Reverse Transcription Mechanism Research Articles

Regulatory mechanisms of human and mouse telomerase reverse transcriptase gene transcription: distinct dependency on c-Myc

Telomerase-a complex ribonucleoprotein enzyme-synthesizes telomeric repeats to avoid telomere loss that accompanies cell division and chromosomal replication. Expression of telomerase is detectable in embryonic cells and cancer cells, but not in normal human cells. On the other hand, in mice, substantial expression of telomerase is detected in normal cells and tissues as well as in immortalized cells. These results suggest that the regulatory mechanisms of telomerase activity in humans and mice differ. Considering these results along with the fact that the expression of the telomerase reverse transcriptase (TERT) gene is a rate-limiting step for telomerase activity, we compared transcriptional regulatory mechanisms of both the species. A series of luciferase assays and RT-PCR analyses demonstrated that c-Myc, a dominant transactivator for human TERT (hTERT), is not involved in the regulation of mouse TERT (mTERT). These results suggest that distinct molecules and pathways are involved in the process of immortalization and tumorigenesis in human and mouse cells.

Rotifer rDNA-specific R9 retrotransposable elements generate an exceptionally long target site duplication upon insertion

Ribosomal DNA genes in many eukaryotes contain insertions of non-LTR retrotransposable elements belonging to the R2 clade. These elements persist in the host genomes by inserting site-specifically into multicopy target sites, thereby avoiding random disruption of single-copy host genes. Here we describe R9 retrotransposons from the R2 clade in the 28S RNA genes of bdelloid rotifers, small freshwater invertebrate animals best known for their long-term asexuality and for their ability to survive repeated cycles of desiccation and rehydration. While the structural organization of R9 elements is highly similar to that of other members of the R2 clade, they are characterized by two distinct features: site-specific insertion into a previously unreported target sequence within the 28S gene, and an unusually long target site duplication of 126 bp. We discuss the implications of these findings in the context of bdelloid genome organization and the mechanisms of target-primed reverse transcription.

Diversity-Generating Retroelement Homing Regenerates Target Sequences for Repeated Rounds of Codon Rewriting and Protein Diversification

Diversity-generating retroelements (DGRs) introduce vast amounts of sequence diversity into target genes. During mutagenic homing, adenine residues are converted to random nucleotides in a unidirectional, reverse transcriptase-dependent transposition process from a donor template repeat (TR) to a recipient variable repeat (VR). Using a Bordetella bacteriophage DGR as a model, we demonstrate that homing occurs through a TR-containing RNA intermediate and is RecA independent. Marker transfer studies show that cDNA integration at the 3' end of VR occurs within a (G/C)(14) element, and deletion analysis demonstrates that the reaction is independent of 5' end cDNA integration. cDNA integration at the 5' end of VR requires only short stretches of sequence homology. We propose that homing occurs through a unique target DNA-primed reverse transcription mechanism that precisely regenerates target sequences. This nonproliferative "copy and replace" mechanism enables repeated rounds of protein diversification and optimization of ligand-receptor interactions.

Hepatitis B viruses: Reverse transcription a different way

Hepatitis B virus (HBV), the causative agent of B-type hepatitis in humans, is the type member of the Hepadnaviridae, hepatotropic DNA viruses that replicate via reverse transcription. Beyond long-established differences to retroviruses in gene expression and overall replication strategy newer work has uncovered additional distinctions in the mechanism of reverse transcription per se. These include protein-priming by the unique extra terminal protein domain of the reverse transcriptase (RT) utilizing an RNA hairpin for de novo initiation of first strand DNA synthesis, and the strict dependence of this process on cellular chaperones. Recent in vitro reconstitution systems enabled first biochemical insights into this multifactorial reaction, complemented by high resolution structural information on the RNA, though not yet the protein, level. Genetic approaches have revealed long-distance interactions in the nucleic acid templates as an important factor enabling the puzzling template switches required to produce the relaxed circular (RC) DNA found in infectious virions. Finally, the failure of even potent HBV RT inhibitors to eliminate nuclear covalently closed circular (ccc) DNA, the functional equivalent of integrated proviral DNA, has spurred a renewed interest in the mechanism of cccDNA generation. These new developments are in the focus of this review.

Novel SINE Families from Salmons Validate Parahucho (Salmonidae) as a Distinct Genus and Give Evidence that SINEs Can Incorporate LINE-related 3′-Tails of Other SINEs

Short interspersed elements (SINEs) constitute a group of retroposons propagating in the genome via a mechanism of reverse transcription, in which they depend on the enzymatic machinery of long retroposons (LINEs). Over 70 SINE families have been described to date from the genomes of various eukaryotes. Here, we characterize two novel SINEs from salmons (Actinopterygii: Salmonoidei). The first family, termed SlmI, was shown to be widespread among all genera of the suborder. These SINEs have a tRNA(Leu)-related promoter region at their 5'-end, a unique central conserved domain with a subfamily-specific region, and an end with RSg-1-LINE-derived 3'-terminus preceding the A/T-rich tail. The same LINE-related segment is also shared by two other salmonid SINEs: HpaI and OS-SINE1. The structural peculiarities and overall sequence identity of the SlmI 3'-terminus suggest that it has been acquired from HpaI SINEs but not directly from the partner LINE. This region plays a crucial role in the process of retrotransposition of short interspersed elements, and the case of its SINE-to-SINE transmission is the first recorded to date. Possible scenarios and potential evolutionary implications of the observed interaction between short retroposons are discussed. Apart from the above, we found a copy of the SlmI SINE in the GenBank entry for the blood fluke, Schistosoma japonicum (Trematoda: Strigeiformes) -- a trematode causing one of the most important human helminth infections, with its genome known to host other groups of salmonoid retroposons. In the present article, we suggest our views with regard to possible ways in which such an intensive horizontal transfer of salmonoid retroposons to the schistosomal genome occurs. The second novel SINE family, termed SlmII, originates from one of the SlmI subfamilies, with which it shares the same tRNA-related region, central domain, and a part of RSg-1-derived segment, but has a different 3'-tail of unidentified origin. Its distribution among salmonids validates Parahucho (Japanese huchen) as a distinct monotypic genus.

The Duck Hepatitis B Virus Reverse Transcriptase Functions as a Full-length Monomer

Hepadnaviral reverse transcription occurs within cytoplasmic capsid particles and is catalyzed by a virally encoded reverse transcriptase, but the primary structure and multimeric state of the polymerase during reverse transcription are poorly understood. We measured these parameters for the duck hepatitis B virus polymerase employing active enzyme translated in vitro and derived from intracellular core particles and mature virions. In vitro-translated polymerase immunoprecipitated as a monomer, and polymerase molecules with complementary defects in the enzymatic active site and tyrosine 96, which primes DNA synthesis, could not complement or inhibit each other in priming assays. Western analysis using antibodies recognizing epitopes throughout the polymerase combined with nuclease digestion of permeabilized virion-derived capsid particles revealed that only full-length polymerase molecules were in virions and that they were all covalently attached to large DNA molecules. Because DNA synthesis is primed by the polymerase itself and only one copy of the viral DNA is in each capsid, the polymerase must function as an uncleaved monomer. Therefore, a single polymerase monomer is encapsidated, primes DNA synthesis, synthesizes both DNA strands, and participates in the three-strand transfers of DNA synthesis, with all steps after DNA priming performed while the polymerase is covalently coupled to the product DNA. Because the N-terminal domain of the polymerase is displaced from the active site on the same molecule by the viral DNA during reverse transcription, P must be structurally dynamic during DNA synthesis. Therefore, non-nucleoside compounds that interfere with this change may be novel antiviral agents.

Molecular Insights into the Mechanisms of HIV-1 Reverse Transcriptase Resistance to Nucleoside Analogs

The causative agent of acquired immunodeficiency syndrome, HIV-1, depends on one of its enzymes, reverse transcriptase, to copy its single stranded RNA genome into a double stranded DNA nucleic acid suitable for integration in the host cell genome. In the last two decades, the advances in the knowledge of the kinetic mechanism of reverse transcription and in the determination of the crystallographic structures for the complexes of the enzyme with substrates and products were huge. However, all of this knowledge resulted in the design of RT inhibitors for which the virus, after a short period of exposure, becomes less susceptible, due to the development of resistance. The development of resistance is caused by the high frequency of viral mutation and the toxicity of those same drugs. Therefore, a closer look at all the available information might shed some light into this subject and help to develop new strategies to overcome the lack of long term clinical efficiency of these drugs. Here, we present a critical atomic level study of all the mutations that have been detected and reported so far, as a reaction of the enzyme to counteract the action of the inhibitors.

Explanation of the mechanism of carcinogenesis and syntheses of anticancer agents with high selectivity

In 1979, the mechanism of chemical carcinogenesis, a challenging and difficult scientific problem pending for a number of years, was explained by Dai Qianhuan. The mechanism named di-region theory predicted that a carcinogen always metabolizes to form a special bi-functional alkylating agent. This agent induces cross-linkages between the complementary base pairs in DNA and switches on initial mutageneses in genomes including point and frameshift mutations. This, in turn, induces further deep mutageneses including the production of various chimeric chromosomes, deletions and other aberrations found in genomes. In the end this initiates carcinogenesis of the whole cell through the reverse transcription mechanism after a lengthy incubation period. Recently, this laboratory has verified that physical carcinogenesis, including the oncogenesis induced by radiation and asbestos as well as the carcinogenesis induced by endogenous factors such as estrogen or diethyl-stilbestrol switch on carcinogenesis by inducing the formation of cross-linkages between the complementary base pairs in DNA. Di-region theory has now been supported by many experimental observations such as mutational spectra of various carcinogens. The potential for carcinogenesis, teratogenesis, sterility and mutagenesis lumped together as genetic toxicity appears to originate almost uniformly from the cross-linking between complementary bases, i.e. malignant cross-linking, which is in accordance with di-region theory. Other forms of cross-linking between non-complementary bases, benign cross-linkings, show bi-functional alkylation anticancer activity but lack genetic toxicity. The predictable design and synthesis of a high selectivity anticancer agent with high efficacy and low genetic toxicity, a goal long pursued in cancer chemotherapy, have been realized for the first time in this laboratory by inhibiting malignant and heightening benign cross-linking using the principles of di-region theory. A series of patented new anticancer platinum complexes called di-regioplatins, based on the above predetermined design, have been reported. In these cancer cell kill rates, tumor-inhibition rates and the ultimate life-span for two mouse carcinoma models using several com-pounds of cis-di-substituted-benzylaminodihaloplatinum (II) are notably higher than those of cisplatin, but their toxicities all are much lower than cisplatin. Based on a predictive design using di-region theory and group theory, a new anticancer complex, cis-diammine-cyclopentane-1,1-dicarboxylato-platinum (II) called minoplatin, has been synthesized in this laboratory by making the minimal structural revision of adding an CH2 unit on the four-member ring of carboplatin. The water solubility of minoplatin is almost double that of carboplatin, yet its lipid solubility is much higher than carboplatin. Animal acute toxicity of minoplatin is only half that of carboplatin, and the curative effect of minoplatin in a rapid growing animal tumor model of the ascites-type is remarkably higher than that of carboplatin. Genetic toxicity of minoplatin as measured by reverse mutagenesis with the TA 102 strain is only 1/200 of cisplatin and 1/10 of carboplatin. Minoplatin, as opposed to cisplatin, shows no teratogenesis to the offspring of pregnant female mice. Additionally, some recent empirical research results on anticancer platinum complexes have been explained by the di-region theory, and comments from the perspective of predicting the selectivity of anticancer agents have been presented. We anticipate that future clinical testing will demonstrate that minoplatin and di-regioplatins are the first examples in a class consisting of highly selective, low toxicity, and tailored second generation anticancer agents.

Patterns of Intron Gain and Loss in Fungi

Little is known about the patterns of intron gain and loss or the relative contributions of these two processes to gene evolution. To investigate the dynamics of intron evolution, we analyzed orthologous genes from four filamentous fungal genomes and determined the pattern of intron conservation. We developed a probabilistic model to estimate the most likely rates of intron gain and loss giving rise to these observed conservation patterns. Our data reveal the surprising importance of intron gain. Between about 150 and 250 gains and between 150 and 350 losses were inferred in each lineage. We discuss one gene in particular (encoding 1-phosphoribosyl-5-pyrophosphate synthetase) that displays an unusually high rate of intron gain in multiple lineages. It has been recognized that introns are biased towards the 5′ ends of genes in intron-poor genomes but are evenly distributed in intron-rich genomes. Current models attribute this bias to 3′ intron loss through a poly-adenosine-primed reverse transcription mechanism. Contrary to standard models, we find no increased frequency of intron loss toward the 3′ ends of genes. Thus, recent intron dynamics do not support a model whereby 5′ intron positional bias is generated solely by 3′-biased intron loss.

A novel single-stranded DNA enzyme expression system using HIV-1 reverse transcriptase

In this study, we exploited a DNA enzyme expression system using the mechanism of HIV-1 reverse transcription in vitro. HIV-1 reverse transcription is initiated when its cognate primer tRNA (Lys-3) binds to the primer binding site (PBS) of the viral RNA template. Therefore, this RNA contains the HIV-1 PBS, the DNA enzyme, and a tRNA (Lys-3) at the 3 ′-end of its RNA transcript, such that a single-stranded DNA (ssDNA) is synthesized by the HIV-1 reverse transcriptase. We constructed RNA expression vectors including the HIV-1 PBS, the DNA enzyme, and either a native tRNA (Lys-3) or one of two truncated tRNAs (Lys-3), ΔtRNA (Lys-3) and ΔΔtRNA (Lys-3). The reactions of the pVAX1-Dz-tRNA (Lys-3), pVAX1-Dz-ΔtRNA (Lys-3), and pVAX1-Dz-ΔΔtRNA (Lys-3) vectors with T7 RNA polymerase in vitro gave the corresponding RNAs. The liberated RNAs were treated with HIV-1 reverse transcriptase (HIV-1 RT) in vitro, which yielded the corresponding ssDNA. The cleavage assay results demonstrated that the expressed DNA enzyme has cleavage ability against the target sequence. Thus, we have found a new DNA enzyme oligonucleotide expression system using the HIV-1 reverse transcriptase in vitro.

A Novel Endogenous Retrovirus-Related Element in the Human Genome Resembles a DNA Transposon: Evidence for an Evolutionary Link?

A significant fraction of the human genome is composed of various types of transposable elements, which are divided into two broad classes based on their mechanism of transposition: via an RNA intermediate (retroelements) or via a DNA intermediate (DNA transposons). The retroelements, which include endogenous retroviruses, are the more prominent group in humans and seem to be restricted to eukaryotes. DNA transposons are known in almost all well-studied organisms, including bacteria. In the human genome, they exist as mere fossils and seem to have been inactive for millions of years. We report here an element in the human genome that is the product of a recent retrotransposition event, yet apparently, by a novel mechanism of reverse transcription, has acquired inverted repeats more characteristic of DNA transposons.

A Novel Endogenous Retrovirus-Related Element in the Human Genome Resembles a DNA Transposon: Evidence for an Evolutionary Link?

A significant fraction of the human genome is composed of various types of transposable elements, which are divided into two broad classes based on their mechanism of transposition: via an RNA intermediate (retroelements) or via a DNA intermediate (DNA transposons). The retroelements, which include endogenous retroviruses, are the more prominent group in humans and seem to be restricted to eukaryotes. DNA transposons are known in almost all well-studied organisms, including bacteria. In the human genome, they exist as mere fossils and seem to have been inactive for millions of years. We report here an element in the human genome that is the product of a recent retrotransposition event, yet apparently, by a novel mechanism of reverse transcription, has acquired inverted repeats more characteristic of DNA transposons.

Dynamics of R1 and R2 elements in the rDNA locus of Drosophila simulans.

The mobile elements R1 and R2 insert specifically into the rRNA gene locus (rDNA locus) of arthropods, a locus known to undergo concerted evolution, the recombinational processes that preserve the sequence homogeneity of all repeats. To monitor how rapidly individual R1 and R2 insertions are turned over in the rDNA locus by these processes, we have taken advantage of the many 5' truncation variants that are generated during the target-primed reverse transcription mechanism used by these non-LTR retrotransposons for their integration. A simple PCR assay was designed to reveal the pattern of the 5' variants present in the rDNA loci of individual X chromosomes in a population of Drosophila simulans. Each rDNA locus in this population was found to have a large, unique collection of 5' variants. Each variant was present at low copy number, usually one copy per chromosome, and was seldom distributed to other chromosomes in the population. The failure of these variants to spread to other units in the same rDNA locus suggests a strong recombinational bias against R1 and R2 that results in the individual copies of these elements being rapidly lost from the rDNA locus. This bias suggests a significantly higher frequency of R1 and R2 retrotransposition than we have previously suggested.

Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position −25 to +19 from the intron-insertion site on the top strand and from −28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5′-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions −12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3′ exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3′ exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

HIV-1 Reverse Transcriptase Interaction with Model RNA–DNA Duplexes

HIV-1 reverse transcriptase (HIV-1 RT) is a multifunctional enzyme responsible for converting viral RNA into preintegrative DNA during the early stages of viral infection. DNA polymerase and RNase H activities are required, and several conformationally distinct primer–templates must be accommodated by the enzyme during the process. Parameters of interaction between model substrates (ligands) and HIV-1 RT (wild type p66/p51 and the RNase H-deficient mutant p66 E478Q/p51) (analytes) were estimated by surface plasmon resonance at 25°C, pH 8.0. Binding of RT to the ligands is specific and can be analyzed using a conventional 1:1 binding algorithm. RNA–DNA hybrids with 5′-template overhangs of 6 and 12 nucleotides bind to RT approximately one order of magnitude stronger than the corresponding 36-mer with blunt ends due to slower dissociation. Immobilization of the latter through either the 5′-end of RNA or DNA strand does not change the equilibrium constant ( K D) for wild-type RT but the values of kinetic constants of association and dissociation differ significantly. For the p66 E478Q/p51 enzyme, orientation effects are notable even alteringthe K D value. Binding of the p66 E478Q/p51 to any RNA–DNA hybrids is slightly stronger compared with wild type. Data can be interpreted in terms of the mechanism of reverse transcription.

The msDNAs of bacteria

msDNAs are small, structurally unique satellite DNAs found in a number of Gram-negative bacteria. Composed of hundreds of copies of single-stranded DNA--hence the name multicopy single-stranded DNA--msDNA is actually a complex of DNA, RNA, and probably protein. These peculiar molecules are synthesized by a reverse transcription mechanism catalyzed by a reverse transcriptase (RT) that is evolutionarily related to the polymerase found in the HIV virus. The genes, including the RT gene, responsible for the synthesis of msDNA are encoded in a retron, a genetic element that is carried on the bacterial chromosome. The retron is, in fact, the first such retroelement to be discovered in prokaryotic cells. This report is a comprehensive review of the many interesting questions raised by this unique DNA and the fascinating answers it has revealed. We have learned a great deal about the structure of msDNA: how it is synthesized, the structure and functions of the RT protein required to make it, its effects on the host cell, the retron element that encodes it, its possible origins and evolution, and even its potential usefulness as a practical genetic tool. Despite the impressive gains in our understanding of the msDNAs, however, the simple, fundamental question of its natural function remains an enduring mystery. Thus, we have much more to learn about the msDNAs of bacteria.

Evidence for the packaging of multiple copies of Tf1 mRNA into particles and the trans priming of reverse transcription.

Long terminal repeat (LTR)-containing retrotransposons and retroviruses are close relatives that possess similar mechanisms of reverse transcription. The particles of retroviruses package two copies of viral mRNA that both function as templates for the reverse transcription of the element. We studied the LTR-retrotransposon Tf1 of Schizosaccharomyces pombe to test whether multiple copies of transposon mRNA participate in the production of cDNA. Using the unique self-priming property of Tf1, we obtained evidence that multiple copies of Tf1 mRNA were packaged into virus-like particles. By coexpressing two distinct versions of Tf1, we found that the bulk of reverse transcription that was initiated on one mRNA template was subsequently transferred to others. In addition, the first 11 nucleotides of one mRNA were able to prime, in trans, the reverse transcription of another mRNA.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64)and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich pallindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mtRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74 A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Target Specificity of the Endonuclease from theXenopus laevisNon-Long Terminal Repeat Retrotransposon, Tx1L

Elements of the Tx1L family are non-long terminal repeat retrotransposons (NLRs) that are dispersed in the genome of Xenopus 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, the Xenopus 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.