Biomedical subjects

L Haren

Publications and source records attributed to L Haren.

8 recordsLinked to original sources

PubMed2001

The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity.

The bacterial insertion sequence IS911 transposes via a covalently closed circular intermediate. Circle formation involves transposase-mediated pairing of both insertion sequence ends. While full-length transposase, OrfAB, binds poorly in vitro to IS911 DNA fragments carrying a copy of the IS911 end, truncated protein derivatives carrying the first 135 (OrfAB[1-135]) or 149 (OrfAB[1-149]) amino acid residues bind efficiently. They generate a paired-end complex containing two such fragments which resembles that expected for the first synaptic complex. Shorter protein derivatives lacking a region involved in multimerisation do not form these complexes but modify the binding of OrfAB[1-135] and OrfAB[1-149]. DNaseI footprinting demonstrated that OrfAB[1-149] protects a sub-terminal (internal) region of the inverted repeats which includes two blocks of sequence (beta and gamma) conserved between the left (IRL) and right (IRR) ends. DNA binding assays in vitro and measurement of recombination activity in vivo of sequential deletion derivatives of the two inverted repeats suggested a model in which the N-terminal region of OrfAB binds the conserved boxes beta and gamma in a sequence-specific manner and anchors the two insertion sequence ends into a paired-end complex. The external region of the inverted repeat is proposed to contact the C-terminal transposase domain carrying the catalytic site.

Base Sequence↗

PubMed2000

IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif.

Efficient intermolecular transposition of bacterial insertion sequence IS911 involves the activities of two element-encoded proteins: the transposase, OrfAB, and a regulatory factor, OrfA. OrfA shares the majority of its amino acid sequence with the N-terminal part of OrfAB. This includes a putative helix-turn-helix and three of four heptads of a leucine zipper motif. OrfA strongly stimulates OrfAB-mediated intermolecular transposition both in vivo and in vitro. The present results support the notion that this is accomplished by direct interaction between these two proteins via the leucine zipper. We used both a genetic approach, based on gene fusions with phage lambda repressor, and a physical approach, involving co-immunoprecipitation, to show that OrfA not only undergoes oligomerisation but is capable of engaging with OrfAB to form heteromultimers, and that the leucine zipper is necessary for both types of interaction. Furthermore, mutation of the leucine zipper in OrfA inactivated its regulatory function. Previous observations demonstrated that the integrity of the leucine zipper motif was also important for OrfAB binding to the IS911 terminal inverted repeats. Here, we show, in gel shift experiments, using a derivative of OrfAB deleted for the C-terminal catalytic domain, OrfAB[1-149], that the protein is capable of pairing two inverted repeats to generate a species resembling a "synaptic complex". Preincubation of OrfAB[1-149] with OrfA dramatically reduced formation of this complex and favored formation of an alternative complex devoid of OrfA. Together these results suggest that OrfA exerts its regulatory effect by interacting transiently with OrfAB via the leucine zipper and modifying OrfAB binding to the inverted repeats.

Amino Acid Sequence↗

PubMed1999

IS911 transposon circles give rise to linear forms that can undergo integration in vitro.

High levels of expression of the transposase OrfAB of bacterial insertion sequence IS911 leads to the formation of excised transposon circles, in which the two abutted ends are separated by 3 bp. Initially, OrfAB catalyses only single-strand cleavage at one 3' transposon end and strand transfer of that end to the other. It is believed that this molecule, in which both transposon ends are held together in a single-strand bridge, is then converted to the circular form by the action of host factors. The transposon circles can be integrated efficiently into an appropriate target in vivo and in vitro in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a transposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut-and-paste mechanism. However, the linear species appeared not to be derived directly from the plasmid-based copy by direct double-strand cleavages at both ends, but from preformed excised transposon circles. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single-strand cleavages at the 3' transposon ends to generate a linear transposon form with a 3'-OH and a three-nucleotide 5' overhang at the ends. Moreover, while significantly less efficient than the transposon circle, a precleaved linear transposon underwent detectable levels of integration in vitro. The possible role of such molecules in the IS911 transposition pathway is discussed.

Bacterial Proteins↗

PubMed1999

Integrating DNA: transposases and retroviral integrases.

Transposable elements appear quite disparate in their organization and in the types of genetic rearrangements they promote. In spite of this diversity, retroviruses and many transposons of both prokaryotes and eukaryotes show clear similarities in the chemical reactions involved in their transposition. This is reflected in the enzymes, integrases and transposases, that catalyze these reactions and that are essential for the mobility of the elements. In this chapter, we examine the structure-function relationships between these enzymes and the different ways in which the individual steps are assembled to produce a complete transposition cycle.

Catalytic Domain↗

PubMed1998

Multiple oligomerisation domains in the IS911 transposase: a leucine zipper motif is essential for activity.

Structure-function relationships involved in oligomerisation of the transposase OrfAB of the bacterial insertion sequence IS911 have been investigated. Site-directed mutagenesis and sequential deletion coupled with immunoprecipitation have led to the definition of three regions of the protein capable of promoting multimerisation. These include a region predicted to assume a coiled-coil conformation, which is shown to be essential for activity, promoting correct multimerisation of the N-terminal domain of OrfAB and sequence-specific binding to the IS911 terminal inverted repeats mediated by this domain. This region presents the structural and functional characteristics of the leucine zipper motif described in eukaryotic proteins. The two other regions are located further towards the C-terminal end of the protein, adjacent to the leucine zipper and in the region that carries the conserved catalytic DD(35)E motif.

Amino Acid Sequence↗

PubMed1997

IS911-mediated intramolecular transposition is naturally temperature sensitive.

It is shown here that the bacterial insertion sequence IS911 exhibits a temperature-sensitive transposition phenotype. Previous results have demonstrated that elevated levels of the IS911 transposase OrfAB generate significant quantities of a figure-eight form, created by cleavage and circularization of one of the transposon strands, and of an excised circular form, in which both transposon strands have been circularized. We show here that the level of both types of molecule observed in vivo was greatly reduced at 42 degrees C compared with 37 degrees C. On the other hand, reducing the temperature to 30 degrees C resulted in a significant increase in production. Transposition activity at this temperature was sufficiently high to permit detection in vivo of an excised circular form of a defective single IS911 chromosomal copy when OrfAB is supplied in trans. A similar temperature-activity profile is observed for a cell-free reaction that uses partially purified OrfAB and generates the figure-eight form uniquely. Moreover, two point mutants of OrfAB were obtained, which render the reactions partially temperature resistant both in vivo and in vitro. These results suggest that some property of transposase itself is sensitive to elevated temperatures.

Bacterial Proteins↗

PubMed1996

IS911-mediated transpositional recombination in vitro.

A cell-free system is described that accomplishes an unusual type of transposition/recombination involving the bacterial insertion sequence IS911. Using a plasmid substrate carrying a derivative of IS911, we show that bacterial cell extracts enriched for the IS911 transposase, OrfAB, carry out a single-strand cleavage and transfer reaction. This results in the formation of a figure-eight molecule in which a single strand of the element is circularized, faithfully reproducing an event previously detected in vivo. Moreover, when presented with a figure-eight substrate, OrfAB is capable of "reversing" strand transfer. This activity is equivalent to the "disintegration" reaction carried out by retroviral integrases. We demonstrate that the domain of OrfAB responsible for this catalytic activity is located in the carboxy-terminal region of the protein, since a peptide composed of this region retains disintegration activity. The OrfAB-mediated excision-circularization process previously observed in vivo was proposed to proceed via a figure-eight intermediate by circularization of the second transposon strand. The absence of transposon circles in cell-free reaction suggests either that the figure-eight form is not an intermediate or that additional host factors are required that are eliminated from the cell extract. Two types of model, replicative and non-replicative, are discussed to explain how the figure-eight molecule could be processed into the transposon circle.

Bacterial Proteins↗

PubMedDate not supplied

Phosphorylation and cytotoxicity of therapeutic nucleoside analogues: a comparison of alpha and gamma herpesvirus thymidine kinase suicide genes.

Thymidine kinase (TK) genes from three alpha-herpesviruses (i.e., human herpes simplex type 1, varicella-zoster virus, equid herpesvirus 4) and two y-herpesviruses (i.e., Epstein-Barr virus and Saimiri herpesvirus 2) were cloned in expression vectors based on zeocin resistance by complementation of a TK-defective Escherichia coli strain. In vivo complementation of an appropriate yeast strain and in vitro enzymatic measurements demonstrated that all viral TKs possess a second phosphorylating activity corresponding to the thymidylate kinase function in contrast to the E coli TK, which is deprived of this activity. When expressed in an engineered E coli strain rendered resistant to purine and pyrimidine nucleoside analogs, the viral TKs sensitize host bacteria to 3'-azido-3'-deoxythymidine (AZT), 3'-deoxy-2',3'-didehydrothymidine (D4T), dideoxyinosine, or fluorodeoxyuridine (5-FUdR). The extent of activation of all these analogs, in this bacterial assay, was found to be greatly superior for the two gamma-virus TKs, compared to the alpha-virus TKs, including the reference suicide gene, HSV1-TK. TK from the two gamma-Epstein-Barr and Saimiri 2 viruses were also found to be more efficient in sensitizing murine melanoma B16 tumor cells to pyrimide nucleoside analogs.

Animals↗