scholarly journals Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities

1999 ◽  
Vol 73 (2) ◽  
pp. 1580-1590 ◽  
Author(s):  
Xiaohuai Zhou ◽  
Irene Zolotukhin ◽  
Dong-Soo Im ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Active Form ◽  
Dna Helicase ◽  
Enzyme Concentration ◽  
Viral Gene ◽  
Helicase Activity ◽  
Enzyme Molecule ◽  
Strand Displacement ◽  
Adeno Associated Virus ◽  
Site Specific

ABSTRACT The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trsendonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.

scholarly journals Adeno-Associated Virus Site-Specific Integration Is Regulated by TRP-185

2006 ◽  
Vol 81 (4) ◽  
pp. 1990-2001 ◽  
Author(s):  
Noriaki Yamamoto ◽  
Masato Suzuki ◽  
Masa-aki Kawano ◽  
Takamasa Inoue ◽  
Ryou-u Takahashi ◽  
...  
Keyword(s):  
Nonstructural Protein ◽  
Integration Site ◽  
Dna Helicase ◽  
Binding Property ◽  
Helicase Activity ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Dna Binding Property ◽  
Aavs1 Locus

ABSTRACT Adeno-associated virus (AAV) integrates site specifically into the AAVS1 locus on human chromosome 19. Although recruitment of the AAV nonstructural protein Rep78/68 to the Rep binding site (RBS) on AAVS1 is thought to be an essential step, the mechanism of the site-specific integration, particularly, how the site of integration is determined, remains largely unknown. Here we describe the identification and characterization of a new cellular regulator of AAV site-specific integration. TAR RNA loop binding protein 185 (TRP-185), previously reported to associate with human immunodeficiency virus type 1 TAR RNA, binds to AAVS1 DNA. Our data suggest that TRP-185 suppresses AAV integration at the AAVS1 RBS and enhances AAV integration into a region downstream of the RBS. TRP-185 bound to Rep68 directly, changing the Rep68 DNA binding property and stimulating Rep68 helicase activity. We present a model in which TRP-185 changes the specificity of the AAV integration site from the RBS to a downstream region by acting as a molecular chaperone that promotes Rep68 complex formation competent for 3′→5′ DNA helicase activity.

scholarly journals Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication

2007 ◽  
Vol 81 (11) ◽  
pp. 5777-5787 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
William F. McDonald ◽  
Xiaohuai Zhou ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Dna Helicase ◽  
Strand Displacement ◽  
Adeno Associated Virus ◽  
Replication System ◽  
Factor C

ABSTRACT Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase δ, but not polymerase ε or α, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase δ complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase δ, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase δ, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

scholarly journals A Role for Single-Stranded Templates in Cell-Free Adeno-Associated Virus DNA Replication

2000 ◽  
Vol 74 (2) ◽  
pp. 744-754 ◽  
Author(s):  
Peter Ward ◽  
R. Michael Linden
Keyword(s):  
Dna Replication ◽  
Infected Cell ◽  
Dna Binding Protein ◽  
Strand Displacement ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.

scholarly journals The Main Role of Srs2 in DNA Repair Depends on Its Helicase Activity, Rather than on Its Interactions with PCNA or Rad51

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Alex Bronstein ◽  
Lihi Gershon ◽  
Gilad Grinberg ◽  
Elisa Alonso-Perez ◽  
Martin Kupiec
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Genome Stability ◽  
Dna Helicase ◽  
Main Role ◽  
Helicase Domain ◽  
Helicase Activity ◽  
C Terminus ◽  
Srs2 Helicase

ABSTRACTHomologous recombination (HR) is a mechanism that repairs a variety of DNA lesions. Under certain circumstances, however, HR can generate intermediates that can interfere with other cellular processes such as DNA transcription or replication. Cells have therefore developed pathways that abolish undesirable HR intermediates. TheSaccharomyces cerevisiaeyeast Srs2 helicase has a major role in one of these pathways. Srs2 also works during DNA replication and interacts with the clamp PCNA. The relative importance of Srs2’s helicase activity, Rad51 removal function, and PCNA interaction in genome stability remains unclear. We created a newSRS2allele [srs2(1-850)] that lacks the whole C terminus, containing the interaction site for Rad51 and PCNA and interactions with many other proteins. Thus, the new allele encodes an Srs2 protein bearing only the activity of the DNA helicase. We find that the interactions of Srs2 with Rad51 and PCNA are dispensable for the main role of Srs2 in the repair of DNA damage in vegetative cells and for proper completion of meiosis. On the other hand, it has been shown that in cells impaired for the DNA damage tolerance (DDT) pathways, Srs2 generates toxic intermediates that lead to DNA damage sensitivity; we show that this negative Srs2 activity requires the C terminus of Srs2. Dissection of the genetic interactions of thesrs2(1-850) allele suggest a role for Srs2’s helicase activity in sister chromatid cohesion. Our results also indicate that Srs2’s function becomes more central in diploid cells.IMPORTANCEHomologous recombination (HR) is a key mechanism that repairs damaged DNA. However, this process has to be tightly regulated; failure to regulate it can lead to genome instability. The Srs2 helicase is considered a regulator of HR; it was shown to be able to evict the recombinase Rad51 from DNA. Cells lacking Srs2 exhibit sensitivity to DNA-damaging agents, and in some cases, they display defects in DNA replication. The relative roles of the helicase and Rad51 removal activities of Srs2 in genome stability remain unclear. To address this question, we created a new Srs2 mutant which has only the DNA helicase domain. Our study shows that only the DNA helicase domain is needed to deal with DNA damage and assist in DNA replication during vegetative growth and in meiosis. Thus, our findings shift the view on the role of Srs2 in the maintenance of genome integrity.

scholarly journals Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

2001 ◽  
Vol 75 (7) ◽  
pp. 3230-3239 ◽  
Author(s):  
Miran Yoon ◽  
Deborah H. Smith ◽  
Peter Ward ◽  
Francisco J. Medrano ◽  
Aneel K. Aggarwal ◽  
...  
Keyword(s):  
Dna Replication ◽  
Catalytic Domain ◽  
Adeno Associated Virus ◽  
Free System ◽  
Site Specific ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Site Specific Integration ◽  
Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

scholarly journals Synthetic DNA Replication Bubbles Bound and Unwound with Twofold Symmetry by a Simian Virus 40 T-Antigen Double Hexamer

1998 ◽  
Vol 72 (11) ◽  
pp. 8676-8681 ◽  
Author(s):  
Natalia V. Smelkova ◽  
James A. Borowiec
Keyword(s):  
Dna Replication ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Dna Helicase ◽  
T Antigen ◽  
Dna Unwinding ◽  
Replication Forks ◽  
Helicase Activity ◽  
Synthetic Dna ◽  
Stimulation Of

ABSTRACT Dimerization of simian virus 40 T-antigen hexamers (TAgH) into double hexamers (TAgDH) on model DNA replication forks has been found to greatly stimulate T-antigen DNA helicase activity. To explore the interaction of TAgDH with DNA during unwinding, we examined the binding of TAgDH to synthetic DNA replication bubbles. Tests of replication bubble substrates containing different single-stranded DNA (ssDNA) lengths indicated that efficient formation of a TAgDH requires ≥40 nucleotides (nt) of ssDNA. DNase I probing of a substrate containing a 60-nt ssDNA bubble complexed with a TAgDH revealed that T antigen bound the substrate with twofold symmetry. The strongest protection was observed over the 5′ junction on each strand, with 5 bp of duplex DNA and ∼17 nt of adjacent ssDNA protected from nuclease cleavage. Stimulation of the T-antigen DNA helicase activity by an increase in ATP concentration caused the protection to extend in the 5′ direction into the duplex region, while resulting in no significant changes to the 3′ edge of strongest protection. Our data indicate that each TAgH encircles one ssDNA strand, with a different strand bound at each junction. The process of DNA unwinding results in each TAgH interacting with a greater length of DNA than was initially bound, suggesting the generation of a more highly processive helicase complex.

scholarly journals Factors Affecting the Terminal Resolution Site Endonuclease, Helicase, and ATPase Activities of Adeno-Associated Virus Type 2 Rep Proteins

1999 ◽  
Vol 73 (10) ◽  
pp. 8235-8244 ◽  
Author(s):  
Jianwen Wu ◽  
Michael D. Davis ◽  
Roland A. Owens
Keyword(s):  
Virus Type ◽  
Helicase Activity ◽  
Endonuclease Activity ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Rabbit Reticulocyte Lysate System ◽  
Wide Range

ABSTRACT The Rep68 and Rep78 proteins (Rep68/78) of adeno-associated virus type 2 (AAV) are critical for AAV replication and site-specific integration. They bind specifically to the AAV inverted terminal repeats (ITRs) and possess ATPase, helicase, and strand-specific/site-specific endonuclease activities. In the present study, we further characterized the AAV Rep68/78 helicase, ATPase, and endonuclease activities by using a maltose binding protein-Rep68 fusion (MBP-Rep68Δ) produced in Escherichia coli cells and Rep78 produced in vitro in a rabbit reticulocyte lysate system. We found that the minimal length of single-stranded DNA capable of stimulating the ATPase activity of MBP-Rep68Δ is 100 to 200 bases. The degree of stimulation correlated positively with the length of single-stranded DNA added to the reaction mixture. We then determined the ATP concentration needed for optimal MBP-Rep68Δ helicase activity and showed that the helicase is active over a wide range of ATP concentrations. We determined the directionality of MBP-Rep68Δ helicase activity and found that it appears to move in a 3′ to 5′ direction, which is consistent with a model in which AAV Rep68/78 participates in AAV DNA replication by unwinding DNA ahead of a cellular DNA polymerase. In this report, we also demonstrate that single-stranded DNA is capable of inhibiting the MBP-Rep68Δ or Rep78 endonuclease activity greater than 10-fold. In addition, we show that removal of the secondary Rep68/78 binding site, which is found only in the hairpin form of the AAV ITR, causes a three- to eightfold reduction in the ability of the ITR to be used as a substrate for the Rep78 or MBP-Rep68Δ endonuclease activity. This suggests that contact between Rep68/78 and this secondary element may play an important role in the Rep-mediated endonuclease activity.

scholarly journals Phosphorylation of MCM4 at Sites Inactivating DNA Helicase Activity of the MCM4-MCM6-MCM7 Complex during Epstein-Barr Virus Productive Replication

2006 ◽  
Vol 80 (20) ◽  
pp. 10064-10072 ◽  
Author(s):  
Ayumi Kudoh ◽  
Tohru Daikoku ◽  
Yukio Ishimi ◽  
Yasushi Kawaguchi ◽  
Noriko Shirata ◽  
...  
Keyword(s):  
Dna Replication ◽  
Epstein Barr Virus ◽  
Cyclin A ◽  
Dna Helicase ◽  
Lytic Replication ◽  
Helicase Activity ◽  
Chromosomal Dna ◽  
Barr Virus ◽  
Mcm Complex ◽  
Epstein Barr

ABSTRACT Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.

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.

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