- Open Access
DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae
© Kahlon et al; licensee BioMed Central Ltd. 2011
Received: 30 August 2010
Accepted: 20 June 2011
Published: 20 June 2011
Determining the mechanisms by which transposable elements move within a genome increases our understanding of how they can shape genome evolution. Class 2 transposable elements transpose via a 'cut-and-paste' mechanism mediated by a transposase that binds to sites at or near the ends of the transposon. Herves is a member of the hAT superfamily of class 2 transposons and was isolated from Anopheles gambiae, a medically important mosquito species that is the major vector of malaria in sub-Saharan Africa. Herves is transpositionally active and intact copies of it are found in field populations of A gambiae. In this study we report the binding activities of the Herves transposase to the sequences at the ends of the Herves transposon and compare these to other sequences recognized by hAT transposases isolated from other organisms.
We identified the specific DNA-binding sites of the Herves transposase. Active Herves transposase was purified using an Escherichia coli expression system and bound in a site-specific manner to the subterminal and terminal sequences of the left and right ends of the element, respectively, and also interacted with the right but not the left terminal inverted repeat. We identified a common subterminal DNA-binding motif (CG/AATTCAT) that is critical and sufficient for Herves transposase binding.
The Herves transposase binds specifically to a short motif located at both ends of the transposon but shows differential binding with respect to the left and right terminal inverted repeats. Despite similarities in the overall structures of hAT transposases, the regions to which they bind in their respective transposons differ in sequence ensuring the specificity of these enzymes to their respective transposon. The asymmetry with which the Herves terminal inverted repeats are bound by the transposase may indicate that these differ in their interactions with the enzyme.
Transposable elements (TEs) are ubiquitous components of genomes in which they impact genomic evolution and maintenance [1–6]. Their mobility properties have resulted in their adoption as genetic tools in modern genetics with one of their many uses in biotechnology being the introduction of foreign genes into insect disease vectors of medical and agricultural importance [7–14]. Anopheles gambiae is the principal vector of the malaria-causing parasite Plasmodium falciparum in sub-equatorial Africa and is a mosquito species in which robust TE-based genetic tools need to be developed. At present there are six reports of successful genetic transformation of this mosquito, one using the P element, and five using the piggyBac element, transformation remaining a low frequency event [9, 15–19]. Isolating active, well adapted, endogenous TEs from A gambiae and understanding their biology is likely to improve the efficiency of genetic transformation in this species since these native active TEs are likely to have adapted to overcome or evade the host response systems that are proposed inactivate mobile DNA [20, 21].
Class 2 transposases typically bind to the TIRs and nearby internal sequences and mediate transposition to a new genomic location by the classical 'cut-and-paste' mechanism . Other cis-acting sequences, which usually consist of short repeat sequence motifs located close to the TIRs, are also important for proper transposase binding and efficient excision and transposition [28–35]. In many cases, native cis elements are not optimized for maximal transposition mobility; thus, new and improved TE gene vectors can be designed by altering these elements to increase or decrease transposase binding [36, 37]. The identification and characterization of these transposase binding sites and of the specific DNA-binding transposase residues is therefore important to our understanding of the biology and post integration behavior of TEs. This study aimed to identify the DNA sequences of the Herves element bound by its transposase.
Purification of Herves transposase and its binding to the Herves-L end
Herves transposase is 603 amino acids in length and is predicted to have a molecular weight of 70 kDa. Herves transposase was purified from an Escherichia coli expression system and its size was confirmed by SDS-PAGE (Figure 1b). To examine the binding of Herves transposase to the Herves-L end, we focused on the terminal 100 bp region. A radioactively labeled Herves-L 1-100 bp probe was incubated in the presence or absence of purified Herves transposase for use with a molar excess (200-fold) of unlabeled specific and non-specific DNA fragments were used as specific and non-specific competitors, respectively, in electrophoretic mobility shift assays (EMSAs). The transposase interacted with the Herves-L 100 bp probe and formed three transposase-DNA complexes (Figure 1c). A specific competitor competed for the transposase, but the non-specific competitor did not affect binding (Figure 1c) implicating a sequence-specific interaction between the transposase and the probe.
To confirm these results, each of the unlabeled 30 bp fragments was tested against the Herves-L bp 12-48 probe for binding to the transposase. Binding to the transposase was observed for this probe (Figure 2b), which resulted in two transposase-DNA complexes. The unlabeled L bp 28-60 fragment specifically competed for binding of the transposase (Figure 2b). Herves-L bp 48-75, bp 61-90, and bp 76-100 competed partially with the probe, indicating weak transposase binding to these regions. These results suggested that Herves-L bp 12-48 and bp 28-60 have strong and equal binding for Herves transposase, leading us to believe that the DNA binding motif lay within the Herves-L bp 28-48 fragment.
Transposase binds to the Herves-R end
To investigate the binding of transposase to the Herves-R end, the Herves-R 1-100 bp fragment was radiolabeled and used in EMSAs. Herves transposase interacted specifically with the probe and formed two transposase-DNA complexes (Figure 1c). Unlabeled specific competitor competed with the probe for transposase and, notably, the addition of a non-specific competitor led to the formation of a single, higher-molecular-weight complex (Figure 1c). The molecular composition of this complex, however, is unknown.
Two transposase-DNA complexes formed with the Herves-R bp 1-30, compared with a single complex each with the Herves-R bp 15-45 and bp 61-90 fragments, implicating the existence of two transposase binding sites within Herves-R bp 1-30 fragment and one site within both the Herves-R bp 15-45 and bp 61-90 fragments (Figure 4a). To determine relative transposase binding preferences, each 30 bp overlapping DNA fragment was allowed to compete against the Herves-R bp 61-90 probe for transposase binding using EMSAs. Fragment bp 15-45 successfully competed against the probe for transposase, whereas the Herves-R bp 1-30 and bp 31-60 fragments had no effect (Figure 4b). These data suggest that the transposase binds strongly to the terminal Herves-R end at positions bp 15-45 and bp 61-90.
We performed DNase I protection assays to identify specific binding motifs in the R end of Herves however these were inconclusive and showed some evidence of protection at bp 23-35 and bp 63-92 (Figure 4c).
Mutational analysis of the Herves transposase binding motif
The CG/AATTCAT motif is conserved between the Herves-L and Herves-R ends
We identified similar potential binding motifs within the Herves-R bp 15-22 and bp 73-86 regions. Furthermore, the bp 1-30 region also contains the R-TIR, a potential candidate for transposase binding. To determine whether the R-TIR or the CG/AATTCAT motif mediated the binding of transposase to the Herves-R bp 1-30 region, we mutated each region (Herves-R TIRmut and Herves-R bp 15-22mut) and subjected them to EMSA. Mutating each potential binding site abolished its ability to compete against the wild-type probe, suggesting that the CG/AATTCAT motif and R-TIR are both important for the transposase binding to the Herves-R end (Figure 5c).
The CGATTCAT motif is sufficient for purified Herves transposase binding
We used Herves-L bp 28-60 as a specific competitor for transposase against the (CGATTCAT)4 probe and found that it outcompeted it for transposase (Figure 7). Furthermore, splitting the CGATTCAT motif in half abolished the binding (data not shown). Together, these data indicated that the CGATTCAT motif was sufficient for the transposase binding.
We also tested the ability of unlabeled sequence variants of the CGATTCAT motif (CGATTCT T/CGATTCAC/CGTTCAT) to compete against radiolabeled CGATTCAT for transposase binding. None of the sequence variants competed fully with CGATTCAT for the transposase, indicating that CGATTCAT is the strongest binding motif (Figure 7). Nevertheless, CGATTCAC competed partially for transposase, suggesting that this variant may also be important for binding of transposase.
We purified active Herves transposase and demonstrated that it site-specifically binds to subterminal and terminal sequences at the Herves-L and Herves-R ends, respectively. Such asymmetrical binding may affect transposition frequency. The Drosophila P element transposase has been shown to bind asymmetrically to the P ends and interchanging the L end sequence with the R end sequence led to fewer transposition events . This phenomenon also occurs for the Ac element in maize and the Tag1 element in Arabidopsis[28, 31].
There was strong transposase binding to the Herves-L bp 12-48 and bp 28-60 regions and relatively weak binding to bp 48-75 as shown by EMSA and DNase I footprinting. None of these fragments however, outcompeted the L bp 1-100 probe for transposase binding, suggesting that the binding was cooperative between two or more regions. Furthermore, the overlapping Herves-L bp 12-48 and bp 28-60 fragments showed similar levels of binding, indicating that the binding motif lies in the overlapping region in Herves-L bp 28-48. In contrast to the L end, the binding occurred toward the terminal sequences on the Herves-R end in regions bp 15-45 and bp 61-90.
EMSA results with the Herves-L bp 28-60 probe and single nucleotide sequence variants indicated that the CGATTCAT motif, or its derivatives, mediated binding of the transposase. The CGATTCAT transposase-binding motif and its derivatives are repeated and conserved in the Herves-L and Herves-R end sequences. Our results suggested that this motif is important and sufficient for transposase binding, because: (1) mutating the CGATTCAT motif at either end abolished binding, and (2) the transposase bound specifically to a synthetic tetramer of the motif.
TEs frequently have multiple transposase binding sites adjacent to their TIRs [33, 39–42]. In other hAT elements, such as Ac, Tol2 and Tag1, their respective transposases bind to short sequence repeats [31, 33, 34, 43]. For Ac and Tol2, the transposase binding sequence motifs differ at the L and R ends [33, 34]. The Herves transposase-binding CGATTCAT motif, however, is highly conserved at both ends with several single nucleotide variants CGATTCAC, CGTTCAT, and CGATTCTT being present. Our results suggest that these additional motifs may also mediate transposase binding. Although these derivatives are related to the CGATTCAT motif, their ability to bind transposase differs. The transposase binds to CGATTCAT, but weakly to the CGATTCAC and CGTTCAT motifs. It is also possible that the transposase only recognizes a subset or a family of related sequences in which GATTC or ATTCA is the central sequence. Similar results have been reported for the Tag1 element, for which the R-TGACCC and L-AAACCC motifs have different affinities for the transposase [31, 43]. The sequences that flank these motifs differ, and although they might fail to influence transposase binding, they may regulate transposition .
We observed no binding to the L-TIR. Several related hAT transposases, such as Ac and Tag1, do not bind their L-TIR and R-TIR sequences [33, 43]. This phenomenon raises the possibility that transposase binding to the L-TIR may require the presence of a host factor however nuclear extracts from a Herves transposase-expressing Drosophila S2 cell line did not bind to L-TIR making the argument for such a factor less compelling. Nevertheless, pure Herves transposase interacted with the R-TIR sequence, the binding at which appeared to be cooperative since both the R-TIR and CGATTCAT motif at 15-22 bp participated in it.
We have identified the sites within the Herves element to which the Herves transposase binds and shown that it binds asymmetrically to sequences at either end of the element. Future work will be directed towards determining whether mutants of Herves which show changes in the binding of the transposase will affect the transpositional activity of Herves in vivo leading to the development of this endogenous TE of A gambiae as a genetic tool in this medically important mosquito species.
We identified the specific DNA-binding sites of the Herves transposase, a member of the hAT transposon superfamily. We found that it displayed an asymmetry of specific binding to the L and R ends of the Herves transposon in that it bound to both subterminal regions but interacted only with the R, but not L, TIR. We identified a common subterminal DNA-binding motif (CG/AATTCAT) that is critical and sufficient for Herves transposase binding. The asymmetry of binding of the transposase to the L and R ends may indicate that these ends differ in their interactions with the enzyme during the transposition reaction. The differences in transposase binding sites between different hAT transposases illustrates that this superfamily provides a fascinating diversity with which to study the biology of transposition.
The Herves ORF was cloned into pBAD myc/HisA (Invitrogen, Carlsbad, CA). The Bsp HI (incorporated into the Herves start codon) and Kpn I restriction sites were used to amplify a 766-bp fragment of the Herves ORF using the Herves F-Bsp HI (GATCAATCATGATGGCTCCAACAAACGCAAC) and Herves R-Kpn I (GTTCAAGGTACCTTGAATCCAATTAGCTATATTCTTACC) primers.
The resulting fragment was cloned into Nco I/Kpn I-digested pBAD myc/HisA to generate pBADHvPCR1. The remaining Herves ORF (1,118 bp) was amplified using the Herves F-Kpn I (CAAGGTACCTTGAACAAATTTGACATAGAGGATAAG) and Herves R-Hin dIII primers (TATCAAGCTTTGAACAAATTTGACATAGAGGATAAG) and cloned into Kpn I/Hin dIII digested pBADHvPCR1 to generate pBADHv1.
Herves transposase purification
Herves transposase was purified by His-tag purification as described . pBADHv1-transformed LMG 194 E coli cells were grown overnight at 30°C in LB media that contained carbenicillin (100 mg/ml). The overnight culture was diluted 1:100 in LB and carbenicillin (100 mg/ml) and grown at 30°C and 230 rpm to an absorbance of 0.6 at 600 nm. The cultures were then induced with 0.1% L-arabinose and shaken at 16°C for 18 h. The cells were harvested and washed by centrifugation with binding buffer (0.5 M NaCl, 20 mM Tris-Cl pH 7.9, 10% glycerol, 10 mM imidazole). The cells were lysed twice using a French press at 20,000 psi. The cell lysate was cleared by centrifugation and by passing through 0.45 μm syringe filters. Cleared lysate was loaded onto Sepharose (Amersham/GE Healthcare, Piscataway, NJ) chromatography columns that were pre-equilibrated with Ni2+. The columns were washed with 10 ml binding buffer and 6 ml wash buffer (0.5 M NaCl, 20 mM Tris-Cl pH 7.9, 10% glycerol, 50 mM imidazole). His-tagged Herves was eluted in five 1 ml fractions of elution buffer (0.5 M NaCl, 20 mM Tris-Cl pH 7.9, 10% glycerol, 200 mM imidazole). The purified Herves transposase was dialyzed overnight in dialysis buffers 1 (0.5 M NaCl, 20 mM Tris base, 10% glycerol pH 8.0) and 2 (0.5 M NaCl, 20 mM Tris base, 2 mM dithiothreitol (DTT), 25% glycerol pH 8.0) for 3 h using a Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific, Waltham, MA). The dialyzed, purified Herves transposase was stored at -80°C.
The DNA fragment (100 nM) that we tested for transposase binding was end labeled using T4 polynucleotide kinase and 32P ATP and purified on a Biospin 30 column (BioRad, Hercules, CA). The labeled DNA fragment (probe) was incubated at 4°C for 45 min with 1 × EMSA binding buffer (16 mM Tris pH 8.0, 0.2 μg bovine serum albumin (BSA), 0.4 μg T3 single-stranded oligo, 0.5 μg poly(dI-dC), 1 mM DTT, 150 mM NaCl, 0.25% Triton X) and 850 nM of Herves transposase. Specific and non-specific DNA fragments were used as specific and non-specific competitors, respectively (if applicable). The reaction was incubated with the probe for an additional 40 min at 4°C. The non-specific competitors were 126 bp gDNA fragment (E1) that flanks Hermes TE from Musca domestica and a 30 bp DNA oligo from Aedes aegypti β2 tubulin. The EMSA reaction products were analyzed on a 5% TBE polyacrylamide gel (Bio-Rad).
DNase I protection assay
DNA fragments (100 bp each) from the Herves-L and Herves-R ends, containing an Eco RV restriction site at the L-end or R-end, were cloned into pJET 1.2 (Fermentas/Thermo Fisher Scientific, Piscataway, NJ) to generate pL5'Eco RV, pL3'Eco RV, pR5'Eco RV, and pR3'Eco RV. The transferred and non-transferred strands from the Herves-L and Herves-R ends were selectively radiolabeled at one end by digesting pL5'Eco RV, pL3'Eco RV, pR5'Eco RV, and pR3'Eco RV with Xho I and Eco RV and labeling them with [32P] dATP using Klenow (NEB, Ipswich, MA). Herves transposase was allowed to bind to 100 nM single end-labeled DNA fragment (probe) under the same binding conditions as in the EMSA. The optimal concentrations of transposase were determined empirically (Additional files 1 and 2). The DNA probe was subjected to DNase I digestion for 2 min at 4°C. The reaction was stopped by adding stop solution (92% ethanol, 0.7 M ammonium acetate, 0.35 μg tRNA) for 15 min in a dry ice/ethanol bath. DNA was extracted with phenol/chloroform and precipitated with ethanol. The reaction products were analyzed on a 10% denaturing polyacrylamide sequencing gel. The DNA sequencing kit 2.0 (USB) was used to construct a nucleotide ladder that was analyzed with the reaction products on the sequencing gel (Additional file 3).
This research was supported by PHS grants AI45741 and GM48102 to PWA and DAO, respectively, and by the Interdepartmental Graduate Program in Cell, Molecular and Developmental Biology at the University of California, Riverside.
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