scholarly journals Mechanism of Rep-Mediated Adeno-Associated Virus Origin Nicking

2000 ◽  
Vol 74 (17) ◽  
pp. 7762-7771 ◽  
Author(s):  
J. Rodney Brister ◽  
Nicholas Muzyczka
Keyword(s):  
Specific Activity ◽  
Dna Helicase ◽  
Loop Structure ◽  
Stem Loop ◽  
Adeno Associated Virus ◽  
Stem Loop Structure ◽  
Virus Origin ◽  
Rep Proteins ◽  
Sequence Elements ◽  
Origin Function

ABSTRACT The single-stranded adeno-associated virus type 2 (AAV) genome is flanked by terminal repeats (TRs) that fold back on themselves to form hairpinned structures. During AAV DNA replication, the TRs are nicked by the virus-encoded Rep proteins at the terminal resolution site (trs). This origin function apparently requires three sequence elements, the Rep binding element (RBE), a small palindrome that comprises a single tip of an internal hairpin within the TR (RBE′), and the trs. Previously, we determined the sequences at the trs required for Rep-mediated cleavage and demonstrated that the trs endonuclease reaction occurs in two discrete steps. In the first step, the Rep DNA helicase activity unwinds the TR, thereby extruding a stem-loop structure at thetrs. In the second step, Rep transesterification activity cleaves the trs. Here we investigate the contribution of the RBE and RBE′ during this process. Our data indicate that Rep is tethered to the RBE in a specific orientation duringtrs nicking. This orientation appears to align Rep on the AAV TR, allowing specific nucleotide contacts with the RBE′ and directing nicking to the trs. Accordingly, alterations in the polarity or position of the RBE relative to the trsgreatly inhibit Rep nicking. Substitutions within the RBE′ also reduce Rep specific activity, but to a lesser extent. Interestingly, Rep interactions with the RBE and RBE′ during nicking seem to be functionally distinct. Rep contacts with the RBE appear necessary for both the DNA helicase and trs cleavage steps of the endonuclease reaction. On the other hand, RBE′ contacts seem to be required primarily for TR unwinding and formation of thetrs stem-loop structure, not cleavage. Together, these results suggest a model of Rep interaction with the AAV TR during origin nicking through a tripartite cleavage signal comprised of the RBE, the RBE′, and the trs.

scholarly journals Rep-Mediated Nicking of the Adeno-Associated Virus Origin Requires Two Biochemical Activities, DNA Helicase Activity and Transesterification

1999 ◽  
Vol 73 (11) ◽  
pp. 9325-9336 ◽  
Author(s):  
J. Rodney Brister ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Helicase ◽  
Regulatory Function ◽  
General Mechanism ◽  
Loop Structure ◽  
Helicase Activity ◽  
Stem Loop ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Stem Loop Structure ◽  
Virus Origin

ABSTRACT The single-stranded adeno-associated virus (AAV) genome is flanked by terminal hairpinned origins of DNA replication (terminal repeats [TRs]) that are nicked at the terminal resolution site (trs) by the AAV Rep protein in an ATP-dependent, site-specific manner. Here we determine the minimal trssequence necessary for Rep cleavage, 3′-CCGGT/TG-5′, and show that this 7-base core sequence is required only on the nicked strand. We also identify a potential stem-loop structure at thetrs. Interestingly, Rep nicking on a TR substrate that fixes this trs stem-loop in the extruded form no longer requires ATP. This suggests that ATP-dependent Rep helicase activity is necessary to unwind the duplex trs and extrude the stem-loop structure, prior to the ATP-independent Rep transesterification reaction. The extrusion of origin stem-loop structures prior to nicking appears to be a general mechanism shared by plant and animal viruses and bacterial plasmids. In the case of AAV, this mechanism of TR nicking would provide a possible regulatory function.

scholarly journals Demonstration of Nicking/Joining Activity at the Origin of DNA Replication Associated with the Rep and Rep′ Proteins of Porcine Circovirus Type 1

2006 ◽  
Vol 80 (13) ◽  
pp. 6225-6234 ◽  
Author(s):  
Tobias Steinfeldt ◽  
Tim Finsterbusch ◽  
Annette Mankertz
Keyword(s):  
Dna Replication ◽  
Virus Replication ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Loop Structure ◽  
Stem Loop ◽  
Single Strand Break ◽  
Stem Loop Structure ◽  
Rep Proteins

ABSTRACT The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3′-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a double-stranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep′ have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep′ is as yet unknown. In this study, the ability of PCV1 Rep and Rep′ to “nick” and “join” strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5′-TAGTATTAC-3′) located at the apex of a putative stem-loop structure. In addition, the Rep and Rep′ proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual “nicking/joining” activities associated with PCV1 Rep and Rep′ are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.

968: Gelsolin Gene Silencing Involving Unusual Chromatin Structures and a Novel Stem Loop Structure of the Promoter Region in Human Bladder Cancer

2004 ◽  
Vol 171 (4S) ◽  
pp. 256-257
Author(s):  
Kazunori Haga ◽  
Ataru Sazawa ◽  
Toru Harabayashi ◽  
Nobuo Shinohara ◽  
Minoru Nomoto ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Gene Silencing ◽  
Promoter Region ◽  
Loop Structure ◽  
Human Bladder Cancer ◽  
Human Bladder ◽  
Stem Loop ◽  
Stem Loop Structure

Detection and sequence analysis of an imperfect stem-loop structure in cis activating gene element from SV40PolyA

2011 ◽  
Vol 33 (4) ◽  
pp. 337-346
Author(s):  
Hong-Gang WANG ◽  
Huan MA ◽  
Zhu LI ◽  
Bin ZHANG ◽  
Xiang-Yang JING ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure ◽  
In Cis ◽  
Gene Element

scholarly journals Locking up the AS1411 Aptamer with a Flanking Duplex: Towards an Improved Nucleolin-Targeting

2021 ◽  
Vol 14 (2) ◽  
pp. 121
Author(s):  
André Miranda ◽  
Tiago Santos ◽  
Eric Largy ◽  
Carla Cruz
Keyword(s):  
Loop Structure ◽  
Binding Properties ◽  
Stem Loop ◽  
Affinity Ligand ◽  
Stem Loop Structure ◽  
Topology Maintenance ◽  
G Quadruplex ◽  
Dimeric Form ◽  
Biophysical Techniques ◽  
Melting Experiments

We have designed AS1411-N6, a derivative of the nucleolin (NCL)-binding aptamer AS1411, by adding six nucleotides to the 5′-end that are complementary to nucleotides at the 3′-end forcing it into a stem-loop structure. We evaluated by several biophysical techniques if AS1411-N6 can adopt one or more conformations, one of which allows NCL binding. We found a decrease of polymorphism of G-quadruplex (G4)-forming sequences comparing to AS1411 and the G4 formation in presence of K+ promotes the duplex folding. We also studied the binding properties of ligands TMPyP4, PhenDC3, PDS, 360A, and BRACO-19 in terms of stability, binding, topology maintenance of AS1411-N6, and NCL recognition. The melting experiments revealed promising stabilizer effects of PhenDC3, 360A, and TMPyP4, and the affinity calculations showed that 360A is the most prominent affinity ligand for AS1411-N6 and AS1411. The affinity determined between AS1411-N6 and NCL denoting a strong interaction and complex formation was assessed by PAGE in which the electrophoretic profile of AS1411-N6 showed bands of the dimeric form in the presence of the ligands and NCL.

scholarly journals Role of a Stem-Loop Structure inHelicobacter pylori cagATranscript Stability

2018 ◽  
Vol 87 (2) ◽  
Author(s):  
John T. Loh ◽  
Aung Soe Lin ◽  
Amber C. Beckett ◽  
Mark S. McClain ◽  
Timothy L. Cover
Keyword(s):  
Steady State ◽  
Untranslated Region ◽  
Loop Structure ◽  
Stem Loop ◽  
Content Type ◽  
Transcript Stability ◽  
Protein Levels ◽  
Caga Protein ◽  
Stem Loop Structure ◽  
Steady State Levels

ABSTRACTHelicobacter pyloriCagA is a secreted effector protein that contributes to gastric carcinogenesis. Previous studies showed that there is variation amongH. pyloristrains in the steady-state levels of CagA and that a strain-specific motif downstream of thecagAtranscriptional start site (the +59 motif) is associated with both high levels of CagA and premalignant gastric histology. ThecagA5′ untranslated region contains a predicted stem-loop-forming structure adjacent to the +59 motif. In the current study, we investigated the effect of the +59 motif and the adjacent stem-loop oncagAtranscript levels andcagAmRNA stability. Using site-directed mutagenesis, we found that mutations predicted to disrupt the stem-loop structure resulted in decreased steady-state levels of both thecagAtranscript and the CagA protein. Additionally, these mutations resulted in a decreasedcagAmRNA half-life. Mutagenesis of the +59 motif without altering the stem-loop structure resulted in reduced steady-statecagAtranscript and CagA protein levels but did not affectcagAtranscript stability.cagAtranscript stability was not affected by increased sodium chloride concentrations, an environmental factor known to augmentcagAtranscript levels and CagA protein levels. These results indicate that both a predicted stem-loop structure and a strain-specific +59 motif in thecagA5′ untranslated region influence the levels ofcagAexpression.

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