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• W2131967018 abstract "We previously analyzed strand transfers catalyzed by human immunodeficiency virus, type 1 reverse transcriptase (RT) in a hairpin-containing RNA template system. In this system, RT produces a series of adjacent RNase H cuts before the hairpin base on the first, or donor template that clears a region of the donor, facilitating invasion by the second, or acceptor RNA. Here we analyze characteristics of the prominent cuts before the hairpin base and their role in strand transfers. Analysis of the template cleavage pattern during synthesis suggested that the RT performs DNA 3′ end-directed primary and secondary cuts while paused at the hairpin base and that these cuts contribute to creation of the invasion site. RT catalyzed similar cleavages on a substrate representing a paused cDNA-template intermediate. DNA 3′ end-directed secondary cuts, which require positioning of the polymerase active site downstream of the primer terminus, had previously not been specifically identified during synthesis. Our findings indicate that during synthesis DNA 3′ end-directed primary and secondary cuts occur at pause sites. RT mutants with substitutions at the His539 residue in the RNase H active site were defective in secondary cleavages. Analysis of the template cleavage pattern generated by the His539 mutants during synthesis revealed inefficient cleavage at the invasion site, correlating with defects in strand transfer. Overall, results indicate RT can catalyze pause-associated DNA 3′ end-directed primary and secondary cuts during synthesis and these cuts can contribute to strand transfer by creation of an invasion site. We previously analyzed strand transfers catalyzed by human immunodeficiency virus, type 1 reverse transcriptase (RT) in a hairpin-containing RNA template system. In this system, RT produces a series of adjacent RNase H cuts before the hairpin base on the first, or donor template that clears a region of the donor, facilitating invasion by the second, or acceptor RNA. Here we analyze characteristics of the prominent cuts before the hairpin base and their role in strand transfers. Analysis of the template cleavage pattern during synthesis suggested that the RT performs DNA 3′ end-directed primary and secondary cuts while paused at the hairpin base and that these cuts contribute to creation of the invasion site. RT catalyzed similar cleavages on a substrate representing a paused cDNA-template intermediate. DNA 3′ end-directed secondary cuts, which require positioning of the polymerase active site downstream of the primer terminus, had previously not been specifically identified during synthesis. Our findings indicate that during synthesis DNA 3′ end-directed primary and secondary cuts occur at pause sites. RT mutants with substitutions at the His539 residue in the RNase H active site were defective in secondary cleavages. Analysis of the template cleavage pattern generated by the His539 mutants during synthesis revealed inefficient cleavage at the invasion site, correlating with defects in strand transfer. Overall, results indicate RT can catalyze pause-associated DNA 3′ end-directed primary and secondary cuts during synthesis and these cuts can contribute to strand transfer by creation of an invasion site. Human immunodeficiency virus, type 1 (HIV-1) 3The abbreviations used are: HIV-1human immunodeficiency virusRTreverse transcriptaseEIAVequine infectious anemia virusFLfull-lengthSPself-primingTPtransfer productWTwild typentnucleotide(s).3The abbreviations used are: HIV-1human immunodeficiency virusRTreverse transcriptaseEIAVequine infectious anemia virusFLfull-lengthSPself-primingTPtransfer productWTwild typentnucleotide(s). reverse transcriptase (RT) is the virally encoded enzyme responsible for conversion of the viral RNA genome into double-stranded DNA. RT is a p66/p51 heterodimer that possesses both RNA- and DNA-dependent DNA polymerase activity and RNase H activity. The polymerase and RNase H active sites reside in the N-terminal and C-terminal portions of the p66 subdomain, respectively. Structural and biochemical studies indicate that the two active sites are separated by a distance corresponding to that covered by 18–19 nt (1Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar, 2Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar, 3Furfine E.S. Reardon J.E. J. Biol. Chem. 1991; 266: 406-412Abstract Full Text PDF PubMed Google Scholar, 4Kohlstaedt L.A. Wang J. Friedman J.M. Rice P.A. Steitz T.A. Science. 1992; 256: 1783-1790Crossref PubMed Scopus (1751) Google Scholar). During minus strand synthesis, the polymerizing RT binds to the 3′ end of the primer and cleaves the RNA template concomitant with DNA synthesis (3Furfine E.S. Reardon J.E. J. Biol. Chem. 1991; 266: 406-412Abstract Full Text PDF PubMed Google Scholar). The relative rate of polymerization is 7–10 times faster than RNase H cleavage and the two activities are uncoupled (5Kati W.M. Johnson K.A. Jerva L.F. Anderson K.S. J. Biol. Chem. 1992; 267: 25988-25997Abstract Full Text PDF PubMed Google Scholar). Biochemical studies indicate that the RNase H cuts generate various sized fragments of RNA some of which are left behind still bound to the newly synthesized cDNA during minus strand synthesis (6DeStefano J.J. Buiser R.G. Mallaber L.M. Bambara R.A. Fay P.J. J. Biol. Chem. 1991; 266: 24295-24301Abstract Full Text PDF PubMed Google Scholar, 7DeStefano J.J. Buiser R.G. Mallaber L.M. Myers T.W. Bambara R.A. Fay P.J. J. Biol. Chem. 1991; 266: 7423-7431Abstract Full Text PDF PubMed Google Scholar).In addition to binding to the 3′ terminus of a primer in an orientation for synthesis, RT has alternate modes of binding that are believed to be biologically relevant during reverse transcription. For example, an RNA oligonucleotide recessed on a longer DNA segment can represent the products that remain behind after the RT has synthesized a segment of minus strand DNA. On such substrates RT preferentially cleaves ∼18 and 8 nt from the 5′ end of the RNA to produce primary and secondary cleavage products, respectively (8Palaniappan C. Fuentes G.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Biol. Chem. 1996; 271: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 9Wisniewski M. Balakrishnan M. Palaniappan C. Fay P.J. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11978-11983Crossref PubMed Scopus (73) Google Scholar, 10DeStefano J.J. Mallaber L.M. Fay P.J. Bambara R.A. Nucleic Acids Res. 1993; 21: 4330-4338Crossref PubMed Scopus (78) Google Scholar). Studies indicate that these RNA 5′ end-directed primary and secondary cleavages are independent of each other and have differing rates (9Wisniewski M. Balakrishnan M. Palaniappan C. Fay P.J. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11978-11983Crossref PubMed Scopus (73) Google Scholar, 11Wisniewski M. Balakrishnan M. Palaniappan C. Fay P.J. Bambara R.A. J. Biol. Chem. 2000; 275: 37664-37671Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). For the RT to make the secondary cut, it must bind ∼10 nt closer to the 5′ end of the RNA relative to positioning for the primary cut. Finally, the RT has also been shown to make internal cleavages not directed by terminus alignment, but occurring with some sequence preference (12Schultz S.J. Zhang M. Champoux J.J. J. Mol. Biol. 2004; 344: 635-652Crossref PubMed Scopus (31) Google Scholar). In vivo, both RNA 5′ end-directed primary and secondary cleavages and internal cleavages are believed to occur on the RNA fragments that remain annealed to the DNA template following synthesis and cleavage by the polymerizing RT.Similar to the primary/secondary cleavage pattern observed when RT utilizes an RNA 5′ end to direct binding, primary and secondary cleavages have also been observed roughly 18 and 8 nt from the DNA 3′ end, respectively (13Wisniewski M. Chen Y. Balakrishnan M. Palaniappan C. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 28400-28410Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Archer R.H. Wisniewski M. Bambara R.A. Demeter L.M. Biochemistry. 2001; 40: 4087-4095Crossref PubMed Scopus (25) Google Scholar, 15Archer R.H. Dykes C. Gerondelis P. Lloyd A. Fay P. Reichman R.C. Bambara R.A. Demeter L.M. J. Virol. 2000; 74: 8390-8401Crossref PubMed Scopus (92) Google Scholar). DNA 3′ end-directed primary cleavage arises from binding of the DNA primer terminus within the polymerase active site resulting in the positioning of the RNase H active site 18–19 nt upstream. Primary cleavages of this type occur concomitant with DNA synthesis. DNA 3′ end-directed secondary cleavage has also been observed (2Gopalakrishnan V. Peliska J.A. Benkovic S.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10763-10767Crossref PubMed Scopus (173) Google Scholar, 13Wisniewski M. Chen Y. Balakrishnan M. Palaniappan C. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 28400-28410Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 14Archer R.H. Wisniewski M. Bambara R.A. Demeter L.M. Biochemistry. 2001; 40: 4087-4095Crossref PubMed Scopus (25) Google Scholar, 15Archer R.H. Dykes C. Gerondelis P. Lloyd A. Fay P. Reichman R.C. Bambara R.A. Demeter L.M. J. Virol. 2000; 74: 8390-8401Crossref PubMed Scopus (92) Google Scholar, 16Schatz O. Mous J. Le Grice S.F. EMBO J. 1990; 9: 1171-1176Crossref PubMed Scopus (126) Google Scholar, 17Wohrl B.M. Volkmann S. Moelling K. J. Mol. Biol. 1991; 220: 801-818Crossref PubMed Scopus (35) Google Scholar). However, these studies were performed in the absence of dNTPs, precluding synthesis. Secondary cuts of this type likely require movement of the polymerase active site away from the primer terminus to allow repositioning of the RNase H active site ∼8 nt behind the primer terminus. Movement of the polymerase active site away from the primer terminus during synthesis is unanticipated, because DNA polymerases are thought to remain bound to the 3′ terminus of the primer during elongation. The possible roles and effects of DNA 3′ end-directed secondary cleavages have not been explored because these cleavages have only been analyzed and characterized as such in the absence of synthesis.In the crystal structure of HIV-1 RT complexed to an RNA:DNA hybrid, the highly conserved histidine at position 539 is shown to interact with the scissile phosphate of the RNA (1Sarafianos S.G. Das K. Tantillo C. Clark Jr., A.D. Ding J. Whitcomb J.M. Boyer P.L. Hughes S.H. Arnold E. EMBO J. 2001; 20: 1449-1461Crossref PubMed Scopus (359) Google Scholar) and is likely a key residue in the RNase H active site. Asparagine and aspartic acid substitutions of His539 produce RT mutants that are particularly defective in secondary cleavage (17Wohrl B.M. Volkmann S. Moelling K. J. Mol. Biol. 1991; 220: 801-818Crossref PubMed Scopus (35) Google Scholar). In addition, substitution of the His539 residue with phenylalanine was shown to reduce RNase H activity, however, inhibition of particular types of RNase H cleavage were not characterized (18Schatz O. Cromme F.V. Gruninger-Leitch F. Le Grice S.F. FEBS Lett. 1989; 257: 311-314Crossref PubMed Scopus (132) Google Scholar, 19Cirino N.M. Kalayjian R.C. Jentoft J.E. Le Grice S.F. J. Biol. Chem. 1993; 268: 14743-14749Abstract Full Text PDF PubMed Google Scholar). Results from these and other studies have shown that mutation of the 539 position has little effect on the polymerase activity of RT, however, the RNase H activity and specificity were altered to varying extents (17Wohrl B.M. Volkmann S. Moelling K. J. Mol. Biol. 1991; 220: 801-818Crossref PubMed Scopus (35) Google Scholar, 18Schatz O. Cromme F.V. Gruninger-Leitch F. Le Grice S.F. FEBS Lett. 1989; 257: 311-314Crossref PubMed Scopus (132) Google Scholar, 20Volkmann S. Wohrl B.M. Tisdale M. Moelling K. J. Biol. Chem. 1993; 268: 2674-2683Abstract Full Text PDF PubMed Google Scholar, 21Tisdale M. Schulze T. Larder B.A. Moelling K. J. Gen. Virol. 1991; 72: 59-66Crossref PubMed Scopus (139) Google Scholar). Additionally, asparagine substitution of the 539 position produced an RT mutant with reduced efficiency of strand transfer in vivo (22Nikolenko G.N. Svarovskaia E.S. Delviks K.A. Pathak V.K. J. Virol. 2004; 78: 8761-8770Crossref PubMed Scopus (69) Google Scholar).RT RNase H activity is essential to several aspects of reverse transcription. One such process is RT-mediated strand transfer (23Smith C.M. Smith J.S. Roth M.J. J. Virol. 1999; 73: 6573-6581Crossref PubMed Google Scholar, 24DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar). Several studies have suggested a correlation between pausing of the RT and strand transfer (24DeStefano J.J. Mallaber L.M. Rodriguez-Rodriguez L. Fay P.J. Bambara R.A. J. Virol. 1992; 66: 6370-6378Crossref PubMed Google Scholar, 25Kim J.K. Palaniappan C. Wu W. Fay P.J. Bambara R.A. J. Biol. Chem. 1997; 272: 16769-16777Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 26Derebail S.S. DeStefano J.J. J. Biol. Chem. 2004; 279: 47446-47454Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 27Wu W. Blumberg B.M. Fay P.J. Bambara R.A. J. Biol. Chem. 1995; 270: 325-332Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 29Roda R.H. Balakrishnan M. Hanson M.N. Wohrl B.M. Le Grice S.F. Roques B.P. Gorelick R.J. Bambara R.A. J. Biol. Chem. 2003; 278: 31536-31546Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Pause sites are positions where synthesis products accumulate because of a slow rate of synthesis, often as a result of an encounter with a secondary structure, such as a hairpin, on the template (30Suo Z. Johnson K.A. Biochemistry. 1997; 36: 12459-12467Crossref PubMed Scopus (92) Google Scholar, 31Klarmann G.J. Schauber C.A. Preston B.D. J. Biol. Chem. 1993; 268: 9793-9802Abstract Full Text PDF PubMed Google Scholar). RT pausing is also correlated with increased RNase H cleavage, an anticipated result of increased residence time of the RT at one position (32Suo Z. Johnson K.A. Biochemistry. 1997; 36: 12468-12476Crossref PubMed Scopus (37) Google Scholar). Recent studies from our laboratory have described a pause-mediated mechanism of strand transfer in which pausing of RT induces cleavage of the RNA template and promotes initial steps of strand transfer (28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 29Roda R.H. Balakrishnan M. Hanson M.N. Wohrl B.M. Le Grice S.F. Roques B.P. Gorelick R.J. Bambara R.A. J. Biol. Chem. 2003; 278: 31536-31546Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33Chen Y. Balakrishnan M. Roques B.P. Bambara R.A. J. Biol. Chem. 2003; 278: 38368-38375Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 34Chen Y. Balakrishnan M. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2003; 278: 8006-8017Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).We have previously analyzed pause-associated transfer by HIV-1 RT using templates containing the equine infectious anemia virus (EIAV) primer binding site hairpin sequence (Fig. 1 and see Fig. 7 in Ref. 28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) (29Roda R.H. Balakrishnan M. Hanson M.N. Wohrl B.M. Le Grice S.F. Roques B.P. Gorelick R.J. Bambara R.A. J. Biol. Chem. 2003; 278: 31536-31546Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The steps in strand transfer with these templates were as follows. During primer extension, RT produced a series of cuts on the first, or donor template, upstream of the hairpin base. This generated an invasion site, a region where the nascent cDNA could interact with the second, or acceptor RNA, to facilitate transfer. Primer extension continued on the donor template and the acceptor:cDNA hybrid propagated toward the primer terminus. At a site downstream of the invasion site, predominantly within the loop region, the primer terminus switched templates from the donor to the acceptor. In this two-step “dock and lock” model for strand transfer an important component of the transfer mechanism is clearing of a region of the donor template (creation of an invasion site) to allow acceptor interaction with the nascent cDNA. In this study we seek to further expand our understanding of strand transfer in this system by determining the characteristics of cleavages that promote efficient creation of an invasion site.EXPERIMENTAL PROCEDURESMaterials—DNA oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA). NotI, HindIII, and Escherichia coli RNase H were purchased from Invitrogen. Poly(rA)-oligo(dT) was obtained from Amersham Biosciences. Radionucleotides were purchased from PerkinElmer Life Sciences. All other enzymes and dNTP solutions were obtained from Roche Applied Science.Preparation of WT RT and His539 Mutants—Expression plasmids (HXB2 strain) pKK-p66(His6) and pKK-p51(His6) (35Lee R. Kaushik N. Modak M.J. Vinayak R. Pandey V.N. Biochemistry. 1998; 37: 900-910Crossref PubMed Scopus (66) Google Scholar) were kindly obtained as a gift from N. Kaushik. PCR primer-based mutagenesis of pKK-p66(His6) was used to create RT clones with TTT, GAC, and AGA coding for phenylalanine, aspartic acid, and arginine at the 539 position, respectively. The entire RT coding region of WT RT and the three mutant clones were sequenced to verify that the sequence was conserved with the exception of codon 539. RT was purified as previously described (29Roda R.H. Balakrishnan M. Hanson M.N. Wohrl B.M. Le Grice S.F. Roques B.P. Gorelick R.J. Bambara R.A. J. Biol. Chem. 2003; 278: 31536-31546Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 36Pandey V.N. Kaushik N. Rege N. Sarafianos S.G. Yadav P.N. Modak M.J. Biochemistry. 1996; 35: 2168-2179Crossref PubMed Scopus (141) Google Scholar) with the minor variation that the p66 and p51 subunits were purified separately and mixed in equal amounts prior to dialysis. The specific activities of the RT preparations were determined by measuring the rate of [α-32P]dTTP incorporation using poly(rA)-oligo(dT) as substrates in a filter binding assay. The specific activities of the RTs were 18,000, 18,000, 21,000, and 24,000 units/mg for WT, H539F, H539D, and H539R RT, respectively. One unit of RT activity is defined as the amount required to incorporate 1 nmol of dTTP into nucleic acid product in a 10-min reaction at 37 °C using poly(rA)-oligo(dT) as template-primer.Generation of RNA Templates—The donor and acceptor constructs pEIAV-Donor and pEIAV-A2 have been previously described (28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). RNA templates were generated by in vitro run-off transcription from linearized plasmids using T7 RNA polymerase as previously described (28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). RNA donor DI was generated from pEIAV-Donor linearized with NotI and RNA AI-2 was generated from pEIAV-A2 linearized with HindIII. Internally labeled donor DI was synthesized by adding [α-32P]CTP to the in vitro transcription reaction. All RNAs were purified by PAGE. To assess the purity of the isolated transcripts, all RNAs were treated with calf alkaline phosphatase, 5′ end radiolabeled with polynucleotide kinase and [γ-32P]ATP, and run on a polyacrylamide gel.Preparation of Substrates—To prepare RNA templates for 5′ end labeling, the 5′ phosphate was removed by calf alkaline phosphatase as per the manufacturer's protocol. RNA and DNA were radiolabeled with polynucleotide kinase as per the manufacturer's protocol using [γ-32P]ATP. Unincorporated ATP was removed by a Bio-Rad P-30 Micro BioSpin size exclusion column. Substrates were prepared by mixing together the RNA and DNA in the appropriate ratio and heating at 95 °C for 5 min followed by slow cooling in a heat block to room temperature. The sequences of the primers used were: dP1, 5′-TACGATTTAGGTGACACTATAG-3′; dP2, 5′-ACACTATAGAATATGCATCACTAGTAAGCTTCAGGTATGGTCTGC-3′; and dP3, 5′-GGCCGCAGATTTAGGTGACACTATAGAATATGCATCAC-3′. Primers ddP2 and ddP3 were the same sequence as dP2 and dP3, respectively, except that the 3′ terminal base was a 2′,3′-dideoxycytidine instead of 2′-deoxycytidine. The 41-mer RNA and 77-mer DNA have been previously described (9Wisniewski M. Balakrishnan M. Palaniappan C. Fay P.J. Bambara R.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11978-11983Crossref PubMed Scopus (73) Google Scholar).Primer Extension and Strand Transfer Assays—Reactions were performed as previously described with slight modifications (28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). DI donor and radiolabeled dP1 primer were mixed in a 1:2 ratio prior to annealing for primer extension experiments. For strand transfer reactions a 1:2:2 ratio of donor:acceptor:primer was used. RT was preincubated with annealed primer and template for 5 min prior to initiation by addition of MgCl2 and dNTPs. The final reaction contained 4 nm primer, 2 nm donor, 4 units of RT, 50 mm Tris-HCl (pH 8.0), 6 mm MgCl2, 50 μm dNTPs, 50 mm NaCl, 1 mm dithiothreitol, and 1 mm EDTA. Strand transfer reactions additionally contained 4 nm acceptor. Reactions were incubated at 37 °C and were terminated at appropriate time points by the addition of 2× termination dye (20 mm EDTA (pH 8.0), 90% formamide, and 0.1% each of bromphenol blue and xylene cyanol). The products were separated by denaturing PAGE and visualized and quantitated by Storm PhosphorImager (GE Healthcare) and ImageQuant software version 1.2 (GE Healthcare). A 10-bp DNA ladder, 5′ end radiolabeled with [γ-32P]ATP using polynucleotide kinase per the manufacturer's protocol, was loaded to serve as a size marker (labeled L in the figures).RNase H Cleavage Assays—Reactions were performed as described above with some minor variations. DNA and radiolabeled RNA were mixed in a 10:1 ratio prior to annealing for donor cleavage experiments. The final reaction contained 2 nm template, 20 nm primer, 4 units of RT, 50 mm Tris-HCl (pH 8.0), 6 mm MgCl2, 50 mm NaCl, 1 mm dithiothreitol, and 1 mm EDTA. The concentration of dNTPs was 50 μm unless otherwise described. To determine the exact position of the cleavage fragments, reaction samples were run alongside RNase T1 and alkaline hydrolysis ladders generated from the donor template. For polymerase trap experiments, 1 μg of poly(rA)-oligo(dT) per 12.5-μl reaction was added when the reaction was initiated.RESULTSWe reasoned that the ability of RT to generate a series of adjacent cuts at the primary invasion site influences the efficiency of acceptor invasion. Experiments were designed to determine the origin of these cuts and their relevance to the transfer process.Primer Extension and Donor RNA Cleavage by WT HIV-1 RT on EIAV Templates—The hairpin-containing template system used in this study has been previously described (28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and is illustrated in Fig. 1. The donor template region from +1 to +78 folds into a stable hairpin as predicted by mfold (37Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10091) Google Scholar, 38Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3198) Google Scholar). The acceptor shares a 97-nt region of homology with the donor that begins 19 nt before the hairpin base and extends to the 5′ end of the hairpin. The primer binds to a unique sequence at the 3′ end of the donor RNA. Primer extension by RT was monitored using a 5′ end-labeled DNA primer annealed to the donor template. Extension of the dP1 primer by WT RT resulted in a full-length product (FL) that was 136 nt long and a self-priming product (SP) that was 194 nt long (Fig. 2A). Early in the reaction (1–3 min) the majority of extended primers were stalled at the base of the hairpin (+58/+59). Over time, synthesis proceeded beyond the base of the hairpin and the amount of FL product increased. However, the +58/+59 pause products remained prominent in these reactions. Aside from the major pause site at the hairpin base, pausing of WT RT was also observed at +44, +80, +93, +120, and numerous minor sites.FIGURE 2Primer extension on donor template. Schematic above the panel illustrates the generation of FL and SP products from dP1 primer extension on donor DI. Primer extension reactions using a 5′ end-labeled primer. A, WT; B, H539F; C, H539D; and D, H539R. The reactions were sampled at the times indicated above the lanes, and the products resolved on an 8% denaturing polyacrylamide gel. The prominent pause site at the hairpin base (+58/+59), other pause sites along the donor template (+44, +80, +93, and +120), as well as the FL and SP products, are indicated to the right of the gel. Lane L, 10-bp DNA ladder.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Concomitant with primer extension, the RNase H activity of RT cleaves the donor template. Degradation of the donor template during primer extension was monitored by use of a 5′ end radiolabeled donor template. Fig. 3A illustrates the 5′ terminal donor fragments generated during synthesis by WT RT. By synthesis, we mean the overall primer extension reaction carried out by RT in the presence of primer-template and dNTPs, including primers that are extended, paused, or terminated. In the first minute of the reaction, extension of the primer to about 80 nt was detected. During this time, progressive cleavages on the donor generated products in the range of 120–60 nt (compare Figs. 2A and 3A). In previous studies using this template system we have shown that the 19-nt region of homology between the donor and acceptor RNA, before the hairpin base is important for transfer (28Roda R.H. Balakrishnan M. Kim J.K. Roques B.P. Fay P.J. Bambara R.A. J. Biol. Chem. 2002; 277: 46900-46911Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 29Roda R.H. Balakrishnan M. Hanson M.N. Wohrl B.M. Le Grice S.F. Roques B.P. Gorelick R.J. Bambara R.A. J. Biol. Chem. 2003; 278: 31536-31546Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Efficient cleavage of the donor within this region creates a site on the cDNA accessible for acceptor interaction. Two prominent cleavages within this region were those at +93 and +83, which corresponds to 15 and 5 nt before the hairpin base, respectively. By 3 min of reaction, the 93-nt product was cleaved and lost, whereas the 83-nt product persisted for the duration of the reaction.FIGURE 3Degradation of donor template during primer extension. Schematic above the panel illustrates the substrate (dP1 primer and DI donor) used in the reaction. Numbers above the donor represent positions of prominent cleavage near the hairpin base. Numbers indicated by brackets represent the distance of a particular cut site from the hairpin base. A–D, RNase H assay of donor DI degradation during dP1 primer extension using a 5′ end-labeled donor. A, WT; B, H539F; C, H539D; and D, H539R. The reactions were sampled at the times indicated above the lanes and resolved on an 8% denaturing polyacrylamide gel. Positions of prominent cleavage are indicated to the left of the gel. E, following the 30-min reaction, 1 unit of E. coli RNase H was added and reactions were incubated for an additional 3 min. a, WT; b, H539F; c, H539D; and d, H539R. Lane L, 10-bp DNA ladder. Lane C, control reaction containing all components except RT incubated for 30 min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Cleavages Generated during Primer Extension Are Similar to DNA 3′ End-directed Cleavages Using a Fixed Length Primer—Whereas there is only a single major pause site at the hairpin base (+58/+59), we observed a series of adjacent cuts within the 19-nt region of homology before the hairpin. We considered that some of the cuts were the result of DNA 3′ end-directed primary and secondary cleavages made at the pause site, during the course of synthesis. To test this idea, we designed substrates that modeled an extended primer paused at the base of the hairpin and examined the cleavages that were made. The dP2 primer anneals to the template such that its 3′ terminus is base paired to the template 1 nt before the hairpin base (+79 on DI) (Fig. 4). To enable detection of 3′ end-directed cleavages associated with the fixed length primer, the reactions were performed in the absence of dNTPs to prevent primer extension. Addition of WT RT to a" @default.
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• W2131967018 date "2005-12-01" @default.
• W2131967018 modified "2023-10-01" @default.
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