Chromosomal environment of a trypanosome metacyclic VSG gene expression site

The gene encoding the metacyclic Variant Surface Glycoprotein (VSG) ILTat 1.22 is expressed in situ from a monocistronic, telomeric transcription unit in metacyclic-derived trypanosomes. The genomic environment upstream of the 1.22 basic copy gene (1.22BC) is composed of single copy, transcriptionally silent sequence. This sequence occurs in an area which, in bloodstream VSG gene expression sites, is thought to be subject to the influence of a developmentally regulated position silencing effect. For this reason, the silent area upstream of the 1.22BC is designated as a 'metacyclic domain'. The metacyclic domain is defined as any single copy, silent sequence linked with the M-VSG gene. In a step towards understanding the nature of the control of M-VSG gene expression, efforts to define the extent of the metacyciic domain for the ILTat 1.22 M-VSG gene were undertaken. Initially, YAC cloning technology was employed to accomplish the required cloning. This proved only partially successful and ultimately unnecessary. X cloning, genomic southern analysis and transcriptional studies were employed to define the extent of the 1.22 metacyclic domain. The metacyclic domain was found to end 21 kb upstream of the 1.22BC gene. Sequence upstream of the metacyclic domain appears to be diploid and transcriptionally active, especially in metacyclic-derived trypanosomes. A gene candidate in this region was partially sequenced and found to have sequences highly homologous to a gene, ESAG 1, that occurs in bloodstream VSG gene expression sites

Hairpin Telomere Resolvases

Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.Canadian Institutes of Health Research (CIHR

Structure, function, and evolution of linear replicons in Borrelia

Spirochetes of the genus Borrelia include important human pathogens that cause Lyme borreliosis and relapsing fever. The genomes of Borrelia species can be composed of up to 24 DNA molecules, most of which are linear. The plasmid content and linear replicon sequence arrangement vary widely between isolates. The linear replicons are terminated by covalently closed DNA hairpins or hairpin telomeres. Replication of these elements involves a unique reaction, called telomere resolution, to produce hairpin telomeres from replicative intermediates. The telomere resolvase, ResT, is thought to contribute to the genetic flux of the linear molecules by promoting stabilized telomere fusions. Telomere resolvases are related to the tyrosine recombinases and ResT can generate the crucial reaction intermediate of this class of enzyme, the Holliday junction. This observation has led to the proposal that telomere resolvases evolved from tyrosine recombinases inducing DNA linearization in the genomes that acquired them.Canadian Institutes of Health Research (CIHR

Hairpin Telomere Resolvases

Covalently closed hairpin ends, also known as hairpin telomeres, provide an unusual solution to the end replication problem. The hairpin telomeres are generated from replication intermediates by a process known as telomere resolution. This is a DNA breakage and reunion reaction promoted by hairpin telomere resolvases (also referred to as protelomerases) found in a limited number of phage and bacteria. The reaction promoted by these enzymes is a chemically isoenergetic two-step transesterification without a requirement for divalent metal ions or high-energy cofactors and uses an active site and mechanism similar to that for type IB topoisomerases and tyrosine recombinases. The small number of unrelated telomere resolvases characterized to date all contain a central, catalytic core domain with the active site, but in addition carry variable C- and N-terminal domains with different functions. Similarities and differences in the structure and function of the telomere resolvases are discussed. Of particular interest are the properties of the Borrelia telomere resolvases, which have been studied most extensively at the biochemical level and appear to play a role in shaping the unusual segmented genomes in these organisms and, perhaps, to play a role in recombinational events.Canadian Institutes of Health Research (CIHR

<i>B</i>. <i>burgdorferi</i> RecA-promoted strand exchange with long substrates.

<p>A) Schematic of the strand exchange assay using ϕX174 virion DNA (closed circular ssDNA; css) and XhoI-linearized ϕX174 duplex DNA (linear dsDNA; lds). As strand exchange initiates, joint molecule (JM) intermediates form. As strand exchange progresses, the final strand exchange products of nicked circular DNA (NC) and linear ssDNA (lss) are formed. Partial, intermolecular strand exchange between multiple donor and target molecules can also give rise to large aggregates (aggr.). B) 0.8% agarose 1X TAE gel analysis of strand exchange reactions performed with RecA and SSB. Strand exchange was assayed in a buffer containing 25 mM HEPES (pH 7.6), 1 mM DTT, 100 μg/ml BSA, 50 mM NaCl, 2 mM ATP and the indicated concentration of MgCl₂ in 120 μL reaction volumes. The assay was conducted as a staged reaction with pre-incubation of RecA with the donor ssDNA (css; 30°C, 5 min) followed by addition of SSB and linear duplex DNA (lds) and continued incubation at 37°C. RecA was present at 2 μM, ϕX174 virion (css) at 5.1 μM (nt) and XhoI-linearized ϕX174 duplex DNA (lds) at 5.1 μM (nt). SSB was added after pre-incubation of RecA with the single stranded donor and was present at 0.9 μM. The concentration of added MgCl₂ is noted in the loading key above the gel. The migration position of the substrate DNA is noted to the left of the gel and of the products to the right. Under our gel conditions css and lss have identical gel mobilities. M denotes mock incubation of the substrate DNAs without added RecA or SSB. C) 0.8% agarose 1X TAE gel analysis of SSB order-of-addition effects and ATP hydrolysis requirements of strand exchange reactions performed with long substrates. Strand exchange reactions were performed as noted in B) using buffer conditions with the optimal 5 mM MgCl₂ concentration and 0.9 μM of added SSB. The order of SSB addition and nucleotide cofactor present is indicated in the loading key above the gel. When added first, SSB was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of RecA and linear ϕX174 duplex DNA followed by continued incubation at 37°C for the times indicated above the gel. When SSB was added second, RecA was incubated with ϕX174 virion at 30°C for 5 min prior to the addition of SSB and linear duplex DNA, followed by continued incubation at 37°C for the times indicated above the gel. M denotes mock incubation of the substrate DNAs without added RecA or SSB.</p