The proliferative response of peripheral blood mononuclear cells (PBMC) to a crude extract from Cryptosporidium parvum (CCE) was studied in persons who acquired cryptosporidiosis in the same outbreak (15 immunocompetent subjects with prior cryptosporidiosis and 22 human immunodeficiency virus [HIV]-positive persons with various levels of immunosuppression and active cryptosporidiosis) and in individual patients (8 HIV-positive patients with active cryptosporidiosis and 15 HIV-positive persons without history of cryptosporidiosis). PBMC from HIV-positive persons showed less proliferation to CCE and mitogens than did PBMC from immunocompetent subjects with prior cryptosporidiosis, independent of CD4 cell count. In immunocompetent subjects, cytokine gene expression was consistent with cytokine production, whereas in HIV-positive subjects it was not. The production of interferon-gamma in CCE-stimulated PBMC from both immunocompetent and HIV-positive subjects with cryptosporidiosis and the lack of interferon-gamma in CCE-stimulated PBMC from HIV-positive subjects without cryptosporidiosis indicate that C. parvum mainly induces a Th1 response.
In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using anin vitro system that closely mimics the initiation of (+)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3′- and 5′-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5′ direction of the RNA strand. DNA synthesis resumes again in the 3′ direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position +12, by treating these with Fe2+ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (+)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex. In this study, we have analyzed the interdependence between the polymerase and RNase H active sites of human immunodeficiency virus-1 reverse transcriptase (RT) using anin vitro system that closely mimics the initiation of (+)-strand DNA synthesis. Time course experiments show that RT pauses after addition of the 12th DNA residue, and at this stage the RNase H activity starts to cleave the RNA primer from newly synthesized DNA. Comparison of cleavage profiles obtained with 3′- and 5′-end-labeled primer strands indicates that RT now translocates in the opposite direction, i.e. in the 5′ direction of the RNA strand. DNA synthesis resumes again in the 3′ direction, after the RNA-DNA junction was efficiently cleaved. Moreover, we further characterized complexes generated before, during, and after position +12, by treating these with Fe2+ to localize the RNase H active site on the DNA template. Initially, when RT binds the RNA/DNA substrate, oxidative strand breaks were seen at a distance of 18 base pairs upstream from the primer terminus, whereas 17 base pairs were observed at later stages when the enzyme binds more and more DNA/DNA. These data show that the initiation of (+)-strand synthesis is accompanied by a conformational change of the polymerase-competent complex. Retroviral RTs 1The abbreviations used are: RT, reverse transcriptase; HIV-1, human immunodeficiency virus type 1; PBS, primer-binding site; PPT, polypurine tract; nt, nucleotides; bp, base pairs; dNTP, 3′-deoxynucleosidetriphosphate; ddNTP, 2′,3′-dideoxynucleosidetriphosphate; ds, double-stranded. are multifunctional enzymes possessing RNA- and DNA-dependent polymerase activities and a ribonuclease H (RNase H) activity that degrades the RNA strand of RNA/DNA hybrids (1Telesnitsky A. Goff S.P. Coffin J.M. Hughes S.H. Varmus H.E. Retroviruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 121-160Google Scholar, 2Gilboa E. Mitra S.W. Goff S.P. Baltimore D. Cell. 1979; 18: 93-100Abstract Full Text PDF PubMed Scopus (421) Google Scholar). Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) uses a cellular tRNA primer to initiate reverse transcription from a complementary primer-binding site (PBS) near the 5′-end of the viral RNA (3Marquet R. Isel C. Ehresmann C. Ehresmann B. Biochimie (Paris). 1995; 77: 113-124Crossref PubMed Scopus (200) Google Scholar, 4Mak J. Kleiman L. J. Virol. 1997; 71: 8087-8095Crossref PubMed Google Scholar, 5Isel C. Ehresmann C. Gérard Keith Ehresmann B. Marquet R. J. Mol. Biol. 1995; 247: 236-250Crossref PubMed Scopus (229) Google Scholar, 6Isel C. Lanchy J.M. Le Grice S.F.J. Ehresmann C. Ehresmann B. Marquet R. EMBO J. 1996; 15: 917-924Crossref PubMed Scopus (178) Google Scholar). Despite changes of binding and kinetic properties, observed concomitant with synthesis of the first DNA strand (7Lanchy J.-M. Ehresmann C. Le Grice S.F.J. Ehresmann B. Marquet R. EMBO J. 1996; 15: 7178-7187Crossref PubMed Scopus (109) Google Scholar), i.e.(−)-strand DNA, complexes with the initially bound RNA/RNA duplex and the newly synthesized DNA/RNA substrates share certain common features. RNase H cleavages on the RNA strand of DNA/RNA primer/template combinations occur at a constant distance of 18 bp upstream of the nascent primer terminus (8Wöhrl B.M. Moelling K. Biochemistry. 1990; 29: 10141-10147Crossref PubMed Scopus (112) Google Scholar, 9Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar). Analogously, RNase H-induced cleavages within the tRNA/RNA duplex, designated as RNase H* activity (10Hostomsky Z. Hughes S.H. Goff S.P. Le Grice S.F.J. J. Virol. 1994; 68: 1970-1971Crossref PubMed Google Scholar), were observed at the same distance from the 3′-end of the primer, although these cuts are restricted to stalled complexes (11Götte M. Fackler S. Hermann T. Perola E. Cellai L. Gross H.J. Le Grice S.F.J. Heumann H. EMBO J. 1995; 14: 833-841Crossref PubMed Scopus (80) Google Scholar). Together, these data provide strong evidence that RT binds to both RNA/RNA and DNA/RNA substrates with the same orientation, and the number of bp between the two active sites is 18 in each case. RT-DNA/DNA complexes, which are generated during (+)-strand synthesis, have been relatively well characterized (12Jacobo-Molina A. Ding J. Nanni R.G. Clark Jr., A.D. Lu X. Tantillo C. Williams R.L. Kamer G. Ferris A.L. Clark P. Hizi A. Hughes S.H. Arnold E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6320-6324Crossref PubMed Scopus (1122) Google Scholar, 13Metzger W. Hermann T. Schatz O. Le Grice S.F.J. Heumann H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5909-5913Crossref PubMed Scopus (59) Google Scholar, 14Wöhrl B.M. Tantillo C. Arnold E. Le Grice S.F.J. Biochemistry. 1995; 34: 5343-5350Crossref PubMed Scopus (80) Google Scholar, 15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The crystal structure of HIV-1 RT complexed to an 18-base primer/19-base template DNA homoduplex (21Schatz O. Mous J. Le Grice S.F.J. EMBO J. 1990; 9: 1171-1176Crossref PubMed Scopus (126) Google Scholar) suggests that the first 7 DNA/DNA base pairs near the polymerase active site adopt an A-type conformation, whereas the region further upstream is in the preferred B-conformation, both structurally distinct segments being separated by a kink. Little information is currently available regarding the interaction between RT and the RNA/DNA primer/template combination that is initially bound during (+)-strand synthesis. A short segment near the 3′-end of viral genomic RNA, termed the polypurine tract (PPT), is resistant to RNase H degradation and, unlike the rest of the genomic RNA, remains intact during synthesis of the (−)-strand DNA. The PPT fragment then serves as a primer for (+)-strand polymerization, whereas the (−)-strand DNA is used as a template to guide synthesis. Later after initiation, the RNA primer is removed by RNase H cuts at the DNA-RNA junction and adjacent positions (16Huber H.E. Richardson C.C. J. Biol. Chem. 1990; 265: 10565-10573Abstract Full Text PDF PubMed Google Scholar, 17Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 28169-28176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Here, we demonstrate that the RNA primer is cleaved precisely after the 12th DNA residue has been incorporated. This is an unexpected result, since the relative positions of the polymerase and RNase H active sites would not allow DNA synthesis and RNase H degradation to occur at the same time on the same strand. In contrast, the spatial relationship between both active sites facilitates temporally coordinated activities on opposite strands, in a distance of about 18 base pairs, when RT is complexed with DNA/RNA primer/templates (9Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar). We now show that RT pauses after addition of the 12th DNA residue during the initiation of (+)-strand synthesis. At this point, another RT molecule (not the one that accomplishes DNA synthesis) binds the substrate in an RNase H-competent binding mode to cleave the RNA primer. The directionality of the latter reaction, i.e. in 5′ direction with respect to the RNA strand, distinguishes this binding mode from the polymerase-competent complex. In the polymerase-competent mode, RT binds its RNA/DNA substrate in the same orientation as that described for DNA/DNA, RNA/RNA, and DNA/RNA primer/templates. This has been shown by treating stalled complexes with Fe2+, which allowed us to localize the RNase H active site on the DNA template (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Together, our data provide a detailed model for the early steps of (+)-strand DNA synthesis. Oligonucleotides used in this study were derived from the polypurine tract located near the 3′-end of the HIV-1 genome (HXB-2 isolate). DNA oligonucleotides as well as RNA and chimeric DNA-RNA primers were synthesized on an Applied Biosystems 392/8 synthesizer using the standard phosphoramidite method, followed by purification on 12% polyacrylamide, 7 m urea gels containing 50 mm Tris borate, pH 8.0, 1 mmEDTA. 5′-End labeling of oligonucleotides (DNA, RNA, or chimera) was conducted with [γ-32P]ATP and T4 polynucleotide kinase; and 3′-end labeling of the DNA template was performed with [α-32P]ddATP using terminal transferase (Boehringer Mannheim) according to the manufacturer's recommendation. The chimeric 3′-end-labeled primer (3D-17R) was generated by extending the pure RNA PPT-primer (17R) with HIV-1 RT in the presence of dATP, dCTP, and [α-32P]dTTP using reaction conditions as described below. All end-labeled nucleic acids were again electrophoretically purified to obtain homogeneous products. Heterodimeric HIV-1 RT (p66/p51) and the RNase H-deficient p66(E478Q)/p51 mutant enzyme were prepared and purified essentially as described (37Le Grice S.F.J. Grüninger-Leitch F. Eur. J. Biochem. 1990; 178: 307-314Crossref Scopus (300) Google Scholar). Primer/template sequences were prehybridized prior to incubation with RT. A mixture containing the template strand (120 nm) and 32P-labeled primer (100 nm) in a buffer containing 50 mm Tris-HCl, pH 7.8, 50 mm NaCl was heated to 95 °C for 2 min followed by incubation at 72 °C for 10 min and cooled for 20 min to room temperature. Complete hybridization was confirmed on native polyacrylamide gels. The pre-annealed primer/template substrate (100 nm) was incubated with HIV-1 RT (150 nm) in a buffer containing 50 mm Tris-HCl, pH 7.8, 50 mmNaCl. Appropriate dNTP/ddNTP combinations (100 and 200 μm, respectively), which permitted control of the extent of DNA synthesis, were included in the preincubation mixture to form ternary RT-primer/template-dNTP complexes. 20-μl reactions were initiated with MgCl2 at a final concentration of 6 mm and allowed to proceed for 15 min at 37 °C. Nucleic acids were subsequently precipitated with ethanol, and reaction products were finally analyzed on 15% polyacrylamide, 7 murea gels, followed by exposure overnight. A list of different primers and the template used to generate registers 3, 4, 6, 11, 12, 13, 14, 15, 16 and 17 are as follows: DNA template 57D, 5′-CGTTGGGAGTGAATTAGCCCTTCCAGTCCCCCCTTTTCTTTTAAAAAGTGGCTAAGA-3′; the chimeric primer 3D-17R and the homologous DNA primer 20D, 5′-UUAAAAGAAAAGGGGGGACT-3′ (RNA residues are written in italics) and 5′-TTAAAAGAAAAGGGGGGACT-3′. Both primers were employed to generate registers 3, 4, 6, 11, and 12 as shown in Fig. 1. The chimeric RNA-DNA oligonucleotides 12D-17R, 5′-UUAAAAGAAAAGGGGGGACTGGAAGGGCT-3′, 14D-17R, 5′-UUAAAAGAAAAGGGGGGACTGGAAGGGCTAA-3′, and 16D-17R, 5′-UUAAAAGAAAAGGGGGGACTGGAAGGGCTAATT-3′, were used to generate registers 12–16. Register 12 was obtained by annealing the 12D-17R primer to the DNA template and incubating the preformed substrate with RT and ddATP (500 μm) as described above. In the latter case, Mg2+ was omitted from the reaction mixture to prevent incorporation of the chain-terminating stop-nucleotide as well as RNase H degradation of the chimeric primer. Register 13 was then generated by addition of Mg2+ (6 mm) to allow incorporation of ddATP. The presence of Mg2+ also resulted in RNase H cleavages at the RNA-DNA junction. Registers 14 and 15 were generated with primer 14D-17R and ddTTP, and registers 16 and 17 were generated with primer 16D-17R and ddCTP using the same procedure. Time course experiments were similarly performed. In these experiments, we lowered the ratio of RT-primer/template to monitor appearance of all reaction products during a time course of 60 min. The pre-annealed primer/template substrate (100 nm) was preincubated for 5 min at 37 °C with HIV-RT (50 nm) in a buffer containing 50 mm Tris-HCl, pH 7.8, and 50 mm NaCl. Polymerization and RNase H degradation was then initiated simultaneously by addition of MgCl2 at a final concentration of 6 mm. Reactions were performed at 37 °C and stopped at different time points by adding 1-μl aliquots of the reaction mixture to 9 μl of 95% formamide containing 40 mm EDTA. Most of the time course experiments were performed with the DNA template 57D and the chimeric 3D-17R primer. We also used the pure RNA primer 17R, 5′-UUAAAAGAAAAGGGGGG-3′, the chimeric 12D-17R primer and its isolated DNA segment 12D, 5′-ACTGGAAGGGCT-3′, for comparative purpose (see Fig. 5). Additionally, to analyze whether the reaction profile depends on the template sequence, we employed another DNA template with a randomly chosen sequence upstream from the PPT binding site: 5′-CAGTGATCTCGAGCTACATGATCGTCACCCCCCTTTTCTTTTAAAAAGTGGCTAAGA-3′. Stalled RT-nucleic acid complexes that contained differentially extended primer strands were prepared as described above. RT was used in a final concentration of 150 nm, and the ratio of primer/template (radiolabeled at the 3′-end or 5′-end) was 120/100 nm. Preformed complexes in a volume of 16 μl were incubated with a mixture of 2 μl of Fe(NH4)2SO4·6H2O (400 μl) and 2 μl of dithiothreitol (50 mm). Reactions were allowed to proceed for 5 min at 37 °C and were stopped with 40 μl of a solution containing 0.1 m thiourea, 200 ng of tRNA, 10 mm EDTA, and 0.6 m sodium acetate. Samples were subsequently precipitated with ethanol and loaded on a 12% polyacrylamide-urea gel. The size of the Fe2+-dependent cleavage fragments were assigned by a T-ladder generated after modifying the DNA with OsO4/bipyridin followed by cleaving the sugar-phosphate backbone with piperidine. Reactions with ONOOK were performed as described previously (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Briefly, RT-primer/template complexes were prepared in a buffer containing 80 mm sodium cacodylate, pH 7, and 20 mm NaCl. Cleavage reactions were conducted by adding 1 μl of a stable alkaline ONOOK solution (pH 12, 90 mm) to the sample solution buffered at pH 7. Reaction products were analyzed as described above. In order to analyze the interplay between RT polymerase and RNase H active sites on a PPT-derived RNA/DNA primer/template substrate, we first generated a series of stalled complexes, termed registers, through use of various dNTP/ddNTP combinations. Instead of employing a pure RNA primer that, because of the sequence 5′-CAGT-3′ immediately flanking the primer, leads to termination of DNA synthesis at positions +1, +2, +3, and +4, we devised a chimeric DNA-RNA primer (Pr + PPT 3D-17R) that yields chain termination at positions +4, +6, +11, and +12 (Fig. 1). This approach allowed us to study early events of the initiation reaction when RT is complexed with its initially bound RNA/DNA substrate as well as with chimeric replication intermediates. We also devised a DNA primer (Pr + PPT 20D) to generate a homologous DNA/DNA substrate for comparative purposes. The polymerization and RNase H cleavage products of differentially arrested RT-nucleic acid complexes are shown in Fig.2. As expected, the 5′-end-labeled DNA primer was precisely elongated by 1, 3, 8, and 9 nt (left panel, lanes 4, 6, 11, and 12). The presence of all four dNTPs yielded a product of 44 nt in length (lane 27). The results obtained with the homologous RNA/DNA substrate are more complex (Fig.2, right panel), since the RT polymerase and RNase H activities both use the same strand as substrate. Consistent with previous reports (16Huber H.E. Richardson C.C. J. Biol. Chem. 1990; 265: 10565-10573Abstract Full Text PDF PubMed Google Scholar, 17Fuentes G.M. Rodrı́guez-Rodrı́guez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 28169-28176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 18Powell M.D. Levin J.G. J. Virol. 1996; 70: 5288-5296Crossref PubMed Google Scholar), RNase H cuts are seen at the DNA-RNA interface and further upstream, adjacent to the junction, at four consecutive positions, and between positions −7 and −8. Precise extension with reasonable yield was observed only in register 4 when a single nt was added to the chimeric primer (Fig. 2, right panel, lane 4). This product was termed 4D-17R, with respect to the number of DNA and RNA residues of the extended primer. The expected reaction product in register 6, i.e. 6D-17R, is visible, but another shorter product, termed 1D-17R (see below), is also seen just above the RNase H cuts. Similar observations were made in registers 11 and 12 (lanes 11 and 12). In either case, the expected reaction products were less pronounced than the shorter ones, i.e. 2D-17R and 3D-17R. To characterize further the origin of the shorter reaction products, we next followed their formation in time course experiments. These were performed with dNTP/ddNTP combinations to yield registers 6 and 11 (Fig.3 A). The 6D-17R product appeared within the first few minutes and increased only slightly with longer reaction times (Fig. 3 A, left panel). RNase H cuts at the junction (17R) and further upstream (16R and 15R) were seen clearly after 3 min, and the above-mentioned 1D-17R product, which migrated a little slower than the 17R cleavage band, first appeared between 12 and 20 min and further increased over longer incubation times. This result shows an order of product formation, i.e. first elongation to yield the 6D-17R product followed by RNase H cleavages and finally the formation of the 1D-17R product. RT thus initiates a second round of (+)-strand synthesis using the cleaved RNA fragment as a primer. We will now use the term "primary reaction" and "secondary reaction" to distinguish between these two types of initiation events (schematically illustrated in Fig. 3 C). During the primary reaction, incorporation of ddATP results in chain termination at position +6, whereas, during the secondary initiation reaction, ddATP is added at position +1 to yield a complex, termed register 1′. Equivalent patterns were seen in registers 11 and 12 (Fig. 3 A, right, and B, left); here, the stop-nucleotides (ddCTP and ddTTP) were added at positions +2 and +3 to yield the secondary 2D-17R and 3D-17R products. The presence of dGTP should, in principal, allow elongation of RNA fragments that are generated by RNase H cleavages at the DNA-RNA junction and adjacent positions. Thus, the shorter secondary products may represent a heterogeneous mixture of chimeric strands, each of which contains a different number of DNA residues at the 3′-end. It can hardly be deduced from the above experiment which of the RNA fragments is preferentially used during the secondary initiation reaction. However, the observation that the 3D-17R product co-migrates exactly with the unextended primer indicates that the 17R cleavage product is most efficiently used (Fig. 3 B, left panel). If shorter cleavage products would have been extended, the 3D-17R fragment would have migrated somewhat faster, due to the increased number of DNA residues in the chain-terminated product (see Fig. 2; the pure DNA oligonucleotide migrates faster than the chimeric DNA-RNA strand of the same length). The above results showed that the initiation of (+)-strand synthesis is a complex process that involves primary and secondary initiation reactions, superimposed on the primer removal reaction. In order to understand better the natural reaction pathways, we next analyzed the order of product formation in the presence of all four dNTPs. The time course (Fig. 3 B, right panel) shows that RT pauses at position +12, before DNA synthesis resumes to yield the run-off product (27D-17R). All reaction products, including the cleavage products (17R, 16R, and 15R), are already seen after the 1st min. The primer removal reaction appears to be equally efficient in the presence or absence of chain terminating nucleotides, as the enzyme encounters the template around position +12. For example compare Fig. 3 A register 11, Fig.3 B register 12, and run-off synthesis. Thus, these data do not provide any information regarding the temporal relationship between the polymerase and RNase H active sites. The primer may be randomly cleaved at any point after initiation, immediately after synthesis of the run-off product, or alternatively the primer may already be removed once the 12th nt has been added. The above data do not enable us to distinguish among these various scenarios, since cleaved 5′-end-labeled RNA fragments migrate at the same position in each case. We therefore followed formation and processing of the initially synthesized product, using a 3′-end-labeled primer (Fig.4). Putative secondary reactions are not detectable in this experiment, since the radiolabel is attached to the third DNA residue. The accumulation of the 12D-17R product, the pausing site, was again seen at early stages after initiation, i.e.1 and 3 min. A relatively small fraction of this product is further extended to yield the unprocessed run-off product, which is later cleaved, as shown by the time-dependent decrease of this band. However, it seems that most of the 12D-17R reaction intermediate is prematurely cleaved to yield the 12D product. The 12D product then accumulates between 3 and 20 min and is later extended to yield the processed run-off product. Taken together, these data demonstrate that the primer is not randomly cleaved at any stage after initiation. The appearance of the single 12D product shows that the RNA primer is efficiently and precisely cleaved at the RNA-DNA junction after the 12th DNA residue has been incorporated. Whether secondary initiation reactions also occur in the absence of chain-terminating stop-nucleotides cannot be answered on the basis of the above time course experiments. Synthesis of a secondary (+)-strand may be initiated after pausing and the following primer removal. Our data point to the existence of three different primer 3′-ends at position +12 that can potentially be recognized by the polymerase active site, i.e. the DNA 3′-end of the elongated unprocessed primer (12D-17R), the DNA 3′-end of the elongated processed primer (12D), and the RNA 3′-end of the processed primer (17R). To determine which of these three 3′-ends might preferentially be used for DNA synthesis, we used a stoichiometric mixture of primer/template combinations with an uncleaved chimeric primer containing 17 RNA and 12 DNA residues and a nicked substrate with a 17-mer RNA primer and a 12-mer DNA primer. The former represents the substrate prior to primer removal, whereas the latter mimics the substrate after the RNase H cut at the RNA-DNA junction. The efficiency of DNA synthesis from each of the three available 3′-ends was compared in a competition experiment, using three separate reaction mixtures with 5′-end-labeled primers and an RNase H-deficient RT (Fig.5 A). The data show the following order of efficiency: 12D > 12D17R ≫ 17R. After a 60-min reaction, 55% of the 12D primer and 45% of the chimeric 12D-17R primer were extended to yield the final run-off product. In contrast, only 25% of the 17R was found to be extended, and most of the extended fraction accumulated after incorporation of the first nucleotide. These data show that secondary initiation reactions are clearly suppressed in the absence of a chain-terminated primary product and that RT preferentially elongates the 3′-end of the newly synthesized and processed DNA fragment. The additional pausing site at position +1 does not necessarily correlate with putative difficulties of RT to strand-displace the annealed 12D fragment, since accumulation of this product is also seen in the absence of the 12D strand. Fig.5 B shows that initiation with the pure 17R primer resulted in dual pausing at position +1 and, in agreement with the data obtained with the 3D-17R primer, also at position +12. Moreover, synthesis of full-length (+)-strand DNA is still very efficient in this circumstance, indicating that the primer removal reaction is not required for polymerization steps after RT has reached position +12. We next analyzed the possible structural requirements for the specific pausing site at position +12. Pausing may be caused by the particular sequence or secondary structures of the single-stranded DNA template that inhibit the translocation of the enzyme. However, the specific pausing site after addition of the 12th nt is not observed with an homologous DNA/DNA substrate (Fig. 6), which indicates that pausing rather depends on the structure of the complexed chimeric substrate and its interaction with RT. This conclusion is additionally supported by the observation that different template sequences, shown under experimental procedures, do not alter the pausing profile as long as DNA synthesis was initiated with the RNA primer (data not shown). To further approach this problem, we wished to define structural characteristics of complexes generated before, during, and after position +12. Studying RT-DNA/DNA complexes, we have recently shown that the interaction between the DNA template and the RT-associated RNase H can be monitored in the presence of Fe2+ and that Fe2+ binds to one of the metal-binding sites of the RNase H domain, thereby generating a high local concentration of hydroxyl radicals that might serve as active species to cause an oxidative strand break (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Here, we have used this tool to determine the number of base pairs between both active sites depending on the bound substrate,i.e. RNA/DNA or DNA/DNA. Although the purine-rich sequence of the dsDNA substrate used in this study differs markedly from the PBS-derived sequence used previously (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), the number of bp between the RT active sites was identical, i.e. 17 in each case (Fig.7 A, lane 1). In contrast, when RT was complexed with the homologous RNA/DNA substrate, a specific cut was seen on the DNA template at a distance of 18 bp upstream of the primer terminus (lane 2). Lower concentrations of cleaved products were seen when RT was bound to the RNA/DNA substrate, suggesting a diminished efficiency of cleavage, but the cleavage profile was exactly the same. However, the major product is shifted exactly by a single nucleotide when RNA/DNA is bound to RT. The small percentage of side products, i.e. less than 10%, can be explained by the oxidative cleavage mechanism that involves diffusible hydroxyl radicals as active species (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). To provide additional information in regard to differences between RT-DNA/DNA and RT-RNA/DNA complexes, we also wished to study the protein-nucleic acid interface using hydroxyl radicals generated via Fe[EDTA]2− as well as via ONOOK, as described previously (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 19Götte M. Marquet R. Isel C. Anderson V.E. Keith G. Gross H.J. Ehresmann C. Ehresmann B. Heumann H. FEBS Lett. 1996; 390: 226-228Crossref PubMed Scopus (21) Google Scholar). However, a clear footprint could not be obtained, presumably reflecting the ability of RT to bind these substrates in both a polymerase-competent mode as well as one that facilitates removal of the primer (see "Discussion"). Despite the lack of a clear footprint, a faint band located on the DNA template strand, 7 bp upstream of the primer 3′-end, was seen when both complexes were treated with ONOOK (Fig. 7 A, lanes 3 and 4). This cut was also seen as part of the ONOOK-dependent footprint on the DNA/DNA substrate derived from the PBS sequence (15Götte M. Maier G. Gross H.J. Heumann H. J. Biol. Chem. 1998; 273: 10139-10146Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This particular reaction is thus a feature of polymerase-competent complexes and can be used as a second marker, in addition to the Fe2+cut, to determine differences in the number of base pairs that fit in the substrate-binding channel. It is interesting to note that the ONOOK cut appeared at the same position, regardless whether DNA/DNA or RNA/DNA w
During initiation of minus-strand synthesis by HIV-1 reverse transcriptase, a 3‘-DNA−RNA-5‘ junction is formed involving the 3‘-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the RNA template strand specifically, opposite the newly synthesized DNA strand. We have determined the crystal structure at 1.9 Å resolution of an eight-base pair hybrid duplex representing the junction to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. The junction octamer is in a global A-type conformation throughout. A base pair step with distinct stacking geometry and variable backbone conformation is located next to the main endonucleolytic cleavage site. This base pair step may serve as a recognition site for HIV-RT RNase H.
Thirty synthetic oligoribonucleotides, 3 to 18 nucleotides (nt) long, were analyzed by capillary electrophoresis, under nondenaturing conditions, using a commercial kit. The migration time t(m) was dependent on nt length and composition, capillary length, operating temperature, and type of sieving polymer. Under fixed experimental conditions, the t(m) proved predictable by the equation: t(m) = [0.22(n-1) + 6.14A/n + 6.86G/n + 3.61 (C+U)/n] min, for n>3, where A/n, G/n, C/n, U/n is the frequency of each type of nt within the oligonucleotide (ONT). The equation accounts for the influence of charge-to-mass ratio on t(m), but not for structural effects, if present. This approximation is acceptable for short ONTs. The possibility of detecting n+1, n-1, n-2 impurities, having predicted the t(m), is of crucial importance in assessing the purity of synthetic ONTs dedicated to structural studies. This appears to be feasible. High resolution was shown among homologous series of ONTs of increasing length, and in some cases, even within groups of ONTs of the same length but different composition. The addition of 7 M urea to the buffer, as denaturing agent, accelerates the t(m) and significantly lowers the resolution for the shortest ONTs. It was also possible to monitor the state of association of mixtures of RNA and DNA sequence-complementary strands.
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The interaction and subsequent photoaddition reaction on UVA irradiation of 4,4'-dimethyl-5-methoxyangelicin and 4,4'-dimethyl-5-methoxypsoralen to twelve synthetic oligonucleotides was studied. A new approach was developed which allows: (a) accurate determination of the kinetics constant of the overall photoaddition process to each oligonucleotide; (b) identification of the different types of intercalation sites, individualized by a combination of two consecutive base pairs and the frequency of their appearance along the oligomer sequences; and (c) the value of the kinetics constant relative to each type of site to be determined. This approach also makes it possible to highlight the influence of sequences flanking each site that participate in the photoaddition process. Thus, the possibility of discriminating which sites are active in the photoaddition reaction and their contribution to the overall process appears to be a useful tool for studying the correlation between furocoumarin photocombination with DNA and the consequent biological effects.
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