Shawn M. Christensen

R2 Target-Primed Reverse Transcription: Ordered Cleavage and Polymerization Steps by Protein Subunits Asymmetrically Bound to the Target DNA

ABSTRACT R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.

RNA from the 5′ end of the R2 retrotransposon controls R2 protein binding to and cleavage of its DNA target site

Non-LTR retrotransposons insert into eukaryotic genomes by target-primed reverse transcription (TPRT), a process in which cleaved DNA targets are used to prime reverse transcription of the elements RNA transcript. Many of the steps in the integration pathway of these elements can be characterized in vitro for the R2 element because of the rigid sequence specificity of R2 for both its DNA target and its RNA template. R2 retrotransposition involves identical subunits of the R2 protein bound to different DNA sequences upstream and downstream of the insertion site. The key determinant regulating which DNA-binding conformation the protein adopts was found to be a 320-nt RNA sequence from near the 5′ end of the R2 element. In the absence of this 5′ RNA the R2 protein binds DNA sequences upstream of the insertion site, cleaves the first DNA strand, and conducts TPRT when RNA containing the 3′ untranslated region of the R2 transcript is present. In the presence of the 320-nt 5′ RNA, the R2 protein binds DNA sequences downstream of the insertion site. Cleavage of the second DNA strand by the downstream subunit does not appear to occur until after the 5′ RNA is removed from this subunit. We postulate that the removal of the 5′ RNA normally occurs during reverse transcription, and thus provides a critical temporal link to first- and second-strand DNA cleavage in the R2 retrotransposition reaction.

Role of the Bombyx mori R2 element N-terminal domain in the target-primed reverse transcription (TPRT) reaction

R2 is a site-specific non-long terminal repeat (non-LTR) retrotransposon encoding a single polypeptide with reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The current model of R2 retrotransposition involves an ordered series of cleavage and polymerization steps carried out by at least two R2 protein subunits, one bound upstream and one bound downstream of the integration site. The role in the retrotransposition reaction of two conserved DNA-binding motifs, a C2H2 zinc finger (ZF) and a Myb motif, located within the N-terminal domain of the protein are explored in this report. These motifs do not appear to play a role in RT or the ability of the protein to bind the R2 RNA transcript. Methylation and missing nucleoside interference-based DNA footprints using polypeptides to the N-terminal domain suggest the ZF and Myb motifs bind to regions −3 to −1 and +10 to +15 with reference to the insertion site. Mutations in these DNA sites or of the N-terminal protein domain blocked binding and the activity of the downstream subunit. Mutations of the protein domain also affected binding of the upstream subunit but not its function, suggesting the primary path to DNA target recognition by R2 involves both upstream and downstream subunits.

Target Specificity of the Endonuclease from the Xenopus laevis Non-Long Terminal Repeat Retrotransposon, Tx1L

ABSTRACT Elements of the Tx1L family are non-long terminal repeat retrotransposons (NLRs) that are dispersed in the genome ofXenopus laevis. Essentially all genomic copies of Tx1L are found inserted at a specific site within another family of transposable elements (Tx1D). This suggests that Tx1L is a site-specific retrotransposon. Like many (but not all) other NLRs, theXenopus element encodes an apparent endonuclease that is related in sequence to the apurinic-apyrimidinic endonucleases that participate in DNA repair. This enzyme is thought to introduce the single-strand break in target DNA that initiates transposition by the target-primed reverse transcription (TPRT) mechanism. To explore the issue of target specificity more fully, we expressed the polypeptide encoded by the endonuclease domain of open reading frame 2 from Tx1L (Tx1L EN) and characterized its cleavage capabilities. This endonuclease makes a specific nick in the bottom strand precisely at one end of the presumed Tx1L target duplication. Because this activity leaves a 5′-phosphate and 3′-hydroxyl at the nick, it has the location and chemistry required to initiate new insertion events by TPRT. Tx1L EN does not make a specific cut at a preferred target site for Tx1D elements, ruling out the alternative possibility that the composite Tx1L-Tx1D element moves as a unit under the control of functions encoded by Tx1L. Further characterization revealed that the endonuclease remains active for many hours at room temperature and that it is capable of enzymatic turnover. Scanning substitution mutagenesis located the recognition site for Tx1L EN within 10 bp surrounding the primary nick site. Implications of these features for natural transposition events are discussed.

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA.

Convergently Recruited Nuclear Transport Retrogenes are Male Biased in Expression and Evolving Under Positive Selection in Drosophila

The analyses of gene duplications by retroposition have revealed an excess of male-biased duplicates generated from X chromosome to autosomes in flies and mammals. Investigating these genes is of primary importance in understanding sexual dimorphism and genome evolution. In a particular instance in Drosophila, X-linked nuclear transport genes (Ntf-2 and ran) have given rise to autosomal retroposed copies three independent times (along the lineages leading to Drosophila melanogaster, D. ananassae, and D. grimshawi). Here we explore in further detail the expression and the mode of evolution of these Drosophila Ntf-2- and ran-derived retrogenes. Five of the six retrogenes show male-biased expression. The ran-like gene of D. melanogaster and D. simulans has undergone recurrent positive selection. Similarly, in D. ananassae and D. atripex, the Ntf-2 and ran retrogenes show evidence of past positive selection. The data suggest that strong selection is acting on the origin and evolution of these retrogenes. Avoiding male meiotic X inactivation, increasing level of expression of X-linked genes in male testes, and/or sexual antagonism might explain the recurrent duplication of retrogenes from X to autosomes. Interestingly, the ran-like in D. yakuba has mostly pseudogenized alleles. Disablement of the ran-like gene in D. yakuba indicates turnover of these duplicates. We discuss the possibility that Dntf-2r and ran-like might be involved in genomic conflicts during spermatogenesis.

Electrical detection of single-base DNA mutation using functionalized nanoparticles

We report an electrical scheme to detect specific DNA. Engineered hairpin probe DNA are immobilized on a silicon chip between gold nanoelectrodes. Hybridization of target DNA to the hairpin melts the stem nucleotides. Gold nanoparticle-conjugated universal reporter sequence detects the open hairpins by annealing to the exposed stem nucleotides. The gold nanoparticles increase charge conduction between the electrodes. Specifically, we report on a hairpin probe designed to detect a medically relevant mutant form of the K-ras oncogene. Direct current measurements show three orders of magnitude increase in conductivity for as low as 2fmol of target molecules.

Nanostructures for medical diagnostics

Nanotechnology is the art of manipulating materials on atomic or molecular scales especially to build nanoscale structures and devices. The field is expanding quickly, and a lot of work is ongoing in the design, characterization, synthesis, and application of materials, structures, devices, and systems by controlling shape and size at nanometer scale. In the last few years, much work has been focused on the use of nanostructures toward problems of biology and medicine. In this paper, we focus on the application of various nanostructures and nanodevices in clinical diagnostics and detection of important biological molecules. The discussion starts by introducing some basic techniques ofmicro-/nanoscale fabrication that have enabled reproducible production of nanostructures. The prospects, benefits, and limitations of using these novel techniques in the fields of biodetection and medical diagnostics are then discussed. Finally, the challenges of mass production and acceptance of nanotechnology by the medical community are considered.

Independently derived targeting of 28S rDNA by A- and D-clade R2 retrotransposons: Plasticity of integration mechanism

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through a target primed reverse transcription mechanism (TPRT). R2 elements have been used as a model system for investigating non-LTR retrotransposon integration. We previously demonstrated that R2 retrotransposons require two subunits of the element-encoded multifunctional protein to integrate—one subunit bound upstream of the insertion site and one bound downstream. R2 elements have been phylogenetically categorized into four clades: R2-A, B, C, and D, that diverged from a common ancestor more than 850 million years ago. All R2 elements target the same sequence within 28S rDNA. The amino-terminal domain of R2Bm, a R2-D clade element, contains a single zinc finger and a Myb motif that are responsible for binding R2 protein downstream of the insertion site. Target site recognition is of interest as it is the first step in the integration reaction and may help elucidate evolutionary history and integration mechanism. The amino-terminal domain of R2-A clade members contains three zinc fingers and a Myb motif. We show here that R2-Lp, an R2-A clade member, uses its amino-terminal DNA binding motifs to bind upstream of the insertion site. Because the R2-A and R2-D clade elements recognize 28S rDNA differently, we conclude the A- and D-clades represent independent targeting events to the 28S site. Our results also indicate a certain plasticity of insertional mechanics exists between the two clades.

Targeting novel sites

Restriction-like endonuclease (RLE) bearing non-LTR retrotransposons are site-specific elements that integrate into the genome through target primed reverse transcription (TPRT). RLE-bearing elements have been used as a model system for investigating non-LTR retrotransposon integration. R2 elements target a specific site in the 28S rDNA gene. We previously demonstrated that the two major sub-classes of R2 (R2-A and R2-D) target the R2 insertion site in an opposing manner with regard to the pairing of known DNA binding domains and bound sequences-indicating that the A- and D-clades represent independently derived modes of targeting that site. Elements have been discovered that group phylogenetically with R2 but do not target the canonical R2 site. Here we extend our earlier studies to show that a separate R2-A clade element, which targets a site other than the canonical R2 site, does so by using the amino-terminal zinc fingers and Myb motifs. We further extend our targeting studies beyond R2 clade elements by investigating the ability of the amino-terminal zinc fingers from the nematode NeSL-1 element to target its integration site. Our data are consistent with the use of an amino-terminal DNA binding domain as one of the major targeting determinants used by RLE-bearing non-LTR retrotransposons to secure a protein subunit near the insertion site. This amino-terminal DNA binding domain can undergo modifications, allowing the element to target novel sites. The binding orientation of the amino-terminal domain relative to the insertion site is quite variable.