Further characterization of the discontinuities in cauliflower mosaic virus DNA

Richards, K.; Guilley, H.; Jonard, G.

doi:10.1016/0014-5793(81)80552-2

Cited by 46 publications

(19 citation statements)

References 13 publications

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“…5A). This is 1 nucleotide upstream of that previously reported by Richards et al (32). In addition to the predominant 5Ј end, lesser secondary ends were observed 1 or 2 nucleotides to the 5Ј side of the major end.…”

Section: Resultssupporting

confidence: 58%

“…Factors Affecting Plus-strand Priming Fidelity in Vivo-The 5Ј ends of the authentic CaMV plus-strands have been mapped previously to fixed positions (32), as is the case with other REs. Therefore, we wanted to determine (i) whether priming fidelity was maintained at the ectopic site and (ii) the effects of mutations on priming precision in vivo.…”

Section: Resultsmentioning

confidence: 99%

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Analysis of Polypurine Tract-associated DNA Plus-strand Priming in Vivo Utilizing a Plant Pararetroviral Vector Carrying Redundant Ectopic Priming Elements

Noad

Al-Kaff

Turner

et al. 1998

Journal of Biological Chemistry

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Initiation of DNA plus-strand synthesis in most reverse-transcribing elements requires primer generation by reverse transcriptase-associated RNase H at one or more template polypurine tracts (PPTs). We have exploited infectious clones of the plant pararetrovirus cauliflower mosaic virus carrying redundant ectopic plus-strand priming elements to study priming in vivo. Ectopic priming generated an additional discontinuity in progeny virion DNA during infection of plants. We found that altering the length of the 13-base pair PPT by ؎25% significantly reduced priming efficiency. A short pyrimidine tract 5 to the PPT, highly conserved among diverse reverse-transcribing elements, was shown to play an important role in PPT recognition in vivo. The predominant DNA plus-strand 5 end remained 3 nucleotides from the PPT 3 end in mutant primers that were longer or shorter than the wild-type primer. Use of an ectopic redundant primer to study replication-dependent priming was validated by demonstrating that it could rescue infectivity following destruction of the wild-type priming elements. We propose a model for plant pararetroviral plus-strand priming in which pyrimidines enhance PPT recognition during polymerasedependent RNase H cleavages, and suggest that fidelity of primer maturation during polymerase-independent cleavages involves PPT length measurement and 3 end recognition by RNase H. Retro-elements (REs),1 the propagation of which involves copying genomic RNA into DNA mediated by reverse transcriptase (RT), are found in organisms representing the major groups of eukaryotes. REs include animal retroviruses, animal and plant pararetroviruses, and retrotransposable elements (1). Although REs are diverse, most share several common features. Important among these is the pol gene product specifying multiple enzyme activities associated with reverse transcriptase (RT) involved in RE DNA synthesis and processing (2, 3).REs have a different origin of replication for synthesis of each strand of the double-stranded (ds)DNA phase. First, reverse transcription of the genomic RNA into minus-strand DNA is initiated in most REs by a host tRNA (4). During minus-strand synthesis, RT-associated RNase H activity degrades the RNA template (5,6). This degradation appears to occur in at least two stages. Processive DNA minus-strand synthesis is accompanied by a polymerase-dependent, non-processive RNase H degradation of the template leaving RNA fragments. These fragments are probably further degraded by subsequent polymerase-independent RNase H cleavages (7-9). The RNA primer for DNA plus-strand synthesis is generated during these steps by RNase H cleavage at a specific site called the polypurine tract (PPT), 13-18 nucleotides long. For REs producing an integrating linear double-stranded DNA with long terminal repeats (LTRs), precision of plus-strand initiation is essential for RE viability as one LTR border is defined by this step. Therefore, understanding the role of the PPT is important, particularly in retroviruses where plus-strand i...

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Section: Resultssupporting

confidence: 58%

Section: Resultsmentioning

confidence: 99%

Analysis of Polypurine Tract-associated DNA Plus-strand Priming in Vivo Utilizing a Plant Pararetroviral Vector Carrying Redundant Ectopic Priming Elements

Noad

Al-Kaff

Turner

et al. 1998

Journal of Biological Chemistry

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“…At each of the three discontinuities of CaMV virion DNA, the 5' and 3' extremities of the interrupted strand overlap one another by some 8-20 nucleotides (Franck et al, 1980;Richards et al, 1981). It is difficult to see how such overlapping sequences could be introduced into a completed, covalently closed duplex CaMV DNA molecule, leading us to suggest that the discontinuities are generated in the course of initiation and termination of DNA replication (Franck et al, 1980).…”

Section: Discussionmentioning

confidence: 99%

Observations concerning the discontinuous DNAs of cauliflower mosaic virus.

1983

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The double-stranded circular DNA encapsidated within cauliflower mosaic virus (CaMV) particles contains three single-stranded discontinuities, two in one strand and one in the other, so that, upon denaturation, three linear singlestranded DNAs are produced. Here we show that a fourth much smaller single-stranded DNA, termed a1, is also present in denatured CaMV DNA preparations. The 5' extremity of a, is identical to that of the a-strand, the strand of DNA possessing only one interruption, while its 3' extremity lies just two nucleotides downstream from a major transcription initiation site. We also show that the interrupted strand at each discontinuity sometimes has a single ribonucleotide in place of a deoxyribonucleotide at its 5' extremity. Oligoribonucleotide chains of eight and 10 residues in length have also been detected at the 5' end of one of the discontinuities. These structures are thought to be the vestiges of primers which have not been completely excised prior to encapsidation of the DNA. The possibility that synthesis of the astrand occurs by reverse transcription of viral RNA using initiator tRNAmet as primer is discussed.

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“…In the virus particle the major form of the DNA (8 kb) is circular, relaxed and contains three singlestranded discontinuities at specific sites, one in the minus strand and two in the plus strand (3,4,5). At the discontinuities the DNA has a triplestranded structure with the 5'-and 3'-extremities of the interrupted strand overlapping for 8 to 20 nucleotides (5,6). Discontinuous molecules of this type co-exist in infected cells with supercoiled viral DNA (7,8,9) which is present as minichromosomes in the nuclei (8,10).…”

Section: Introductionmentioning

confidence: 99%