RESUMEN
DNA segregation ensures that cell offspring receive at least one copy of each DNA molecule, or replicon, after their replication. This important cellular process includes different phases leading to the physical separation of the replicons and their movement toward the future daughter cells. Here, we review these phases and processes in enterobacteria with emphasis on the molecular mechanisms at play and their controls.
Asunto(s)
Cromosomas Bacterianos , Enterobacteriaceae , Enterobacteriaceae/genética , Cromosomas Bacterianos/genética , ADN , Replicón , Replicación del ADNRESUMEN
Low-copy-number plasmids require sophisticated genetic devices to achieve efficient segregation of plasmid copies during cell division. Plasmid R388 uses a unique segregation mechanism, based on StbA, a small multifunctional protein. StbA is the key protein in a segregation system not involving a plasmid-encoded NTPase partner, it regulates the expression of several plasmid operons, and it is the main regulator of plasmid conjugation. The mechanisms by which StbA, together with the centromere-like sequence stbS, achieves segregation, is largely uncharacterized. To better understand the molecular basis of R388 segregation, we determined the crystal structure of the conserved N-terminal domain of StbA to 1.9 Å resolution. It folds into an HTH DNA-binding domain, structurally related to that of the PadR subfamily II of transcriptional regulators. StbA is organized in two domains. Its N-terminal domain carries the specific stbS DNA binding activity. A truncated version of StbA, deleted of its C-terminal domain, displays only partial activities in vivo, indicating that the non-conserved C-terminal domain is required for efficient segregation and subcellular plasmid positioning. The structure of StbA DNA-binding domain also provides some insight into how StbA monomers cooperate to repress transcription by binding to the stbDR and to form the segregation complex with stbS.
Asunto(s)
Proteínas Bacterianas , Segregación Cromosómica , Nucleósido-Trifosfatasa , Plásmidos , Proteínas Bacterianas/química , ADN/química , ADN/metabolismo , Nucleósido-Trifosfatasa/química , Nucleósido-Trifosfatasa/metabolismo , Operón , Plásmidos/genética , Dominios ProteicosRESUMEN
REP, diverse palindromic DNA sequences found at high copy number in many bacterial genomes, have been attributed important roles in cell physiology but their dissemination mechanisms are poorly understood. They might represent non-autonomous transposable elements mobilizable by TnpAREP, the first prokaryotic domesticated transposase associated with REP. TnpAREP, fundamentally different from classical transposases, are members of the HuH superfamily and closely related to the transposases of the IS200/IS605 family. We previously showed that Escherichia coli TnpAREP processes cognate single stranded REP in vitro and that this activity requires the integrity of the REP structure, in particular imperfect palindromes interrupted by a bulge and preceded by a conserved DNA motif. A second group of REPs rather carry perfect palindromes, raising questions about how the latter are recognized by their cognate TnpAREP. To get insight into the importance of REP structural and sequence determinants in these two groups, we developed an in vitro activity assay coupled to a mutational analysis for three different TnpAREP/REP duos via a SELEX approach. We also tackled the question of how the cleavage site is selected. This study revealed that two TnpAREP groups have co-evolved with their cognate REPs and use different strategies to recognize their REP substrates.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/química , Genoma Bacteriano , Secuencias Invertidas Repetidas , Transposasas/metabolismo , Escherichia coli/genética , Marinomonas/genética , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Técnica SELEX de Producción de Aptámeros , Stenotrophomonas maltophilia/genéticaRESUMEN
Bacterial transposons, through their ability to transfer DNA sequences from one position in the genome to another, play a central role in the shape and the evolution of genomes. Extensive studies have been performed during the last five decades to understand the molecular mechanisms involved in the transposition of a variety of elements. Among the methods used, the papillation and the mating out coupled to arbitrary primed PCR assays described in this chapter are widely used as very powerful approaches to detect and characterize transposition events in vivo.
Asunto(s)
Bacterias/genética , Técnicas Bacteriológicas , Elementos Transponibles de ADN , Expresión Génica , Genes Reporteros , Plásmidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Transposons are found in a wide variety of forms throughout the prokaryotic world where they actively contribute to the adaptive strategies of bacterial communities and hence, to the continuous emergence of new multiresistant pathogens. Contrasting with their biological and societal impact, only a few bacterial transposons have been the subject of detailed molecular studies. In this chapter, we propose a set of reliable biochemical methods as a primary route for studying new transposition mechanisms. These methods include (a) a straightforward approach termed "thermal shift induction" to produce the transposase in a soluble and properly folded configuration prior to its purification, (b) an adaptation of classical electrophoretic mobility shift assays (EMSA) combined to fluorescently labeled DNA substrates to determine the DNA content of different complexes assembled by the transposase, and (c) a highly sensitive "in-gel" DNA footprinting assay to further characterize these complexes at the base pair resolution level. A combination of these approaches was recently applied to decipher the molecular organization of key intermediates in the Tn3-family transposition pathway, a mechanism that has long been refractory to biochemical studies.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Elementos Transponibles de ADN , Transposasas/metabolismo , Fenómenos Fisiológicos Bacterianos , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Expresión Génica , Sustancias Macromoleculares/metabolismo , Unión Proteica , Coloración y Etiquetado , Temperatura , Transposasas/genéticaRESUMEN
Members of the IS200/IS605 insertion sequence family differ fundamentally from classical IS essentially by their specific single-strand (ss) transposition mechanism, orchestrated by the Y1 transposase, TnpA, a small HuH enzyme which recognizes and processes ss DNA substrates. Transposition occurs by the 'peel and paste' pathway composed of two steps: precise excision of the top strand as a circular ss DNA intermediate; and subsequent integration into a specific ssDNA target. Transposition of family members was experimentally shown or suggested by in silico high-throughput analysis to be intimately coupled to the lagging strand template of the replication fork. In this study, we investigated factors involved in replication fork targeting and analysed DNA-binding properties of the transposase which can assist localization of ss DNA substrates on the replication fork. We showed that TnpA interacts with the ß sliding clamp, DnaN and recognizes DNA which mimics replication fork structures. We also showed that dsDNA can facilitate TnpA targeting ssDNA substrates. We analysed the effect of Ssb and RecA proteins on TnpA activity in vitro and showed that while RecA does not show a notable effect, Ssb inhibits integration. Finally we discuss the way(s) in which integration may be directed into ssDNA at the replication fork.
Asunto(s)
Replicación del ADN , Elementos Transponibles de ADN/genética , ADN de Cadena Simple/metabolismo , Cromosomas Bacterianos/metabolismo , ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Cinética , Mutagénesis Insercional/genética , Rec A Recombinasas/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
IS911 has provided a powerful model for studying the transposition of members of a large class of transposable element: the IS3 family of bacterial Insertion Sequences (IS). These transpose by a Copy-out-Paste-in mechanism in which a double-strand IS circle transposition intermediate is generated from the donor site by replication and proceeds to integrate into a suitable double strand DNA target. This is perhaps one of the most common transposition mechanisms known to date. Copy-out-Paste-in transposition has been adopted by members of at least eight large IS families. This chapter details the different steps of the Copy-out-Paste-in mechanism involved in IS911 transposition. At a more biological level it also describes various aspects of regulation of the transposition process. These include transposase production by programmed translational frameshifting, transposase expression from the circular intermediate using a specialized promoter assembled at the circle junction and binding of the nascent transposase while it remains attached to the ribosome during translation (co-translational binding). This co-translational binding of the transposase to neighboring IS ends provides an explanation for the longstanding observation that transposases show a cis-preference for their activities.
Asunto(s)
Bacterias/genética , Elementos Transponibles de ADN , Bacterias/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Recombinación Genética , Transposasas/genética , Transposasas/metabolismoRESUMEN
The number and diversity of known prokaryotic insertion sequences (IS) have increased enormously since their discovery in the late 1960s. At present the sequences of more than 4000 different IS have been deposited in the specialized ISfinder database. Over time it has become increasingly apparent that they are important actors in the evolution of their host genomes and are involved in sequestering, transmitting, mutating and activating genes, and in the rearrangement of both plasmids and chromosomes. This review presents an overview of our current understanding of these transposable elements (TE), their organization and their transposition mechanism as well as their distribution and genomic impact. In spite of their diversity, they share only a very limited number of transposition mechanisms which we outline here. Prokaryotic IS are but one example of a variety of diverse TE which are being revealed due to the advent of extensive genome sequencing projects. A major conclusion from sequence comparisons of various TE is that frontiers between the different types are becoming less clear. We detail these receding frontiers between different IS-related TE. Several, more specialized chapters in this volume include additional detailed information concerning a number of these.In a second section of the review, we provide a detailed description of the expanding variety of IS, which we have divided into families for convenience. Our perception of these families continues to evolve and families emerge regularly as more IS are identified. This section is designed as an aid and a source of information for consultation by interested specialist readers.
Asunto(s)
Bacterias/genética , Elementos Transponibles de ADN , Reordenamiento Génico , Variación Genética , Recombinación GenéticaRESUMEN
HUH endonucleases are numerous and widespread in all three domains of life. The major function of these enzymes is processing a range of mobile genetic elements by catalysing cleavage and rejoining of single-stranded DNA using an active-site Tyr residue to make a transient 5'-phosphotyrosine bond with the DNA substrate. These enzymes have a key role in rolling-circle replication of plasmids and bacteriophages, in plasmid transfer, in the replication of several eukaryotic viruses and in various types of transposition. They have also been appropriated for cellular processes such as intron homing and the processing of bacterial repeated extragenic palindromes. Here, we provide an overview of these fascinating enzymes and their functions, using well-characterized examples of Rep proteins, relaxases and transposases, and we explore the molecular mechanisms used in their diverse activities.
Asunto(s)
Replicación del ADN , ADN de Cadena Simple/metabolismo , Endonucleasas/metabolismo , Bacterias/enzimología , Bacterias/genética , Bacterias/virología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófagos/enzimología , Bacteriófagos/genética , Bacteriófagos/fisiología , Conjugación Genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , ADN Circular , ADN de Cadena Simple/genética , ADN Viral/genética , Dependovirus/enzimología , Dependovirus/genética , Dependovirus/fisiología , Endonucleasas/genética , Plásmidos/genética , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single-stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ-irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB-mediated inhibition does not involve Deinococcus-specific factors. TnpB therefore appears to play a regulatory role in ISDra2 transposition.
Asunto(s)
Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN/genética , ADN Bacteriano/metabolismo , Deinococcus/genética , Deinococcus/efectos de la radiación , Regulación hacia Abajo , Transposasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Deinococcus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Mutagénesis Insercional , Transposasas/química , Transposasas/genéticaRESUMEN
Transposase, TnpA, of the IS200/IS605 family member IS608, catalyses single-strand DNA transposition and is dimeric with hybrid catalytic sites composed of an HUH motif from one monomer and a catalytic Y127 present in an α-helix (αD) from the other (trans configuration). αD is attached to the main body by a flexible loop. Although the reactions leading to excision of a transposition intermediate are well characterized, little is known about the dynamic behaviour of the transpososome that drives this process. We provide evidence strongly supporting a strand transfer model involving rotation of both αD helices from the trans to the cis configuration (HUH and Y residues from the same monomer). Studies with TnpA heterodimers suggest that TnpA cleaves DNA in the trans configuration, and that the catalytic tyrosines linked to the 5'-phosphates exchange positions to allow rejoining of the cleaved strands (strand transfer) in the cis configuration. They further imply that, after excision of the transposon junction, TnpA should be reset to a trans configuration before the cleavage required for integration. Analysis also suggests that this mechanism is conserved among members of the IS200/IS605 family.
Asunto(s)
Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , ADN Bacteriano/genética , Helicobacter pylori/enzimología , Transposasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Dominio Catalítico , Secuencia de Consenso , División del ADN , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli , Helicobacter pylori/genética , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/genética , Transposasas/química , Transposasas/genéticaRESUMEN
Extragenic sequences in genomes, such as microRNA and CRISPR, are vital players in the cell. Repetitive extragenic palindromic sequences (REPs) are a class of extragenic sequences, which form nucleotide stem-loop structures. REPs are found in many bacterial species at a high copy number and are important in regulation of certain bacterial functions, such as Integration Host Factor recruitment and mRNA turnover. Although a new clade of putative transposases (RAYTs or TnpA(REP)) is often associated with an increase in these repeats, it is not clear how these proteins might have directed amplification of REPs. We report here the structure to 2.6 Å of TnpA(REP) from Escherichia coli MG1655 bound to a REP. Sequence analysis showed that TnpA(REP) is highly related to the IS200/IS605 family, but in contrast to IS200/IS605 transposases, TnpA(REP) is a monomer, is auto-inhibited and is active only in manganese. These features suggest that, relative to IS200/IS605 transposases, it has evolved a different mechanism for the movement of discrete segments of DNA and has been severely down-regulated, perhaps to prevent REPs from sweeping through genomes.
Asunto(s)
ADN Bacteriano/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Secuencias Invertidas Repetidas , Transposasas/química , Secuencia de Aminoácidos , Dominio Catalítico , División del ADN , ADN Bacteriano/metabolismo , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Transposasas/metabolismoRESUMEN
REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Secuencias Invertidas Repetidas , Transposasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , ADN/química , ADN/metabolismo , División del ADN , ADN Circular/metabolismo , ADN de Cadena Simple/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Datos de Secuencia Molecular , Filogenia , Recombinación Genética , Shigella/enzimología , Shigella/genética , Transposasas/clasificación , Transposasas/genéticaRESUMEN
Single-stranded (ss) transposition, a recently identified mechanism adopted by members of the widespread IS200/IS605 family of insertion sequences (IS), is catalysed by the transposase, TnpA. The transposase of IS608, recognizes subterminal imperfect palindromes (IP) at both IS ends and cleaves at sites located at some distance. The cleavage sites, C, are not recognized directly by the protein but by short sequences 5' to the foot of each IP, guide (G) sequences, using a network of canonical ('Watson-Crick') base interactions. In addition a set of non-canonical base interactions similar to those found in RNA structures are also involved. We have reconstituted a biologically relevant complex, the transpososome, including both left and right ends and TnpA, which catalyses excision of a ss DNA circle intermediate. We provide a detailed picture of the way in which the IS608 transpososome is assembled and demonstrate that both C and G sequences are essential for forming a robust transpososome detectable by EMSA. We also address several questions central to the organization and function of the ss transpososome and demonstrate the essential role of non-canonical base interactions in the IS608 ends for its stability by using point mutations which destroy individual non-canonical base interactions.
Asunto(s)
Elementos Transponibles de ADN , Transposasas/metabolismo , Emparejamiento Base , Secuencia de Bases , División del ADN , ADN de Cadena Simple/metabolismo , Magnesio/química , Nucleoproteínas/metabolismoRESUMEN
Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd²(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.
Asunto(s)
ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Deinococcus/enzimología , Transposasas/química , Transposasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN de Cadena Simple/química , Deinococcus/química , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Alineación de Secuencia , Transposasas/genética , Zinc/metabolismoRESUMEN
DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the "top" IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication.
Asunto(s)
Replicación del ADN , Elementos Transponibles de ADN , ADN de Cadena Simple/metabolismo , Deinococcus/metabolismo , Escherichia coli/metabolismo , ADN Helicasas/metabolismo , ADN Primasa/metabolismo , Deinococcus/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Transactivadores/metabolismoRESUMEN
Stress-induced transposition is an attractive notion since it is potentially important in creating diversity to facilitate adaptation of the host to severe environmental conditions. One common major stress is radiation-induced DNA damage. Deinococcus radiodurans has an exceptional ability to withstand the lethal effects of DNA-damaging agents (ionizing radiation, UV light, and desiccation). High radiation levels result in genome fragmentation and reassembly in a process which generates significant amounts of single-stranded DNA. This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions. A recent study analyzed the mutational profile in the thyA gene following irradiation. The majority of thyA mutants resulted from transposition of one particular Insertion Sequence (IS), ISDra2, of the many different ISs in the D. radiodurans genome. ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences.
Asunto(s)
Fragmentación del ADN/efectos de la radiación , Elementos Transponibles de ADN , Deinococcus/genética , Deinococcus/efectos de la radiación , Genoma Bacteriano/efectos de la radiación , Elementos Transponibles de ADN/efectos de la radiación , Rayos gamma , Mutagénesis Insercional/efectos de la radiaciónRESUMEN
Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any other known transposition system, they strictly require single-strand DNA. TnpA, the shortest identified transposase, uses a nucleophilic tyrosine for these reactions. It recognizes and cleaves only the IS608 "top strand." The results support a transposition model involving excision of a single-strand circle with abutted left (LE) and right (RE) IS ends. Insertion occurs site specifically 3' to conserved and essential TTAC tetranucleotide and appears to be driven by LE. This single-strand transposition mode has important implications not only for dispersion of IS608 but also for the other members of this very large IS family.
Asunto(s)
Elementos Transponibles de ADN/genética , ADN de Cadena Simple/genética , Helicobacter pylori/genética , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , ADN Circular/genética , ADN Circular/metabolismo , ADN de Cadena Simple/metabolismo , Dimerización , Helicobacter pylori/enzimología , Técnicas In Vitro , Cinética , Datos de Secuencia Molecular , Plásmidos , Recombinación Genética , Especificidad por Sustrato , Transposasas/química , Transposasas/genética , Transposasas/metabolismo , Tirosina/genética , Tirosina/metabolismoRESUMEN
The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.
Asunto(s)
Elementos Transponibles de ADN/genética , Transposasas/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Catálisis , Cristalización , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Dimerización , Activación Enzimática , Helicobacter pylori/enzimología , Enlace de Hidrógeno , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Transposasas/química , Transposasas/genética , Tirosina/genética , Tirosina/metabolismoRESUMEN
IS911 transposition involves a closed circular insertion sequence intermediate (IS-circle) and two IS-encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N-terminal region with OrfA. This includes a helix-turn-helix (HTH) motif and the first three of four heptads of a leucine zipper (LZ). OrfAB binds specifically to IS911 ends via its HTH whereas OrfA does not. We show here: that OrfA binds DNA non-specifically and that this requires the HTH; that OrfA LZ is required for its multimerization; and that both motifs are essential for OrfA activity. We propose that these OrfA properties are required to assemble a nucleoprotein complex committed to random IS911 insertion. This control of IS911 insertion activity by OrfA in this way would assure its dispersion.