme, facilitates CTX phage-integration in vivo. In vitro, we show that it prevents futile Xer recombination cycles by impeding new rounds of strand exchanges once the pseudo-HJ is formed. We further demonstrate that this activity relies on the unexpected ability of Endo III to bind to HJs even in the absence of the recombinases. These results explain how tandem copies of the phage genome can be created, which is crucial for subsequent virion production.


Assuntos
Bacteriófagos/metabolismo , Toxina da Cólera/metabolismo , Reparo do DNA , DNA Cruciforme , Desoxirribonuclease (Dímero de Pirimidina)/genética , Proteínas de Escherichia coli/genética , Vibrio cholerae/metabolismo , Catálise , DNA/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Biblioteca Gênica , Genoma , Glicosilação , Lisogenia , Modelos Genéticos , Mutagênese , Mutação , Oligonucleotídeos/genética , Fases de Leitura Aberta , Recombinases/metabolismo , Recombinação Genética
9.
Int J Evol Biol ; 2012: 436196, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22888464

RESUMO

Sequences related to transposons constitute a large fraction of extant genomes, but insertions within coding sequences have generally not been tolerated during evolution. Thanks to their unique nuclear dimorphism and to their original mechanism of programmed DNA elimination from their somatic nucleus (macronucleus), ciliates are emerging model organisms for the study of the impact of transposable elements on genomes. The germline genome of the ciliate Paramecium, located in its micronucleus, contains thousands of short intervening sequences, the IESs, which interrupt 47% of genes. Recent data provided support to the hypothesis that an evolutionary link exists between Paramecium IESs and Tc1/mariner transposons. During development of the macronucleus, IESs are excised precisely thanks to the coordinated action of PiggyMac, a domesticated piggyBac transposase, and of the NHEJ double-strand break repair pathway. A PiggyMac homolog is also required for developmentally programmed DNA elimination in another ciliate, Tetrahymena. Here, we present an overview of the life cycle of these unicellular eukaryotes and of the developmentally programmed genome rearrangements that take place at each sexual cycle. We discuss how ancient domestication of a piggyBac transposase might have allowed Tc1/mariner elements to spread throughout the germline genome of Paramecium, without strong counterselection against insertion within genes.

10.
Indian J Med Res ; 133: 195-200, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21415494

RESUMO

One of the major pathogenic determinants of Vibrio cholerae, the cholera toxin, is encoded in the genome of a filamentous phage, CTXφ. CTXφ makes use of the chromosome dimer resolution system of V. cholerae to integrate its single stranded genome into one, the other, or both V. cholerae chromosomes. Here, we review current knowledge about this smart integration process.


Assuntos
Bacteriófagos/genética , Toxina da Cólera/genética , Cólera/microbiologia , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Integração Viral , Sequência de Bases , Genoma Bacteriano , Genoma Viral , Vibrio cholerae/química
11.
Proc Natl Acad Sci U S A ; 108(6): 2516-21, 2011 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-21262799

RESUMO

Most strains of Vibrio cholerae are not pathogenic or cause only local outbreaks of gastroenteritis. Acquisition of the capacity to produce the cholera toxin results from a lysogenic conversion event due to a filamentous bacteriophage, CTX. Two V. cholerae tyrosine recombinases that normally serve to resolve chromosome dimers, XerC and XerD, promote CTX integration by directly recombining the ssDNA genome of the phage with the dimer resolution site of either or both V. cholerae chromosomes. This smart mechanism renders the process irreversible. Many other filamentous vibriophages seem to attach to chromosome dimer resolution sites and participate in the rapid and continuous evolution of toxigenic V. cholerae strains. We analyzed the molecular mechanism of integration of VGJ, a representative of the largest family of these phages. We found that XerC and XerD promote the integration of VGJ into a specific chromosome dimer resolution site, and that the dsDNA replicative form of the phage is recombined. We show that XerC and XerD can promote excision of the integrated prophage, and that this participates in the production of new extrachromosomal copies of the phage genome. We further show how hybrid molecules harboring the concatenated genomes of CTX and VGJ can be produced efficiently. Finally, we discuss how the integration and excision mechanisms of VGJ can explain the origin of recent epidemic V. cholerae strains.


Assuntos
Bacteriófagos/fisiologia , Cromossomos Bacterianos , Variação Genética , Vibrio cholerae , Integração Viral/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cólera/epidemiologia , Cólera/genética , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/virologia , Genoma Viral/fisiologia , Humanos , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Vibrio cholerae/virologia
12.
Proc Natl Acad Sci U S A ; 107(9): 4377-82, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20133778

RESUMO

Cholera toxin is encoded in the genome of CTXvarphi, a lysogenic filamentous phage of Vibrio cholerae. CTXvarphi variants contribute to the genetic diversity of cholera epidemic strains. It has been shown that the El Tor variant of CTXvarphi hijacks XerC and XerD, two host-encoded tyrosine recombinases that normally function to resolve chromosome dimers, to integrate at dif1, the dimer resolution site of the larger of the two V. cholerae chromosomes. However, the exact mechanism of integration of CTXvarphi and the rules governing its integration remained puzzling, with phage variants integrated at either or both dimer resolution sites of the two V. cholerae chromosomes. We designed a genetic system to determine experimentally the tropism of integration of CTXvarphi and thus define rules of compatibility between phage variants and dimer resolution sites. We then showed in vitro how these rules are explained by the direct integration of the single-stranded phage genome into the double-stranded bacterial genome. Finally, we showed how the evolution of phage attachment and chromosome dimer resolution sites contributes to the generation of genetic diversity among cholera epidemic strains.


Assuntos
Bacteriófagos/fisiologia , Toxina da Cólera/genética , Vibrio cholerae/virologia , Tropismo Viral , Integração Viral , Sequência de Bases , Cromossomos Bacterianos , Recombinação Genética
13.
Mol Cell Biol ; 29(21): 5889-99, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19720743

RESUMO

Tn5 transposase cleaves the transposon end using a hairpin intermediate on the transposon end. This involves a flipped base that is stacked against a tryptophan residue in the protein. However, many other members of the cut-and-paste transposase family, including the RAG1 protein, produce a hairpin on the flanking DNA. We have investigated the reversed polarity of the reaction for RAG recombination. Although the RAG proteins appear to employ a base-flipping mechanism using aromatic residues, the putatively flipped base is not at the expected location and does not appear to stack against any of the said aromatic residues. We propose an alternative model in which a flipped base is accommodated in a nonspecific pocket or cleft within the recombinase. This is consistent with the location of the flipped base at position -1 in the coding flank, which can be occupied by purine or pyrimidine bases that would be difficult to stabilize using a single, highly specific, interaction. Finally, during this work we noticed that the putative base-flipping events on either side of the 12/23 recombination signal sequence paired complex are coupled to the nicking steps and serve to coordinate the double-strand breaks on either side of the complex.


Assuntos
Pareamento de Bases/genética , Quebras de DNA de Cadeia Dupla , Modelos Genéticos , Conformação de Ácido Nucleico , Recombinação Genética/genética , Éxons VDJ/genética , Animais , Pareamento Cromossômico/efeitos dos fármacos , Reagentes de Ligações Cruzadas/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteínas de Homeodomínio/metabolismo , Compostos de Manganês/farmacologia , Camundongos , Mutação/genética , Conformação de Ácido Nucleico/efeitos dos fármacos , Óxidos/farmacologia , Dímeros de Pirimidina , Recombinação Genética/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
14.
PLoS One ; 4(7): e6201, 2009 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-19593448

RESUMO

The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposon ends. This is achieved using a hairpin intermediate that requires the DNA to change conformation during the reaction. In Tn5 these changes are controlled in part by a flipped nucleoside that is stacked on a tryptophan residue in a hydrophobic pocket of the transposase. Here we have investigated the base flipping mechanism in Tn10 transposition. As in Tn5 transposition, we find that base flipping takes place after the first nick and is required for efficient hairpin formation and resolution. Experiments with an abasic substrate show that the role of base flipping in hairpin formation is to remove the base from the DNA helix. Specific interactions between the flipped base and the stacking tryptophan residue are required for hairpin resolution later in the reaction. We show that base flipping in Tn10 transposition is not a passive reaction in which a spontaneously flipped base is captured and retained by the protein. Rather, it is driven in part by a methionine probe residue that helps to force the flipped base from the base stack. Overall, it appears that base flipping in Tn10 transposition is similar to that in Tn5 transposition.


Assuntos
Pareamento de Bases , Elementos de DNA Transponíveis , DNA/metabolismo , Conformação de Ácido Nucleico , Transposases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Biocatálise , DNA/química , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Homologia de Sequência de Aminoácidos
15.
J Mol Biol ; 382(3): 567-72, 2008 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-18675277

RESUMO

Although mariner transposons are widespread in animal genomes, the vast majority harbor multiple inactivating mutations and only two naturally occurring elements are known to be active. Previously, we discovered a mariner-family transposon, Mboumar, in the satellite DNA of the ant Messor bouvieri. Several copies of the transposon contain a full-length open reading frame, including Mboumar-9, which has 64% nucleotide identity to Mos1 of Drosophila mauritiana. To determine whether Mboumar is currently active, we expressed and purified the Mboumar-9 transposase and demonstrate that it is able to catalyze the movement of a transposon from one plasmid to another in a genetic in vitro hop assay. The efficiency is comparable to that of the well-characterized mariner transposon Mos1. Transposon insertions were precise and were flanked by TA duplications, a hallmark of mariner transposition. Mboumar has been proposed to have a role in the evolution and maintenance of satellite DNA in M. bouvieri and its activity provides a means to examine the involvement of the transposon in the genome dynamics of this organism.


Assuntos
Formigas , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/metabolismo , Genes de Insetos , Proteínas de Insetos/metabolismo , Transposases/metabolismo , Animais , Formigas/genética , Formigas/metabolismo , Sequência de Bases , DNA Satélite/genética , Proteínas de Ligação a DNA/genética , Evolução Molecular , Proteínas de Insetos/genética , Dados de Sequência Molecular , Plasmídeos/genética , Plasmídeos/metabolismo , Alinhamento de Sequência , Transposases/genética
16.
Nucleic Acids Res ; 35(8): 2584-95, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17412704

RESUMO

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein-DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle.


Assuntos
Elementos de DNA Transponíveis , DNA/química , Transposases/química , Substituição de Aminoácidos , Catálise , DNA/metabolismo , Modelos Químicos , Conformação de Ácido Nucleico , Timidina/química , Transposases/genética , Transposases/metabolismo , Triptofano/química
17.
Mol Cell Biol ; 27(3): 1125-32, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17130240

RESUMO

Transposons have contributed protein coding sequences to a unexpectedly large number of human genes. Except for the V(D)J recombinase and telomerase, all remain of unknown function. Here we investigate the activity of the human SETMAR protein, a highly expressed fusion between a histone H3 methylase and a mariner family transposase. Although SETMAR has demonstrated methylase activity and a DNA repair phenotype, its mode of action and the role of the transposase domain remain obscure. As a starting point to address this problem, we have dissected the activity of the transposase domain in the context of the full-length protein and the isolated transposase domain. Complete transposition of an engineered Hsmar1 transposon by the transposase domain was detected, although the extent of the reaction was limited by a severe defect for cleavage at the 3' ends of the element. Despite this problem, SETMAR retains robust activity for the other stages of the Hsmar1 transposition reaction, namely, site-specific DNA binding to the transposon ends, assembly of a paired-ends complex, cleavage of the 5' end of the element in Mn(2+), and integration at a TA dinucleotide target site. SETMAR is unlikely to catalyze transposition in the human genome, although the nicking activity may have a role in the DNA repair phenotype. The key activity for the mariner domain is therefore the robust DNA-binding and looping activity which has a high potential for targeting the histone methylase domain to the many thousands of specific binding sites in the human genome provided by copies of the Hsmar1 transposon.


Assuntos
Elementos de DNA Transponíveis/genética , Histona-Lisina N-Metiltransferase/metabolismo , Metiltransferases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Éxons/genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência Molecular , Proteínas Metiltransferases , Estrutura Terciária de Proteína
18.
Nucleic Acids Res ; 31(10): 2694-702, 2003 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-12736319

RESUMO

Viral integrase (IN) and Vpr are both components of the human immunodeficiency virus type 1 (HIV-1) pre-integration complex. To investigate whether these proteins interact within this complex, we investigated the effects of Vpr and its subdomains on IN activity in vitro. When a 21mer oligonucleotide was used as a donor and acceptor, both Vpr and its C-terminal DNA-binding domain [(52-96)Vpr] inhibited the integration reaction, whereas the (1-51)Vpr domain did not affect IN activity. Steady-state fluorescence anisotropy showed that both full-length and (52-96)Vpr bind to the short oligonucleotide, thereby extending previous observations with long DNA. The concentrations of the two proteins required to inhibit IN activity were consistent with their affinities for the oligonucleotide. The use of a 492 bp mini-viral substrate confirmed that Vpr can inhibit the IN-mediated reaction. However, the activity of (52-96)Vpr differed notably since it stimulated specifically integration events involving two homologous mini-viral DNAs. Order of addition experiments indicated that the stimulation was maximal when IN, (50-96)Vpr and the mini-viral DNA were allowed to form a complex. Furthermore, in the presence of (50-96)Vpr, the binding of IN to the mini-viral DNA was dramatically enhanced. Taken together, these data suggest that (52-96)Vpr stimulates the formation of a specific complex between IN and the mini-viral DNA.


Assuntos
DNA Viral/genética , Produtos do Gene vpr/metabolismo , Integrase de HIV/metabolismo , HIV-1/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , Ensaio de Desvio de Mobilidade Eletroforética , Polarização de Fluorescência , Produtos do Gene vpr/química , Produtos do Gene vpr/genética , HIV-1/enzimologia , HIV-1/metabolismo , Humanos , Cinética , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Recombinação Genética , Integração Viral/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana
19.
J Virol ; 77(1): 135-41, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12477818

RESUMO

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.


Assuntos
Dissulfetos/química , Integrase de HIV/química , HIV-1/fisiologia , Replicação Viral , Cisteína , Dimerização , Integrase de HIV/fisiologia , Células HeLa , Humanos , Vírion/química
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