Inverted Repeat DNA Research Articles

SwrA-mediated Multimerization of DegU and an Upstream Activation Sequence Enhance Flagellar Gene Expression in Bacillus subtilis

The earliest genes in bacterial flagellar assembly are activated by narrowly-conserved proteins called master regulators that often act as heteromeric complexes. A complex of SwrA and the response-regulator transcription factor DegU is thought to form the master flagellar regulator in Bacillus subtilis but how the two proteins co-operate to activate gene expression is poorly-understood. Here we find using ChIP-Seq that SwrA interacts with a subset of DegU binding sites in the chromosome and does so in a DegU-dependent manner. Using this information, we identify a DegU-specific inverted repeat DNA sequence in the Pflache promoter region and show that SwrA synergizes with DegU phosphorylation to increase binding affinity. We further demonstrate that the SwrA/DegU footprint extends from the DegU binding site towards the promoter, likely through SwrA-induced DegU multimerization. The location of the DegU inverted repeat was critical and moving the binding site closer to the promoter impaired transcription by disrupting a previously-unrecognized upstream activation sequence (UAS). Thus, the SwrA-DegU heteromeric complex likely enables both remote binding and interaction between the activator and RNA polymerase. Small co-activator proteins like SwrA may allow selective activation of subsets of genes where activator multimerization is needed. Why some promoters require activator multimerization and some require UAS sequences is unknown.

Characterization of the complete chloroplast genome of Gypsophila huashanensis Y. W. Tsui & D. Q. Lu, an endemic herb species in China

Gypsophila huashanensis Y. W. Tsui & D. Q. Lu (Caryophyllaceae) is an endemic herb species to the Qinling Mountains in China. In this study, we characterized its whole plastid genome using the Illumina sequencing platform. The complete plastid genome of G. huashanensis is 152,457 bp in length, including a large single-copy DNA region of 83,476 bp, a small single-copy DNA region of 17,345 bp, and a pair of inverted repeat DNA sequences of 25,818 bp. The genome contains 130 genes comprising 85 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Evolutionary analysis showed that the non-coding regions of Caryophyllaceae exhibit a higher level of divergence than the exon regions. Gene site selection analysis suggested that 11 coding protein genes (accD, atpF, ndhA, ndhB, petB, petD, rpoCl, rpoC2, rps16, ycfl, and ycf2) have some sites under protein sequence evolution. Phylogenetic analysis showed that G. huashanensis is most closely related to the congeneric species G. oldhamiana. These results are very useful for studying phylogenetic evolution and species divergence in the family Caryophyllaceae.

Structural basis of the farnesoid X receptor/retinoid X receptor heterodimer on inverted repeat DNA

Farnesoid X receptor (FXR) is a ligand-activated transcription factor known as bile acid receptor (BAR). FXR plays critical roles in various biological processes, including metabolism, immune inflammation, liver regeneration and liver carcinogenesis. FXR forms a heterodimer with the retinoid X receptor (RXR) and binds to diverse FXR response elements (FXREs) to exert its various biological functions. However, the mechanism by which the FXR/RXR heterodimer binds the DNA elements remains unclear. In this study, we aimed to use structural, biochemical and bioinformatics analyses to study the mechanism of FXR binding to the typical FXRE, such as the IR1 site, and the heterodimer interactions in the FXR-DBD/RXR-DBD complex. Further biochemical assays showed that RAR, THR and NR4A2 do not form heterodimers with RXR when bound to the IR1 sites, which indicates that IR1 may be a unique binding site for the FXR/RXR heterodimer. Our studies may provide a further understanding of the dimerization specificity of nuclear receptors.

RF15 | PMON291 Crystal Structures of Androgen DNA Binding Domain Interacting with its Response Elements (C3(1) ARE and MMTV-GRE)

Abstract Prostate cancer (PCa) is the second leading cancer in the world in men. The major therapeutic target in advanced PCa is the ligand-binding pocket (LBP) of the androgen receptor (AR). The anti-androgens used to treat cancer are binding to this domain and compete with the natural agonist. Over time the cancer grows resistant towards this therapy. Nevertheless, the cancer still typically depends on the AR, indicating other sites of the AR may still serve as drug targets. The DNA binding domain (DBD) is a likely good therapeutic target as it is involved in essential processes such AR dimerization, translocation to the nucleus and binding to the androgen response elements (AREs). However the structure and the DNA binding interactions with AREs are not fully explored yet. Only one crystal structure (pdb: 1R4I) is available. In this structure, the DBD is bound to an artificial direct repeat DNA sequences (ADR3), resulting in an unexpected dimeric head-to-head arrangement. A better understanding on how the AR recognizes and binds to different native DNA sequences which are mainly organized as semi inverted repeats is crucial for rational drug design. Here, we performed protein crystallography of the DBD in complex with various natural ARE sequences, including the mouse mammary tumour virus (MMTV-GRE) motif. Surprisingly, this latter element is still able to be activated by a dimerization deficient AR. Our first crystal structures were hallmarked by a high degree of disorder and appeared to bind both in a head-to-head as a head-to-tail fashion. Rational engineering of the DNA duplexes used for crystallography yielded well diffracting crystals for two different complexes. These two structures were diffracted to a range of 2 to 2.7 Å. Both structures with natural sequences appeared to bind in the head-to-head arrangement including the MMTV-GRE which consist out of semi inverted repeat DNA, whereas the C3(1)ARE contains the canonical fully inverted repeats. The structure reveals a slightly bend arrangement from the previously reported structure. The displacement is most visible at the DNA recognition helix bound to the half site and the so-called lever arm, which positions this helix in this region with a RMSD difference of 1.3 A. This indicates that the structural conformation changes of the protein is driven towards improved DNA-base pair recognition. Indeed the protein is able to make an additional set of non-covalent interactions with the base pairs of MMTV-GRE, leading to higher affinity. Both these extra interactions and conformational change lead to a higher affinity binding of protein-DNA, which seems to overcome the barrier introduced by the dimerization breaking mutations. This interaction surface could be a druggable site for structure-based drug design in the future, targeting AR-DBD dimerization. Presentation: Sunday, June 12, 2022 12:30 p.m. - 12:35 p.m., Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Migrating bubble synthesis promotes mutagenesis through lesions in its template.

Break-induced replication (BIR) proceeds via a migrating D-loop for hundreds of kilobases and is highly mutagenic. Previous studies identified long single-stranded (ss) nascent DNA that accumulates during leading strand synthesis to be a target for DNA damage and a primary source of BIR-induced mutagenesis. Here, we describe a new important source of mutagenic ssDNA formed during BIR: the ssDNA template for leading strand BIR synthesis formed during D-loop migration. Specifically, we demonstrate that this D-loop bottom template strand (D-BTS) is susceptible to APOBEC3A (A3A)-induced DNA lesions leading to mutations associated with BIR. Also, we demonstrate that BIR-associated ssDNA promotes an additional type of genetic instability: replication slippage between microhomologies stimulated by inverted DNA repeats. Based on our results we propose that these events are stimulated by both known sources of ssDNA formed during BIR, nascent DNA formed by leading strand synthesis, and the D-BTS that we describe here. Together we report a new source of mutagenesis during BIR that may also be shared by other homologous recombination pathways driven by D-loop repair synthesis.

Interaction of Proteins with Inverted Repeats and Cruciform Structures in Nucleic Acids.

Cruciforms occur when inverted repeat sequences in double-stranded DNA adopt intra-strand hairpins on opposing strands. Biophysical and molecular studies of these structures confirm their characterization as four-way junctions and have demonstrated that several factors influence their stability, including overall chromatin structure and DNA supercoiling. Here, we review our understanding of processes that influence the formation and stability of cruciforms in genomes, covering the range of sequences shown to have biological significance. It is challenging to accurately sequence repetitive DNA sequences, but recent advances in sequencing methods have deepened understanding about the amounts of inverted repeats in genomes from all forms of life. We highlight that, in the majority of genomes, inverted repeats are present in higher numbers than is expected from a random occurrence. It is, therefore, becoming clear that inverted repeats play important roles in regulating many aspects of DNA metabolism, including replication, gene expression, and recombination. Cruciforms are targets for many architectural and regulatory proteins, including topoisomerases, p53, Rif1, and others. Notably, some of these proteins can induce the formation of cruciform structures when they bind to DNA. Inverted repeat sequences also influence the evolution of genomes, and growing evidence highlights their significance in several human diseases, suggesting that the inverted repeat sequences and/or DNA cruciforms could be useful therapeutic targets in some cases.

DNA transposons mediate duplications via transposition-independent and -dependent mechanisms in metazoans

Despite long being considered as “junk”, transposable elements (TEs) are now accepted as catalysts of evolution. One example is Mutator-like elements (MULEs, one type of terminal inverted repeat DNA TEs, or TIR TEs) capturing sequences as Pack-MULEs in plants. However, their origination mechanism remains perplexing, and whether TIR TEs mediate duplication in animals is almost unexplored. Here we identify 370 Pack-TIRs in 100 animal reference genomes and one Pack-TIR (Ssk-FB4) family in fly populations. We find that single-copy Pack-TIRs are mostly generated via transposition-independent gap filling, and multicopy Pack-TIRs are likely generated by transposition after replication fork switching. We show that a proportion of Pack-TIRs are transcribed and often form chimeras with hosts. We also find that Ssk-FB4s represent a young protein family, as supported by proteomics and signatures of positive selection. Thus, TIR TEs catalyze new gene structures and new genes in animals via both transposition-independent and -dependent mechanisms.

Downregulating of hemB via synthetic antisense RNAs for improving 5-aminolevulinic acid production in Escherichia coli.

The online version contains supplementary material available at 10.1007/s13205-021-02733-8.

Identifying Small Molecules That Promote Quasipalindrome-Associated Template-Switch Mutations in Escherichia coli.

DNA can assemble into non-B form structures that stall replication and cause genomic instability. One such secondary structure results from an inverted DNA repeat that can assemble into hairpin and cruciform structures during DNA replication. Quasipalindromes (QP), imperfect inverted repeats, are sites of mutational hotspots. Quasipalindrome-associated mutations (QPMs) occur through a template-switch mechanism in which the replicative polymerase stalls at a QP site and uses the nascent strand as a template instead of the correct template strand. This mutational event causes the QP to become a perfect or more perfect inverted repeat. Since it is not fully understood how template-switch events are stimulated or repressed, we designed a high-throughput screen to discover drugs that affect these events. QP reporters were engineered in the Escherichia coli lacZ gene to allow us to study template-switch events specifically. We tested 700 compounds from the NIH Clinical Collection through a disk diffusion assay and identified 11 positive hits. One of the hits was azidothymidine (zidovudine, AZT), a thymidine analog and DNA chain terminator. The other ten were found to be fluoroquinolone antibiotics, which induce DNA-protein crosslinks. This work shows that our screen is useful in identifying small molecules that affect quasipalindrome-associated template-switch mutations. We are currently assessing more small molecule libraries and applying this method to study other types of mutations.

Single-strand annealing between inverted DNA repeats: Pathway choice, participating proteins, and genome destabilizing consequences.

Double strand DNA breaks (DSBs) are dangerous events that can result from various causes including environmental assaults or the collapse of DNA replication. While the efficient and precise repair of DSBs is essential for cell survival, faulty repair can lead to genetic instability, making the choice of DSB repair an important step. Here we report that inverted DNA repeats (IRs) placed near a DSB can channel its repair from an accurate pathway that leads to gene conversion to instead a break-induced replication (BIR) pathway that leads to genetic instabilities. The effect of IRs is explained by their ability to form unusual DNA structures when present in ssDNA that is formed by DSB resection. We demonstrate that IRs can form two types of unusual DNA structures, and the choice between these structures depends on the length of the spacer separating IRs. In particular, IRs separated by a long (1-kb) spacer are predominantly involved in inter-molecular single-strand annealing (SSA) leading to the formation of inverted dimers; IRs separated by a short (12-bp) spacer participate in intra-molecular SSA, leading to the formation of fold-back (FB) structures. Both of these structures interfere with an accurate DSB repair by gene conversion and channel DSB repair into BIR, which promotes genomic destabilization. We also report that different protein complexes participate in the processing of FBs containing short (12-bp) versus long (1-kb) ssDNA loops. Specifically, FBs with short loops are processed by the MRX-Sae2 complex, whereas the Rad1-Rad10 complex is responsible for the processing of long loops. Overall, our studies uncover the mechanisms of genomic destabilization resulting from re-routing DSB repair into unusual pathways by IRs. Given the high abundance of IRs in the human genome, our findings may contribute to the understanding of IR-mediated genomic destabilization associated with human disease.

Characterization of the regulation of a plant polysaccharide utilization operon and its role in biofilm formation in Bacillus subtilis.

The soil bacterium Bacillus subtilis is often found in association with plants in the rhizosphere. Previously, plant polysaccharides have been shown to stimulate formation of root-associated multicellular communities, or biofilms, in this bacterium, yet the underlying mechanism is not fully understood. A five-gene gan operon (ganSPQAB) in B. subtilis has recently been shown to be involved in utilization of the plant-derived polysaccharide galactan. Despite these findings, molecular details about the regulation of the operon and the role of the operon in biofilm formation remain elusive. In this study, we performed comprehensive genetic analyses on the regulation of the gan operon. We show that this operon is regulated both by a LacI-like transcription repressor (GanR), which directly binds to pairs of inverted DNA repeats in the promoter region of the operon, and by the catabolite control protein A (CcpA). Derepression can be triggered by the presence of the inducer β-1,4-galactobiose, a hydrolysis product of galactan, or in situ when B. subtilis cells are associated with plant roots. In addition to the transcriptional regulation, the encoded ß-galactosidase GanA (by ganA), which hydrolyzes ß-1,4-galactobiose into galactose, is inhibited at the enzymatic level by the catalytic product galactose. Thus, the galactan utilization pathway is under complex regulation involving both positive and negative feedback mechanisms in B. subtilis. We discuss about the biological significance of such complex regulation as well as a hypothesis of biofilm induction by galactan via multiple mechanisms.

Entire plastid phylogeny of the carrot genus (Daucus, Apiaceae): Concordance with nuclear data and mitochondrial and nuclear DNA insertions to the plastid.

We explored the phylogenetic utility of entire plastid DNA sequences in Daucus and compared the results with prior phylogenetic results using plastid and nuclear DNA sequences. We used Illumina sequencing to obtain full plastid sequences of 37 accessions of 20 Daucus taxa and outgroups, analyzed the data with phylogenetic methods, and examined evidence for mitochondrial DNA transfer to the plastid (DcMP). Our phylogenetic trees of the entire data set were highly resolved, with 100% bootstrap support for most of the external and many of the internal clades, except for the clade of D. carota and its most closely related species D. syrticus. Subsets of the data, including regions traditionally used as phylogenetically informative regions, provide various degrees of soft congruence with the entire data set. There are areas of hard incongruence, however, with phylogenies using nuclear data. We extended knowledge of a mitochondrial to plastid DNA insertion sequence previously named DcMP and identified the first instance in flowering plants of a sequence of potential nuclear genome origin inserted into the plastid genome. There is a relationship of inverted repeat junction classes and repeat DNA to phylogeny, but no such relationship with nonsynonymous mutations. Our data have allowed us to (1) produce a well-resolved plastid phylogeny of Daucus, (2) evaluate subsets of the entire plastid data for phylogeny, (3) examine evidence for plastid and nuclear DNA phylogenetic incongruence, and (4) examine mitochondrial and nuclear DNA insertion into the plastid.

Long-Term Expression of Von Willebrand Factor Via Piggybac-Mediated Gene Transfer

von Willebrand disease (vWD), caused by a deficiency in von Willebrand factor (vWF), is the most common inherited bleeding disorder, affecting up to 1% of the population; those affected have a varied bleeding phenotype. von Willebrand factor plays two important roles in hemostasis including platelet adhesion and stability of factor VIII (FVIII). As such, patients with von Willebrand disease type 3 have no measurable vWF protein and demonstrate low levels of FVIII. To date, persistent long-term expression of full-length vWF using gene transfer strategies has not been demonstrated, largely due to the immense size of vWF cDNA (8.4 kb). Non-viral vector systems, such as piggyBac, are used increasingly in gene targeting technologies and as tools for gene transfer applications. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a "cut and paste" mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Advantages of this nonviral vector system include a large transgene cassette capacity, ease of production and purification, and potential for site-specific integration. We hypothesize PB-mediated vWF gene transfer will confer long-term expression and improve the bleeding phenotype in an animal model of vWD. We engineered PB transposon to carry a human von Willebrand factor cDNA (PB vWF). When transfected with PB-vWF and a hyperactive transposase, iPB7, the hepatocarcinoma cell line, HepG2, demonstrated secretion of vWF into supernatants compared to mock transfected controls (p < 0.01). Next, we evaluated the in vivo gene transfer efficiency in von Willebrand deficient mice by hydrodynamic tail-vein injection using PB-vWF driven by the murine albumin enhancer/human alpha anti-trypsin promoter. vWF null mice received 25 or 50 micrograms each of the PB-vWF transposon and iPB7 to determine long-term expression and phenotypic correction. vWF levels were measured prior to injection and then every 4 weeks for up to 24 weeks. Results revealed therapeutic levels (on average 50% normal mice) of vWF post gene transfer with stable expression for 24 weeks in most mice. These data indicate the PB transposon vector system may provide a long-term gene transfer strategy for vWD. To evaluate phenotypic correction, a tail clip assay was performed at the end of the study. PB-vWF gene transfer resulted in at least partial bleeding phenotype correction via tail clip. Additionally, treated mice demonstrated a rescue of the FVIII activity. These data show that the PB vector can be used to deliver this large transgene expression cassette to the liver and achieve long-term expression and phenotypic correction in vivo. DisclosuresStaber:Emergent BioSolutions: Honoraria; Baxalta: Honoraria.

DNA uptake sequences in Neisseria gonorrhoeae as intrinsic transcriptional terminators and markers of horizontal gene transfer.

DNA uptake sequences are widespread throughout the Neisseria gonorrhoeae genome. These short, conserved sequences facilitate the exchange of endogenous DNA between members of the genus Neisseria. Often the DNA uptake sequences are present as inverted repeats that are able to form hairpin structures. It has been suggested previously that DNA uptake sequence inverted repeats present 3' of genes play a role in rho-independent termination and attenuation. However, there is conflicting experimental evidence to support this role. The aim of this study was to determine the role of DNA uptake sequences in transcriptional termination. Both bioinformatics predictions, conducted using TransTermHP, and experimental evidence, from RNA-seq data, were used to determine which inverted repeat DNA uptake sequences are transcriptional terminators and in which direction. Here we show that DNA uptake sequences in the inverted repeat configuration occur in N. gonorrhoeae both where the DNA uptake sequence precedes the inverted version of the sequence and also, albeit less frequently, in reverse order. Due to their symmetrical configuration, inverted repeat DNA uptake sequences can potentially act as bi-directional terminators, therefore affecting transcription on both DNA strands. This work also provides evidence that gaps in DNA uptake sequence density in the gonococcal genome coincide with areas of DNA that are foreign in origin, such as prophage. This study differentiates for the first time, to our knowledge, between DNA uptake sequences that form intrinsic transcriptional terminators and those that do not, providing characteristic features within the flanking inverted repeat that can be identified.

The Cloning and Propagation of an Inverted Repeat DNA in M13 Bacteriophage in Escherichia coli

The Cloning and Propagation of an Inverted Repeat DNA in M13 Bacteriophage in Escherichia coli

Adeno-associated virus inverted terminal repeats stimulate gene editing.

Advancements in genome editing have relied on technologies to specifically damage DNA which, in turn, stimulates DNA repair including homologous recombination (HR). As off-target concerns complicate the therapeutic translation of site-specific DNA endonucleases, an alternative strategy to stimulate gene editing based on fragile DNA was investigated. To do this, an episomal gene-editing reporter was generated by a disruptive insertion of the adeno-associated virus (AAV) inverted terminal repeat (ITR) into the egfp gene. Compared with a non-structured DNA control sequence, the ITR induced DNA damage as evidenced by increased gamma-H2AX and Mre11 foci formation. As local DNA damage stimulates HR, ITR-mediated gene editing was investigated using DNA oligonucleotides as repair substrates. The AAV ITR stimulated gene editing >1000-fold in a replication-independent manner and was not biased by the polarity of the repair oligonucleotide. Analysis of additional human DNA sequences demonstrated stimulation of gene editing to varying degrees. In particular, inverted yet not direct, Alu repeats induced gene editing, suggesting a role for DNA structure in the repair event. Collectively, the results demonstrate that inverted DNA repeats stimulate gene editing via double-strand break repair in an episomal context and allude to efficient gene editing of the human chromosome using fragile DNA sequences.

Preliminary crystallographic analysis of SETMAR bound to DNA

SETMAR, a recently identified double strand break (DSB) repair enzyme in the human genome, contains an N-terminal SET domain and a C-terminal MAR domain. This chimeric protein arose through the fusion of a mariner-family DNA transposase gene, Hsmar1, downstream of a SET histone methyltransferase gene approximately 50 million years ago [1]. Although the SETMAR transposase domain retains the ability to bind terminal inverted repeat (TIR) DNA sequence, which is the hallmark of DNA transposons, it is no longer a functional transposase [2]. Nonetheless, the transposase domain with only 19 amino acid substitutions as compared to the ancestral Hsmar1 transposase has been under a strong selective evolutionary pressure suggesting that the transposase domain is functionally important. Determining how SETMAR interacts with DNA is central to understanding the molecular basis of its evolved DNA repair activity. Toward this goal, we have focused initially on the interaction of the DNA-binding domain (DBD) of the SETMAR transposase domain with TIR DNA. The DBD of SETMAR has been overexpressed and purified. A complex formed between SETMAR DBD and its transposon TIR DNA has been crystallized by using the hanging-drop vapor diffusion method. The crystals diffract to 3.15 Å resolution and exhibit orthorhombic symmetry (C2221), with unit-cell dimensions of a=72.233 Å, b=164.385 Å, and c=67.957 Å. As there is no suitable search model available, we are currently pursuing experimental phasing approaches in order to solve this structure. We anticipate the structural analysis of DBD of SETMAR bound to transposon DNA will provide insight into the mechanism by which SETMAR recognizes both TIR and non-TIR DNA.

Distinct and concurrent pathways of Pol II- and Pol IV-dependent siRNA biogenesis at a repetitive trans-silencer locus in Arabidopsis thaliana.

Short interfering RNAs (siRNAs) homologous to transcriptional regulatory regions can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS) of target genes. In our system, siRNAs are produced by transcribing an inverted DNA repeat (IR) of enhancer sequences, yielding a hairpin RNA that is processed by several Dicer activities into siRNAs of 21-24 nt. Primarily 24-nt siRNAs trigger RdDM of the target enhancer in trans and TGS of a downstream GFP reporter gene. We analyzed siRNA accumulation from two different structural forms of a trans-silencer locus in which tandem repeats are embedded in the enhancer IR and distinguished distinct RNA polymerase II (Pol II)- and Pol IV-dependent pathways of siRNA biogenesis. At the original silencer locus, Pol-II transcription of the IR from a 35S promoter produces a hairpin RNA that is diced into abundant siRNAs of 21-24 nt. A silencer variant lacking the 35S promoter revealed a normally masked Pol IV-dependent pathway that produces low levels of 24-nt siRNAs from the tandem repeats. Both pathways operate concurrently at the original silencer locus. siRNAs accrue only from specific regions of the enhancer and embedded tandem repeat. Analysis of these sequences and endogenous tandem repeats producing siRNAs revealed the preferential accumulation of siRNAs at GC-rich regions containing methylated CG dinucleotides. In addition to supporting a correlation between base composition, DNA methylation and siRNA accumulation, our results highlight the complexity of siRNA biogenesis at repetitive loci and show that Pol II and Pol IV use different promoters to transcribe the same template.

Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.

Author SummaryVariations in the copy number of DNA segments account for a substantial amount of genome diversity of most organisms. DNA amplification, a contributor to copy number variation, can occur in response to various stresses or after altered growth conditions, leading to extensive and often reversible genetic variation. DNA amplification in the parasite Leishmania occurs outside the normal chromosomes and arises by DNA rearrangements involving homologous repeated sequences. We show here that such repeated sequences are widespread in the Leishmania genome and that most of the Leishmania genome is subject to stochastic gene rearrangements mediated by these low-copy repeat sequences. Thus, although cells in the population have a common core genome, many individual cells will differ from the rest of the population by carrying one or more distinct extrachromosomal amplicon. Upon selection with either drugs or culture conditions, a subpopulation can emerge where the amplicon copy number per cell increases, and this clone of cells can then expand to dominate the population. We propose that Leishmania uses adaptive gene amplification at a genome-wide scale as one strategy to adapt to a changing environment.

Biochemical Characterization and Comparison of Two Closely Related Active mariner Transposases

Most DNA transposons move from one genomic location to another by a cut-and-paste mechanism and are useful tools for genomic manipulations. Short inverted repeat (IR) DNA sequences marking each end of the transposon are recognized by a DNA transposase (encoded by the transposon itself). This enzyme cleaves the transposon ends and integrates them at a new genomic location. We report here a comparison of the biophysical and biochemical properties of two closely related and active mariner/Tc1 family DNA transposases: Mboumar-9 and Mos1. We compared the in vitro cleavage activities of the enzymes on their own IR sequences, as well as cross-recognition of their inverted repeat sequences. We found that, like Mos1, untagged recombinant Mboumar-9 transposase is a dimer and forms a stable complex with inverted repeat DNA in the presence of Mg2+ ions. Mboumar-9 transposase cleaves its inverted repeat DNA in the manner observed for Mos1 transposase. There was minimal cross-recognition of IR sequences between Mos1 and Mboumar-9 transposases, despite these enzymes having 68% identical amino acid sequences. Transposases sharing common biophysical and biochemical properties, but retaining recognition specificity toward their own IR, are a promising platform for the design of chimeric transposases with predicted and improved sequence recognition.