Real-time DNA sequencing from single polymerase molecules.

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

DNA-directed DNA polymerase and strand displacement activity of the reverse transcriptase encoded by the R2 retrotransposon.

R2 elements are non-long terminal repeat (non-LTR) retrotransposons with a single open reading-frame encoding reverse transcriptase, DNA endonuclease and nucleic acid-binding domains. The elements are specialized for insertion into the 28 S rRNA genes of many animal phyla. The R2-encoded activities initiate retrotransposition by sequence-specific cleavage of the 28 S gene target site and the utilization of the released DNA 3' end to prime reverse transcription (target primed reverse transcription). The activity of the R2 polymerase on RNA templates has been shown to differ from retroviral reverse transcriptases (RTs) in a number of properties. We demonstrate that the R2-RT is capable of efficiently utilizing single-stranded DNA (ssDNA) as a template. The processivity of the enzyme on ssDNA templates is higher than its processivity on RNA templates. This finding suggests that R2-RT is also capable of synthesizing the second DNA strand during retrotransposition. However, R2-RT lacks the RNAse H activity that is typically used by retroviral and LTR-retrotransposon RTs to remove the RNA strand before the first DNA strand is used as template. Remarkably, R2-RT can displace RNA strands that are annealed to ssDNA templates with essentially no loss of processivity. Such strand displacement activity is highly unusual for a DNA polymerase. Thus the single R2 protein contains all the activities needed to make a double-stranded DNA product from an RNA transcript. Finally, during these studies we found an unexpected property of the highly sequence-specific R2 endonuclease domain. The endonuclease can non-specifically cleave ssDNA at a junction with double-stranded DNA. This activity suggests that second-strand cleavage of the target site may not be sequence specific, but rather is specified by a single-stranded region generated when the first DNA strand is used to prime reverse transcription.

Nucleotide excision repair and template-independent addition by HIV-1 reverse transcriptase in the presence of nucleocapsid protein.

During HIV replication, reverse transcriptase (RT), assisted by the nucleocapsid protein (NC), converts the genomic RNA into proviral DNA. This process appears to be the major source of genetic variability, as RT can misincorporate nucleotides during minus and plus strand DNA synthesis. To investigate nucleotide addition or substitution by RT, we set up in vitro models containing HIV-1 RNA, cDNA, NC, and various RTs. We used the wild type RT and azidothymidine- and didanosine-resistant RTs, because they represent the major forms of resistant RTs selected in patients undergoing therapies. Results show that all RTs can add nucleotides in a non-template fashion at the cDNA 3'-end, a reaction stimulated by NC. Nucleotide substitutions were examined using in vitro systems where 3'-mutated cDNAs were extended by RT on an HIV-1 RNA template. With NC, RT extension of the mutated cDNAs was efficient, and surprisingly, mutations were frequently corrected. These results suggest for the first time that RT has excision-repair activity that is triggered by NC. Chaperoning of RT by NC might be explained by the fact that NC stabilizes an RT-DNA binary complex. In conclusion, RT-NC interactions appear to play critical roles in HIV-1 variability.

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.

Interaction of the Ty3 reverse transcriptase thumb subdomain with template-primer.

Amino acid sequence alignment was used to identify the putative thumb subdomain of reverse transcriptase (RT) from the Saccharomyces cerevisiae long terminal repeat-containing retrotransposon Ty3. The counterpart to helix alphaH of human immunodeficiency virus type 1 (HIV-1) RT, which mediates important interactions with a duplex nucleic acid approximately 3-6 bp behind the DNA polymerase catalytic center, was identified between amino acids 290 and 298 of the Ty3 enzyme. The consequences of substituting Ty3 RT Gln290, Phe292, Gly294, Asn297, and Tyr298 (the counterparts of HIV-1 RT Gln258, Leu260, Gly262, Asn265, and Trp266, respectively) for both DNA polymerase and RNase H activities were examined. DNA-dependent DNA synthesis was evaluated on unmodified substrates and on duplexes containing targeted insertion of locked nucleic acid analogs and abasic lesions in either the template or primer. Based on this combined strategy, our data suggest an interaction of Ty3 RT Tyr298 with primer nucleotide -3, Gly294 with primer nucleotide -4, and Asn297 with template nucleotide -6. Substitution of Ala for Gln290 was well tolerated, despite the high degree of conservation at this position. Mutations in the thumb subdomain of Ty3 also affected RNase H activity, suggesting a closer spatial relationship between its N- and C-terminal catalytic centers compared with HIV-1 RT.

Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase.

Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151-->Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151-->Asn and Asp213-->Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition.

Secondary structure models of the 3' untranslated regions of diverse R2 RNAs.

The RNA structure of the 3' untranslated region (UTR) of the R2 retrotransposable element is recognized by the R2-encoded reverse transcriptase in a reaction called target primed reverse transcription (TPRT). To provide insight into structure-function relationships important for TPRT, we have created alignments that reveal the secondary structure for 22 Drosophila and five silkmoth 3' UTR R2 sequences. In addition, free energy minimization has been used to predict the secondary structure for the 3' UTR R2 RNA of Forficula auricularia. The predicted structures for Bombyx mori and F. auricularia are consistent with chemical modification data obtained with beta-ethoxy-alpha-ketobutyraldehyde (kethoxal), dimethyl sulfate, and 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate. The structures appear to have common helices that are likely important for function.

End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon.

The reverse transcriptase encoded by the non-long terminal repeat retrotransposon R2 has been shown to be able to jump from the 5'-end of one RNA template (the donor) to the 3'-end of a second RNA template (the acceptor) in the absence of preexisting sequence identity between the two templates. These jumps between RNA templates have similarity to the end-to-end template jumps described for the RNA-directed RNA polymerases encoded by certain RNA viruses. Here we describe for the first time the mechanism by which such end-to-end template jumps can occur. Most template jumps by the R2 reverse transcriptase are brought about by the enzyme's ability to add nontemplated (overhanging) nucleotides to the cDNA when it reaches the end of the donor RNA. The enzyme then anneals these overhanging nucleotides to sequences at the 3'-end of the acceptor RNA. The annealing is most efficient if it involves the terminal nucleotide(s) of the acceptor RNA but can occur to sites at least 5 nucleotides from the 3'-end. These end-to-end jumps are similar to steps proposed to be part of the integration reaction of non-long terminal repeat retrotransposons and can explain chimeric integration products derived from multiple RNA templates.

High processivity of the reverse transcriptase from a non-long terminal repeat retrotransposon.

R2 is a retrotransposable element that specifically inserts into the 28 S rRNA genes of arthropods. The element encodes a single protein with endonuclease activity that cleaves the 28 S gene target site and reverse transcriptase (RT) activity that uses the cleaved DNA to prime reverse transcription. Here we compare various properties of the R2 RT activity with those of the well characterized retroviral RT, avian myeloblastosis virus (AMV). In processivity assays using heterogeneous RNA templates, R2 RT can synthesize cDNA over twice the length of that synthesized by AMV RT and can synthesize cDNA over 4 times longer than AMV RT in assays with poly(rA) templates. The higher processivity of R2 RT compared with retroviral RTs is a result of the slower rate of dissociation of the enzyme from RNA templates. The elongation rates of the two enzymes are similar. Finally, a highly distinct property of the R2 RT, compared with retroviral enzymes, is its ability to displace RNA strands annealed to RNA templates during cDNA synthesis. We suggest that both the higher processivity and displacement properties of R2 RT compared with retroviral RT result from the greater affinity of the R2 protein for the RNA template upstream of its active site.

The reverse transcriptase of the R2 non-LTR retrotransposon: continuous synthesis of cDNA on non-continuous RNA templates.

R2 is a non-long terminal repeat (non-LTR) retrotransposon that inserts into the 28 S rRNA genes of arthropods. The element encodes two enzymatic activities: an endonuclease that specifically cleaves the 28 S gene target site, and a reverse transcriptase (RT) that can use the 3' end of the cleaved DNA to prime reverse transcription. R2 RT only utilizes RNA templates that contain the 3' untranslated region of the R2 element as templates in this target primed reverse transcription (TPRT) reaction. Here, detailed biochemical characterization of the R2 RT indicates that the enzyme is capable of making multiple, consecutive jumps between RNA templates. The terminal 3' nucleotide of the "acceptor" RNA and the 5' nucleotide of the "donor" RNA are frequently reverse transcribed in these jumps, indicating that the acceptor RNA does not anneal to the cDNA derived from the donor RNA template. These template jumps occur during TPRT as well as in non-specific extension reactions in which reverse transcription is primed by an oligonucleotide annealed to the RNA template. Analysis of these RT assays done in the absence of the target DNA also revealed that the R2 RT can initiate reverse transcription near the 3' end of any RNA molecule using the 3' end of a second RNA molecule as primer. Again there is no requirement for sequence complementarity between the RNA used as template and the RNA used as primer. These properties of the R2 RT differ substantially from those of retroviral RTs but have similarities to the RT of the Mauriceville retroplasmid of Neurospora crassa. We present a model which relates these unusual properties of the R2 RT to structural differences from retroviral RTs as well as correlates these properties to the likely retrotransposition mechanism of R2 and other non-LTR retrotransposons.