Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase

Champoux, James J.; Gilboa, Eli; Baltimore, David

doi:10.1128/jvi.49.3.686-691.1984

Cited by 79 publications

(41 citation statements)

References 26 publications

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“…Note that this proposition does not preclude RNAs from being used as primers after first strand DNA synthesis. Evidence conclusively shows that second strand DNA synthesis is initiated from a unique RNA primer termed the 'polypurine tract' (8)(9)(10)(11)(12)(13)(14) and perhaps from other RNAs (29). The results shown here and those ofothers (10,22,28) suggest that the use ofRNAs as primers is likely inefficient.…”

Section: Discussionmentioning

confidence: 61%

“…The RNase H activity of the RT is proposed to be required at several stages of viral genomic replication. These include degradation ofthe RNA template after synthesis ofthe first strand of DNA (7), generation of a specific oligopurine ribonucleotide primer from which second strand DNA synthesis will initiate and subsequent removal of the oligopurine primer (8)(9)(10)(11)(12)(13)(14).…”

Section: Introductionmentioning

confidence: 99%

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The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

DeStefano

1995

Nucl Acids Res

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The binding of HIV reverse transcriptase (RT) to heteroduplexes was examined using a substrate consisting of a 42 nt chimeric nucleic acid composed. (5'-->3') of 23 nt of RNA and 19 of DNA. This chimera was hybridized to an internal region of a relatively long complementary DNA or RNA. When the chimera was bound to DNA and conditions limiting cleavage to a single binding event between the enzyme and substrate were employed initial RNase H-directed cleavages occurred 19-21 nt from the chimera 5'-terminus. A 42 nt strand identical in sequence to the chimera and composed of only RNA was cleaved at the same locations. Reducing the length of the DNA portion of the chimera from 19 to 7 nt did not alter the cleavage positions, suggesting that cleavage was not coordinated by the DNA 3'-terminus. Under the same conditions cleavage was not detected when the chimera was bound to RNA. In contrast, addition of dNTPs to the DNA 3'-terminus of the chimera occurred only when the chimera was bound to RNA. The results support preferable binding of RT to RNA-DNA versus DNA-DNA hybrid regions and a model in which the orientation of binding to heteroduplexes is 5'-->3' (relative to the RNA strand), polymerase to RNase H active site, with sites associated with the DNA and RNA strand respectively.

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Section: Discussionmentioning

confidence: 61%

Section: Introductionmentioning

confidence: 99%

The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

DeStefano

1995

Nucl Acids Res

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“…In vitro studies demonstrated that initiation of plus-strand synthesis by the 3Ј PPT involves selective cleavage of viral RNA to produce the correct 3Ј end for subsequent extension by RT (13,24,47,48,53,62). The PPT primer is removed from plus-strand DNA by specific cleavages at or near the 5Ј terminus of the PPT and at the 3Ј RNA-DNA junction (5,24,46). Ultimately, the proper processing of the 3Ј PPT is of great importance since it precisely defines the left end of the linear DNA, which serves as a recognition and cleavage site for viral integrase (58).…”

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confidence: 99%

Sequence and structural determinants required for priming of plus-strand DNA synthesis by the human immunodeficiency virus type 1 polypurine tract

Powell

Levin

1996

J Virol

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At the 3 end of all retroviral genomes there is a short, highly conserved sequence known as the polypurine tract (PPT), which serves as the primer for plus-strand DNA synthesis. We have identified the determinants for in vitro priming by the human immunodeficiency virus type 1 (HIV-1) PPT. We show that when the PPT is removed and placed into different nucleotide contexts, new priming sites are produced at the precise 3 end of the PPT. In addition, we find that a hybrid consisting of a 15-or 20-nucleotide RNA primer annealed to a 35-nucleotide DNA template is competent for initiation of plus-strand synthesis with HIV-1 reverse transcriptase. Thus, no cis-acting elements appear to be required for priming activity. Changes at the 5 end of the PPT have no effect on primer function, whereas the identity of bases at the 3 end is crucial. A primer containing only the 6 G residues from the 3 end of the wild-type PPT sequence and 9 bases of random sequence at the 5 end functions like a wild-type PPT. A short hybrid having a similar helical structure but a primary sequence different from that of the PPT is cleaved imprecisely, resulting in initiation of synthesis at multiple sites; however, total primer extension is close to the wild-type level. We conclude that helical structure as well as the presence of particular bases at the 3 end of the PPT is essential for PPT function.of short RNA-DNA hybrids, we find that bases at the 3Ј end of the PPT are essential for priming activity. The helical structure of the hybrid may play a role in primer recognition by RT, but it is not the sole determinant of activity. MATERIALS AND METHODS Materials. Restriction enzymes, Taq polymerase, T4 ligase, and T4 DNA polymerase were purchased from Boehringer Mannheim (Indianapolis, Ind.). T4 polynucleotide kinase, RNasin, and RNase ONE, an RNase which degrades RNA without base specificity, were obtained from Promega Biotec (Madison, Wis.). HIV-1 RT was purchased from Worthington Biochemical Corp. (Freehold, N.J.). Radioactively labeled nucleotides [␣-32 P]dATP (3,000 Ci/mmol), [␥-32 P]ATP (3,000 Ci/mmol), and [␣-35 S]dATP␣S were purchased from Amersham Life Science Inc. (Arlington Heights, Ill.). RNA oligonucleotides were purchased from Oligos Etc., Inc. (Wilsonville, Oreg.). Sequence ladders were prepared with reagents from a Sequenase kit (U.S. Biochemicals Corp.) according to the manufacturer's instructions.Preparation of minus-strand single-stranded DNA. Circular minus-strand single-stranded DNA templates (3,352 nt in length), which contain a 185-nt region complementary to the PPT and the surrounding region, were prepared from an HIV-1 LAI clone (pKP8115). This clone was derived from a plasmid containing the HIV-1 LAI 3Ј long terminal repeat and part of nef (gift of Keith Peden, Center for Biologics Evaluation and Research, Food and Drug Administration) and contains nt 8587 to 8772 (60, numbering according to GenBank accession number K02013) inserted between the EcoRI and XbaI sites of the pGEM 3Zf(ϩ) vector (Promega Biotec). The PPT is lo...

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“…Omer and Faras (25) have shown that the avian myeloblastosis virus (AMV) RNase H can remove the tRNA primer from minus strong-stop DNA strands by a single cleavage at the junction between the RNA and the DNA chains. Similar studies with M-MuLV plus strands which had been synthesized in vitro and which retained an RNA primer revealed that the AMV RNase H removes the priming RNA fragment intact by cleaving at the junction between the RNA and the DNA (5). In both of these situations the basis for the recognition of the cleavage sequence may simply be the site at which the polynucleotide chain switches from ribonucleotides to deoxyribonucleotides.…”

Section: Dna-rna Hybrids Containing M-mulv Plus-strand Originmentioning

confidence: 87%

RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro

Finston¹,

Champoux²

1984

J Virol

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A 190-base-pair DNA-RNA hybrid containing the Moloney murine leukemia virus origin of plus-strand DNA synthesis was constructed and used as a source of template-primer for the reverse transcriptase in vitro. Synthesis was shown to initiate precisely at the known plus-strand origin. The observation that some of the origin fragments retained ribonucleotide residues on their 5' ends suggests that the primer for chain initiation is an RNA molecule left behind by RNase H during the degradation of the RNA moiety of the DNA-RNA hybrid. If the RNase H is responsible for creating the correct primer terminus, then it must possess a specific endonucleolytic activity capable of recognizing the sequence in the RNA where plus strands are initiated. The 16-base RNase A-resistant fragment which spans the plus-strand origin can also serve as a source of the specific plus-strand primer RNA. Evidence is presented that some of the plus-strand origin fragments synthesized in the endogenous reaction contain 5' ribonucleotides, suggesting that specific RNA primers for plus-strand initiation may be generated during reverse transcription in vivo as well. 26 on July 6, 2020 by guest

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Mechanism of RNA primer removal by the RNase H activity of avian myeloblastosis virus reverse transcriptase

Cited by 79 publications

References 26 publications

The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

The orientation of binding of human immunodeficiency virus reverse transcriptase on nucleic acid hybrids

Sequence and structural determinants required for priming of plus-strand DNA synthesis by the human immunodeficiency virus type 1 polypurine tract

RNA-primed initiation of Moloney murine leukemia virus plus strands by reverse transcriptase in vitro

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