RNA-DNA Duplexes Research Articles

Topoisomerase Action on Short DNA Duplexes Reveals Requirements for Gate and Transfer DNA Segments

Type II topoisomerases change DNA topology by passage of one DNA duplex (the transfer, T-segment) through a transient double-stranded break in another (the gate, G-segment). Here we monitor the passage between short double-stranded DNA segments within long single-stranded DNA circles that leads to catenation of the circles. To facilitate catenation, the circles were brought into close proximity using a tethering oligonucleotide, which was removed after the reaction was complete. We varied the length and the composition of the reacting DNA segments. The minimal DNA duplex length at which we detected catenation was 50-60 bp for DNA gyrase and 40 bp for topoisomerase IV (Topo IV). For Topo IV, catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex. Topo IV cleaved the DNA-DNA duplex, but not the DNA-RNA duplex implying that the DNA-RNA duplex can be a T-segment but not a G-segment.

Feline Lentivirus Evolution in Cross-Species Infection Reveals Extensive G-to-A Mutation and Selection on Key Residues in the Viral Polymerase

Factors that restrict a virus from establishing productive infection in a new host species are important to understand because cross-species transmission events are often associated with emergent viral diseases. To determine the evolutionary pressures on viruses in new host species, we evaluated the molecular evolution of a feline immunodeficiency virus derived from a wild cougar, Puma concolor, during infection of domestic cats. Analyses were based on the coding portion of genome sequences recovered at intervals over 37 weeks of infection of six cats inoculated by either intravenous or oral-nasal routes. All cats inoculated intravenously, but only one inoculated orally-nasally, became persistently viremic. There were notable accumulations of lethal errors and predominance of G-to-A alterations throughout the genome, which were marked in the viral polymerase gene, pol. Viral structural (env and gag) and accessory (vif and orfA) genes evolved neutrally or were under purifying selection. However, sites under positive selection were identified in reverse transcriptase that involved residues in the nucleotide binding pocket or those contacting the RNA-DNA duplex. The findings of extensive G-to-A alterations in this cross-species infection are consistent with the recently described editing of host cytidine deaminase on lentivirus genomes. Additionally, we demonstrate that the primary site of hypermutation is the viral pol gene and the dominant selective force acting on this feline immunodeficiency virus as it replicates in a new host species is on key residues of the virus polymerase.

Purification and characterization of a novel mammalian endoribonuclease

Endonuclease-mediated mRNA decay appears to be a common mode of mRNA degradation in mammalian cells, but yet only a few mRNA endonucleases have been described. Here, we report the existence of a second mammalian endonuclease that is capable of cleaving c-myc mRNA within the coding region in vitro. This study describes the partial purification and biochemical characterization of this enzyme. Five major proteins of approximately 10-35 kDa size co-purified with the endonuclease activity, a finding supported by gel filtration and glycerol gradient centrifugation analysis. The enzyme is an RNA-specific endonuclease that degrades single-stranded RNA, but not double-stranded RNA, DNA or DNA-RNA duplexes. It preferentially cleaves RNA in between the pyrimidine and purine dinucleotides UA, UG, and CA, at the coding region determinant (CRD) of c-myc RNA. The enzyme generates products with a 3'hydroxyl group, and it appears to be a protein-only endonuclease. It does not possess RNase A-like activity. The enzyme is capable of cleaving RNAs other than c-myc CRD RNA in vitro. It is Mg(2+)-independent and is resistant to EDTA. The endonuclease is inactivated at and above 70 degrees C. These properties distinguished the enzyme from other previously described vertebrate endonucleases.

Induction of the Human Oxidized Base-specific DNA Glycosylase NEIL1 by Reactive Oxygen Species

NEIL1, a mammalian DNA glycosylase and ortholog of Escherichia coli Nei/Fpg, is involved in the repair of oxidatively damaged bases in mammalian cells. Exposure of HCT116 human colon carcinoma cells to reactive oxygen species, generated by glucose oxidase (GO), enhanced the levels of NEIL1 mRNA and polypeptide by 2-4-fold by 6 h after GO treatment. A similar oxidative stress-induced increase in human NEIL1 (hNEIL1) promoter-dependent luciferase expression in HCT116 cells indicates that reactive oxygen species activates NEIL1 transcription. The transcriptional start site of hNEIL1 was mapped, and the upstream promoter sequence was characterized via luciferase reporter assay. Two identical CRE/AP-1-binding sites were identified in the promoter that binds transcription factors c-Jun and CREB/ATF2. This binding was significantly enhanced in extracts of cells treated with GO. Furthermore, a simultaneous increase in the level of phosphorylated c-Jun suggests its involvement in up-regulating the NEIL1 promoter. Oxidative stress-induced activation of NEIL1 appears to be involved in the feedback regulation of cellular repair activity needed to handle an increase in the level of oxidative base damage.

Application of RNA–DNA duplex base triplets to antisense drugs

The 64 RNA–DNA duplex base triplets should be helpful for the study of RNA–DNA annealing, the basis of nucleic acids interactions, and some peculiar motifs for design antisense oligonucleotides. Our research result reveal that the energies (such as E, E c, E b, E t, and E nr) of DNA/asRNA hybrids are lower than those of RNA/asDNA hybrids while the energies (such as E n and E nd) of DNA/asRNA hybrids are higher than those of RNA/asDNA hybrids for most binary complex and ternary complex, especially E, E c, E b, and E n in evidence. And the total energy of GGG/CCC hybrid is the lowest of all the hybrids, the more of number of G/C base pair, the lower of energy of the triplet hybrid, and (G/C) 3<U(G/C) 2<A(G/C) 2<U 2(G/C)<AU(G/C)<A 2(G/C) in turn; the U-including hybrid system stability, the more of number of uracil (U), the lower of energy of the triplet hybrid; the energy of GU-including hybrid is lower than that of CU-including hybrid no matter for binary complex or ternary complex. G/A/U bases often deviate from base pair planes, which can form hydrogen bonds with neighboring base pairs and affects the stabilities of triplets. Moreover, some peculiar sequence motifs that could be derived from useful triplet hybrids above, namely CCAC, TCCC, ACTC, ATCC, GCCA, CTCT, CACC, CCCA, TGCT, TGCC, TCTT, and TCCTT, are positively correlated with antisense activities. The former six motifs are the same of Gesteland's results, and the later six motifs are new discovered motifs by us, which proved by Arnedo's, Zewert's, and Zhang's results, which play an important role in designing antisense drugs.

Energy and structural analysis of double nucleic acid triplets

In order to investigate the energy and structural character of RNA–RNA triplets and RNA–DNA duplex base triplets, 64 sets of three-dimensional models of RNA–DNA duplex base triplets and mRNA–tRNA triplex base triplets were constructed and optimized by homologous modeling method using the software InsightII. The comparative statistical method and cluster analysis were adopted to study these features. The result showed: (i) all energy parameters of monomer RNA–DNA hybrid triplets and ternary complexes appeared significantly different; and some parameters related with overall molecules such as overall energy, bond energy and coulomb energy have statistically significant correlations between the structures in vacuum and aquatic solutions while other parameters, including theta energy, phi energy, hydrogen bond energy and non-bond energy, changed significantly, but not continuously. (ii) However, the case of mRNA–tRNA triplets was much more complicated in that only the bond energy's correlation coefficient is −0.8. Typically, the main contribution of GC pairs and G/A/U bases were interesting. The models of RNA–DNA hybrid triplets and mRNA–tRNA triplet should be helpful for the study of base pairing in codons and the biological effectiveness of antisense nucleic acids.

Fine-Structure Analysis of Activation-Induced Deaminase Accessibility to Class Switch Region R-Loops

Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.

Application of RNA-DNA Duplex Base Triplets to Antisense Drugs

Sixty-four sets of three-dimensional models of RNA-DNA duplex base triplets were constructed based on codons by homology modeling method using software InsightII on Indigo workstation, which should be helpful for the study of RNA-DNA annealing, the basis of nucleic acids interactions and some peculiar motifs for design antisense oligonucleotides. Our research result reveal that the energies (such as E, Ec, Eb, Et and Enr) of DNA/asRNA hybrids are lower than those of RNA/asDNA hybrids while the energies (such as En and End) of DNA/asRNA hybrids are higher than those of RNA/asDNA hybrids for most binary complex and ternary complex, especially E, Ec, Eb and En in evidence. And the total energy of GGG/CCC hybrid is the lowest of all the hybrids, the more G/C base pairs, the lower of energy of the triplet hybrid and (G/C)3<U(G/C)2<A(G/C)2 <U2(G/C)<AU(G/C)<A2(G/C) in turn; the U-including hybrid system stability, the more of number of uracil (U), the lower of energy of the triplet hybrid; the energy of GU-including hybrid is lower than that of CU-including hybrid no matter for binary complex or ternary complex. G/A/U bases often deviate from base pair planes, which can form hydrogen bonds with neighboring base pairs and affects the stabilities of triplets. Moreover, some peculiar oligodeoxynucleotide sequence motifs that could be derived from corresponding triplet hybrids are divided into two groups: four-member motif and five - member motif, where the former comprises eight motifs, namely 5’-TCTT-3’,5’-TGCT-3’,5’-CCTT-3’,5’-CCAT-3’, 5’-CATC-3’, 5’-ATCT-3’, 5’-GCTG-3’ and 5’-GTCT-3’; and the later consists of four motifs, viz 5’-TGCTG-3’, 5’-GTCTT-3’, 5’-CCATC-3’ and 5’-CATCT-3’, which are positively correlated with antisense activities and play an important role in designing antisense drugs.

Stabilisation of nucleic acid secondary structures by oligonucleotides with an additional nucleobase; synthesis and incorporation of 2′-deoxy-2′-C-(2-(thymine-1-yl)ethyl)uridine

A nucleoside with two nucleobases is incorporated into oligonucleotides. The synthetic building block, 2'-deoxy-2'-C-(2-(thymine-1-yl)ethyl)uridine, 2, is prepared from uridine via 5',3'-TIPDS-protected 2'-deoxy-2'-C-allyluridine by an oxidative cleavage of the allyl group, a Mitsunobu reaction for the introduction of thymine and appropriate deprotection reactions. This compound is converted into a DMT-protected phosphoramidite and incorporated once into a 13-mer oligodeoxynucleotide sequence, once in an isosequential LNA-modified oligodeoxynucleotide and four times in the middle of a 12-mer oligodeoxynucleotide. These sequences are mixed with different complementary DNA and RNA sequences in order to study the effect of the additional nucleobase in duplexes, in bulged duplexes and in three-way junctions. The first additional thymine is found to be well-accommodated in a DNA-RNA duplex, whereas a DNA-DNA duplex was slightly destabilised. A three-way junction with the additional thymine in the branching point is found to be stabilised in both a DNA-DNA and a DNA-RNA context but destabilised where the modified LNA-sequence is used. In a Mg2+-containing buffer, however, the relative stability of the three-way junctions is found to be opposite with especially the LNA-modified DNA-DNA complex being significantly stabilised by the additional nucleobase.

Competition between Polysaccharide/Polynucleotide Complexation vs. Polynucleotide Hybridization; Salt Concentration Dependence of the Reaction Direction

Schizophyllan is a natural β-(1→3)-D-glucan existing as a triple helix in water and as a single chain in dimethylsulfoxide (DMSO), respectively. As we already reported, when a homo-polynucleotide [ex. poly(A) or poly(dA)] was added to the schizophyllan/DMSO solution and subsequently DMSO was exchanged for water, the single chain of schizophyllan formed a complex with the polynucleotide. In this paper, using an adenine-thymine (or uracil) double strand as a model system, we explored whether s-SPG can unzip the polynucleotide duplexes, examining the competition between the schizophyllan/polynucleotide complexation and the polynucleotide hybridization with circular dichroism and fluorescence spectroscopy at various NaCl concentrations. The unzipping reaction happened for the hetero-duplexes (i.e., RNA-DNA duplexes) at low salt concentrations, on the other hand, it was not observed for the homo-duplexes at all the salt concentrations. When we compared dissociation temperatures, the unzipping reaction of the homo-duplexes was expected at low salt concentrations. This discrepancy between the experimental results and the expectation, as well as the different behavior between the hetero- and homo- duplexes, can be ascribed to the difference in the activation energy of intermediate states. [DOI: 10.1380/ejssnt.2005.38]

The 5-Me of thyminyl (T) interaction with the neighboring nucleobases dictate the relative stability of isosequential DNA–RNA hybrid duplexes

Relative energetic contributions from the base-pairing [DeltaG(degrees)(bp)] vis-a-vis stacking [DeltaG(degrees)(stacking)] to the total free-energy of stabilization [DeltaG(degrees)(37)] for 14 pairs of isosequential hybrid DNA-RNA duplexes (taken from E. A. Lesnik and S. M. Freier, Biochemistry ,1995, 34, 10807) have been dissected in order to understand the differences in the intrinsic nature of the electrostatic forces that are responsible for the self-assembly of the heteroduplexes compared to homoduplexes. The pKa differences between the monomeric nucleotide 3'-ethylphosphates [(d/rN)pEt] as well as nucleotide 3',5'-bis-ethylphosphates [Etp(d/rN)pEt] in both 2'-deoxy (dN) and ribo (rN) series (N = A/G/C/T/U), as the model donor and acceptor (in which stacking is completely eliminated) mimicking those of the internucleotide monomer building blocks of a duplex, can be qualitatively used (P. Acharya, P. Cheruku, S. Chatterjee, S. Acharya and J. Chattopadhyaya, J. Am. Chem. Soc., 2004, 126, 2862) to understand the strength of base-pairing energies in different DNA-RNA (DR), RNA-DNA (RD), DNA-DNA (DD), and RNA-RNA (RR) duplexes. The study has led us to show the following. (1) As the number of excess %T in DR duplexes compared to the isosequential RD duplexes increase the differences in their thermal stabilization [DeltaT(m)](DR-RD) increase and vice-versa(2) The total relative stabilizations, [DeltaDeltaG(degrees)(37)](DR-RD) among the 14 pairs of isosequential DR and RD duplexes (E. A. Lesnik and S. M. Freier, Biochemistry ,1995, 34, 10807) are wholly dependent on the differences in the number of 5-Me(T) stacking interactions with the nearest-neighbors in the D strands of DR duplexes compared to that of the RD duplexes (3) In the relative stabilization of the DR or RD duplexes differences in the free-energy of stackings [DeltaDeltaG(degrees)(stacking)](DR-RD) play a more significant role than the differences in the free-energy of base-pairing, [DeltaDeltaG(degrees)(bp)](DR-RD). In contradistinction, our experimental data shows that RNA-RNA duplexes are more stable than DNA-DNA duplexes because of larger energy gain from the base-pairing in the former compared to the latter (P. Acharya, P. Cheruku, S. Chatterjee, S. Acharya and J. Chattopadhyaya, J. Am. Chem. Soc., 2004, 126, 2862).

Specific recognition of napthyridine-based ligands toward guanine-containing bulges in RNA duplexes and RNA–DNA heteroduplexes

Mismatched bulges in nucleic acid constructs are important in the recognition event between biological molecules. Herein, it is observed that napthyridine dimer 2 is able to specifically bind G–G mismatches in all nucleic acid constructs comprising of RNA–RNA, RNA–DNA and DNA–DNA duplexes. However, the binding affinity of 2 is strongest toward DNA duplex, followed by RNA–DNA heteroduplex and RNA duplex being the weakest binding partner. Nonetheless, this binding behavior suggests that the binding process primarily occurs between the guanine base pairs and the napthyridine moiety, and is independent of the tertiary structure of the nucleic acid duplexes.

Antisense-Induced Fas mRNA Degradation Produces Site-Specific Stable 3'-mRNA Fragment by Exonuclease Cleavage at the Complementary Sequence

Antisense-mediated degradation of target mRNA is achieved by the enzymatic action of nuclease RNase H. The enzyme recognizes hybrid RNA-DNA duplexes and hydrolyzes the RNA strand. Here, we compared six different phosphorothioate oligonucleotides for their ability to induce target-specific mRNA degradation in cultured mouse AML12 cells. We targeted transcripts of the cell surface receptor Fas and analyzed the levels of mRNA by Northern blotting and ribonuclease protection assay (RPA). Four of the tested antisense oligonucleotides reduced the mRNA levels significantly. Cultures treated with one of the antisense molecules resulted in a shifted band on Northern blots. This band of lower molecular weight was not detected after 6 hours of transfection but appeared at 24 hours. By RPA, the product was shown to be a 3'-cleavage fragment of the full-length Fas mRNA. The RPA also mapped the stable fragment to start within the antisense complementary sequence.

Effect of LNA Modifications on Small Molecule Binding to Nucleic Acids

Locked nucleic acid (LNA) is a conformationally constrained DNA analogue that exhibits exceptionally high affinity for complementary DNA and RNA strands. The deoxyribose sugar is modified by a 2′-O, 4′-C oxymethylene bridge, which projects into the minor groove. In addition to changing the distribution of functional groups in the groove and the overall helical geometry relative to unmodified DNA, the bridge likely alters the hydration of the groove. Each of these factors will impact the ability of small molecules, proteins and other nucleic acids to recognize LNA-containing hybrids. This report describes the ability of several DNA-intercalating ligands and one minor groove binder to recognize LNA-DNA and LNA-RNA hybrid duplexes. Using UV-vis, fluorescence and circular dichroism spectroscopies, we find that the minor groove binder as well as the intercalators exhibit significantly lower affinity for LNA-containing duplexes. The lone exception is the alkaloid ellipticine, which intercalates into LNA-DNA and LNA-RNA duplexes with affinities comparable to unmodified DNA-DNA and RNA-DNA duplexes.

Simple cDNA normalization using kamchatka crab duplex-specific nuclease.

We developed a novel simple cDNA normalization method [termed duplex-specific nuclease (DSN) normalization] that may be effectively used for samples enriched with full-length cDNA sequences. DSN normalization involves the denaturation-reassociation of cDNA, degradation of the double-stranded (ds) fraction formed by abundant transcripts and PCR amplification of the equalized single-stranded (ss) DNA fraction. The key element of this method is the degradation of the ds fraction formed during reassociation of cDNA using the kamchatka crab DSN, as described recently. This thermostable enzyme displays a strong preference for cleaving ds DNA and DNA in DNA-RNA hybrid duplexes compared with ss DNA and RNA, irrespective of sequence length. We developed normalization protocols for both first-strand cDNA [when poly(A)+ RNA is available] and amplified cDNA (when only total RNA can be obtained). Both protocols were evaluated in model experiments using human skeletal muscle cDNA. We also employed DSN normalization to normalize cDNA from nervous tissues of the marine mollusc Aplysia californica (a popular model organism in neuroscience) to illustrate further the efficiency of the normalization technique.

Yeast telomerase is capable of limited repeat addition processivity.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Telomerase-mediated sequence addition is dictated by a short 'template' region of the RNA component. Despite the short template segment, telomerases from many organisms have been shown to mediate the synthesis of long extension products. This synthesis presumably depends on two types of translocation events: simultaneous translocation of the RNA-DNA duplex relative to the active site after each nucleotide incorporation (type I or nucleotide addition processivity), and translocation of the RNA relative to the DNA product after each round of repeat synthesis (type II or repeat addition processivity). In contrast, telomerases from yeasts have been shown to synthesize mostly short products, implying a defect in one or both types of translocation. In this report, we analyzed the processivity of yeast telomerase in vitro, and identified two position-specific elongation barriers within the 5' region of the RNA template that can account for the synthesis of incomplete first round products. These barriers respond differently to variations in nucleotide concentration, primer sequence and mutations in the catalytic protein subunit, consistent with their having distinct mechanistic bases. In addition, by using optimal primers and high concentrations of dGTP, we were able to detect significant type II translocation by the yeast enzyme. Thus, the difference between the elongation property of yeast and other telomerases appears to be quantitative rather than qualitative. Our results suggest that yeast may be a useful system for investigating the physiologic significance of repeat addition processivity.

Uridine 2′‐Carbamates: Facile Tools for Oligonucleotide 2′‐Functionalization

A facile method for preparation of uridine 2'-carbamate derivatives based on reaction of 3',5'-disilyl-protected uridine with 1,1'-carbonyldiimidazole followed by treatment with an aliphatic amine is presented. A phosphoramidite monomer suitable for automated oligonucleotide synthesis is obtained in a few steps. The compounds are useful for the introduction of various labels and modifications into an oligonucleotide chain. Although 2'-carbamate modification is somewhat destabilizing for DNA-DNA and DNA-RNA duplexes, it is suitable for the direction of ligands into the minor groove of duplexes or at non-base-paired sites (e.g., loops and bulges) of oligonucleotides. Pyrene-modified oligonucleotide 2'-carbamates show a considerable increase in fluorescence intensity upon hybridization to a complementary RNA (but not DNA).

An error-prone family Y DNA polymerase (DinB homolog from Sulfolobus solfataricus) uses a 'steric gate' residue for discrimination against ribonucleotides.

DNA polymerases of the A and B families, and reverse transcriptases, share a common mechanism for preventing incorporation of ribonucleotides: a highly conserved active site residue obstructing the position that would be occupied by a 2' hydroxyl group on the incoming nucleotide. In the family Y (lesion bypass) polymerases, the enzyme active site is more open, with fewer contacts to the DNA and nucleotide substrates. Nevertheless, ribonucleotide discrimination by the DinB homolog (Dbh) DNA polymerase of Sulfolobus solfataricus is as stringent as in other polymerases. A highly conserved aromatic residue (Phe12 in Dbh) occupies a position analogous to the residues responsible for excluding ribonucleotides in other DNA polymerases. The F12A mutant of Dbh incorporates ribonucleoside triphosphates almost as efficiently as deoxyribonucleoside triphosphates, and, unlike analogous mutants in other polymerase families, shows no barrier to adding multiple ribonucleotides, suggesting that Dbh can readily accommodate a DNA-RNA duplex product. Like other members of the DinB group of bypass polymerases, Dbh makes single-base deletion errors at high frequency in particular sequence contexts. When making a deletion error, ribonucleotide discrimination by wild-type and F12A Dbh is the same as in normal DNA synthesis, indicating that the geometry of nucleotide binding is similar in both circumstances.

Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides.

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected. The modified triplexes are stable at pH 7. Their thermal stability depends on the number of C(G-C) triplets and, for G-rich RNA sequences, it is comparable with the stability of native DNA-RNA duplexes. The CD spectra of triplexes formed by OXNs with purine RNA targets are similar to spectra of A-type helices. A pyrimidine OXN having a clamp structure efficiently inhibits reverse transcription of murine pim-1 mRNA in vitro mediated by the Mo-MuLV reverse transcriptase.

Incorporation of Uracil into Minus Strand DNA Affects the Specificity of Plus Strand Synthesis Initiation during Lentiviral Reverse Transcription

Many retroviruses either encode dUTP pyrophosphatase (dUTPase) or package host-derived uracil DNA glycosylase as a means to limit the accumulation of uracil in DNA strands, suggesting that uracil is detrimental to one or more steps in the viral life cycle. In the present study, the effects of DNA uracilation on (-) strand DNA synthesis, RNase H activity, and (+) strand DNA synthesis were investigated in a cell-free system. This system uses the activities of purified human immunodeficiency virus type 1 (HIV-1) reverse transcriptase to convert single-stranded RNA to double-stranded DNA in a single reaction mixture. Substitution of dUTP for dTTP had no effect on (-) strand synthesis but significantly decreased yields of (+) strand DNA. Mapping of nascent (+) strand 5' ends revealed that this was due to decreased initiation from polypurine tracts with a concomitant increase in initiation at non-polypurine tract sites. Aberrant initiation correlated with a change in RNase H cleavage specificity when assayed on preformed RNA-DNA duplexes containing uracilated DNA, suggesting that appropriate "selection" of the (+) strand primer is affected. Collectively, these data suggest that accumulation of uracil in retroviral DNA may disrupt the viral life cycle by altering the specificity of (+) strand DNA synthesis initiation during reverse transcription.