Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H

Watson, Kenneth F.; Schendel, Peter L.; Rosok, M J; Ramsey, Leonard R.

doi:10.1021/bi00582a004

Cited by 24 publications

(15 citation statements)

References 41 publications

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“…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%

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: 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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“…The viral enzyme is responsible for degradation of genomic viral RNA after synthesis of minusstrand DNA has occurred. Upon removal of the RNA moiety of the DNARNA hybrid, minus-strand DNA becomes available as a template for synthesis of plus-strand DNA (3). The RNase H activity of reverse transcriptase has also been implicated in releasing the tRNA primer that is covalently bound to minus-strand DNA (4), in nicking viral RNA to generate the polypurine-rich oligoribonucleotide that primes synthesis of plus-strand DNA, and in removing the polypurine tract from the DNA extension to which it is linked (5)(6)(7)(8)(9).…”

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confidence: 99%

Ribonuclease H activities associated with viral reverse transcriptases are endonucleases.

Krug

Berger

1989

Proc. Natl. Acad. Sci. U.S.A.

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A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy-that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H. including circular poly(A)-linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNARNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes.The functions encoded in the genomes of retroviruses have come under increasingly intense scrutiny since the human immunodeficiency virus (HIV) was isolated and recognized as the causative agent of AIDS. However, the most promising drugs or vaccines target only two HIV functions; 3'-azido-3'-deoxythymidine (AZT) acts predominantly as an inhibitor of the polymerase activity of reverse transcriptase (1) and recombinant CD4 inhibits viral attachment by binding to products of the env gene (reviewed in ref.2).A retroviral function that has attracted relatively little attention, despite its being carried out by one of the better characterized proteins, is the ribonuclease H (RNase H) activity of reverse transcriptase. An RNase H cleaves RNA in a DNARNA hybrid. The viral enzyme is responsible for degradation of genomic viral RNA after synthesis of minusstrand DNA has occurred. Upon removal of the RNA moiety of the DNARNA hybrid, minus-strand DNA becomes available as a template for synthesis of plus-strand DNA (3). The RNase H activity of reverse transcriptase has also been implicated in releasing the tRNA primer that is covalently bound to minus-strand DNA (4), in nicking viral RNA to generate the polypurine-rich oligoribonucleotide that primes synthesis of plus-strand DNA, and in removing the polypurine tract from the DNA extension to which it is linked (5-9).The RNase H activities of reverse transcriptases are believed to be exonucleases that can attack RNA from either the 5' or the 3' end, whereas the normal cellular RNase H activities are endonucleases (10-12). These conclusions were based on experiments with molecules of two types: synthetic hybrids with blocked termini and circles. Both were cleaved by the RNase H isolated either from Escherichia coli or from mammalian tissues, but neither served as a substrate for ...

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“…Reverse transcriptase contains a ribonuclease H activity as an integral part of its structure (1,10,16,25). This enzyme degrades the RNA moiety of RNA-DNA hybrids and is therefore thought to degrade genome RNA after it has served as a template for the synthesis of minus-strand DNA (4,5,7,26). The enzyme could, however, have other activities.…”

mentioning

confidence: 99%

“…Any RNA primers involved in the synthesis of plus-strand DNA could be similarly removed. Second, to assist the initiation of plus strands of DNA the RNase H activity could create the proper primer RNA by selective degradation of the RNA template (17,18,26). Recent evidence indicates that the site of initiation of plus strands is highly specific (14,15), so that if an RNA primer is generated by this mechanism, the enzyme must recognize the correct sequence and precisely cleave the RNA.…”

mentioning

confidence: 99%

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

Champoux¹,

Gilboa²,

Baltimore³

1984

J Virol

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The single-stranded DNA containing the Moloney murine leukemia virus origin for plus-strand synthesis was cloned in M13mp2 and used as a template for avian myeloblastosis virus reverse transcriptase in the presence of Moloney RNA which had been treated with pancreatic RNase A. The RNA pieces containing the polypurine stretch near the plus-strand origin were processed, presumably by RNase H, to generate primers for DNA synthesis which initiated both at the correct origin site and at one nucleotide downstream from the correct site. Approximately 50% of the labeled DNA fragments synthesized under these conditions retained the priming RNA on their 5' ends. When the isolated fragments were hybridized back to the template DNA and again treated with the reverse transcriptase, all of the RNA was removed from the labeled DNA. By using 5'-end-labeled pancreatic RNase A-resistant fragments, it was possible to show that the RNA primers were removed intact. It appears from these results that the RNase H activity associated with the enzyme shows a preference for cutting at the junction between the RNA and DNA moieties of such complexes and therefore is ideally suited for removing RNA primers.

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Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H

Cited by 24 publications

References 41 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

Ribonuclease H activities associated with viral reverse transcriptases are endonucleases.

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

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