The Experts below are selected from a list of 190410 Experts worldwide ranked by ideXlab platform
Marc S Wold - One of the best experts on this subject based on the ideXlab platform.
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Single-Molecule Analysis of Replication Protein A-DNA Interactions.
Methods in enzymology, 2018Co-Authors: Fletcher E. Bain, Ran Chen, Laura A. Fischer, Marc S WoldAbstract:Abstract Replication Protein A (RPA) is a highly conserved, eukaryotic ssDNA-binding Protein essential for genome stability. RPA interacts with ssDNA and with Protein partners to coordinate DNA Replication, repair, and recombination. Single-molecule analysis of RPA–DNA interactions is leading to a better understanding of the molecular interactions and dynamics responsible for RPA function in cells. Here, we first describe how to express, purify, and label RPA. We then describe how to prepare materials and carry out single-molecule experiments examining RPA–DNA interactions using total internal reflection fluorescence microscopy (TIRFM). Finally, the last section describes how to analyze TIRFM data. This chapter will focus on human RPA. However, these methods can be applied to RPA homologs from other species.
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dynamic binding of Replication Protein a is required for dna repair
Nucleic Acids Research, 2016Co-Authors: Ran Chen, Shyamal Subramanyam, Adrian H Elcock, Maria Spies, Marc S WoldAbstract:Replication Protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding Protein, is essential for Replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA Replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA Replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA-DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions.
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Replication Protein a single stranded dna s first responder dynamic dna interactions allow Replication Protein a to direct single strand dna intermediates into different pathways for synthesis or repair
BioEssays, 2014Co-Authors: Ran Chen, Marc S WoldAbstract:Replication Protein A (RPA), the major single-stranded DNA-binding Protein in eukaryotic cells, is required for processing of single-stranded DNA (ssDNA) intermediates found in Replication, repair and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high-affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other Proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error-prone repair of double-stranded breaks and promote error-free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis.
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diffusion of human Replication Protein a along single stranded dna
Journal of Molecular Biology, 2014Co-Authors: Binh Nguyen, Marc S Wold, Joshua E Sokoloski, Roberto Galletto, Elliot L Elson, Timothy M LohmanAbstract:Abstract Replication Protein A (RPA) is a eukaryotic single-stranded DNA (ssDNA) binding Protein that plays critical roles in most aspects of genome maintenance, including Replication, recombination and repair. RPA binds ssDNA with high affinity, destabilizes DNA secondary structure and facilitates binding of other Proteins to ssDNA. However, RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of RPA–DNA interactions, we combined ensemble and single-molecule fluorescence approaches to examine human RPA (hRPA) diffusion along ssDNA and find that an hRPA heterotrimer can diffuse rapidly along ssDNA. Diffusion of hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a one-dimensional diffusion coefficient of hRPA on ssDNA of D 1 ~ 5000 nt 2 s − 1 at 37 °C. Diffusion of hRPA while bound to ssDNA enables it to be readily repositioned to allow other Proteins access to ssDNA.
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human Replication Protein a rpa can diffuse along single stranded dna
Biophysical Journal, 2014Co-Authors: Binh Nguyen, Marc S Wold, Joshua E Sokoloski, Roberto Galletto, Elliot L Elson, Timothy M LohmanAbstract:Replication Protein A (RPA) is a hetero-trimeric Protein that plays critical roles in many cellular processes. The main function of RPA is to bind to single stranded (ss) DNA and to regulate its availability during DNA metabolic processes. RPA is also known to interact with an array of other Proteins in DNA Replication, repair and recombination processes in eukaryotic organisms. RPA binds to the transient ssDNA that forms during nearly all aspects of DNA metabolism and protects ssDNA from nucleases. Although RPA binds to ssDNA with a very high affinity, it must be dissociated from or redistributed along ssDNA during DNA Replication. To probe this re-arrangement of RPA along ssDNA, ensemble and single molecule studies have been used as complementary techniques to investigate human RPA diffusion along fluorescently labeled ssDNA oligomers. The dynamics of human RPA (hRPA) along fluorescently labeled DNA oligomers was also studied with fluorophore - labeled hRPA. These experiments illustrate that hRPA can spontaneously re-arrange along ssDNA by diffusing while remaining tightly bound. In addition, hRPA can also transiently melt DNA hairpin structures by diffusing in from ssDNA that is adjacent to the DNA hairpin. This ability of hRPA to diffuse along ssDNA means that directional DNA motor Proteins such as polymerases or translocases can push RPA and re-organize it along ssDNA. This diffusion property of RPA is shared with the bacterial analogue of RPA, the E. coli SSB Protein that also has previously been shown to diffuse along ssDNA. (GM030498 (TML), GM098509 (RG), GM044721 (MSW)).
Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.
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direct role for the Replication Protein treslin ticrr in the atr kinase mediated checkpoint response
Journal of Biological Chemistry, 2013Co-Authors: Bachar H Hassan, Michael G Kemp, Laura A Lindseyboltz, Aziz SancarAbstract:TopBP1 (topoisomerase IIβ-binding Protein 1) is a dual Replication/checkpoint Protein. Treslin/Ticrr, an essential Replication Protein, was discovered as a binding partner for TopBP1 and also in a genetic screen for checkpoint regulators in zebrafish. Treslin is phosphorylated by CDK2/cyclin E in a cell cycle-dependent manner, and its phosphorylation state dictates its interaction with TopBP1. The role of Treslin in the initiation of DNA Replication has been partially elucidated; however, its role in the checkpoint response remained elusive. In this study, we show that Treslin stimulates ATR phosphorylation of Chk1 both in vitro and in vivo in a TopBP1-dependent manner. Moreover, we show that the phosphorylation state of Treslin at Ser-1000 is important for its checkpoint activity. Overall, our results indicate that, like TopBP1, Treslin is a dual Replication/checkpoint Protein that directly participates in ATR-mediated checkpoint signaling.
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in vitro analysis of the role of Replication Protein a rpa and rpa phosphorylation in atr mediated checkpoint signaling
Journal of Biological Chemistry, 2012Co-Authors: Laura A Lindseyboltz, Marc S Wold, Joyce T Reardon, Aziz SancarAbstract:Abstract Replication Protein A (RPA) plays essential roles in DNA metabolism, including Replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated- and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17-RFC concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function while a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.
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In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling
The Journal of biological chemistry, 2012Co-Authors: Laura A. Lindsey-boltz, Marc S Wold, Joyce T Reardon, Aziz SancarAbstract:Replication Protein A (RPA) plays essential roles in DNA metabolism, including Replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.
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tipin Replication Protein a interaction mediates chk1 phosphorylation by atr in response to genotoxic stress
Journal of Biological Chemistry, 2010Co-Authors: Michael G Kemp, Zafer Akan, Secil Yilmaz, Mary Grillo, Stephanie L Smithroe, Taehong Kang, Marila Cordeirostone, William K Kaufmann, Robert T Abraham, Aziz SancarAbstract:Mammalian Timeless is a multifunctional Protein that performs essential roles in the circadian clock, chromosome cohesion, DNA Replication fork protection, and DNA Replication/DNA damage checkpoint pathways. The human Timeless exists in a tight complex with a smaller Protein called Tipin (Timeless-interacting Protein). Here we investigated the mechanism by which the Timeless-Tipin complex functions as a mediator in the ATR-Chk1 DNA damage checkpoint pathway. We find that the Timeless-Tipin complex specifically mediates Chk1 phosphorylation by ATR in response to DNA damage and Replication stress through interaction of Tipin with the 34-kDa subunit of Replication Protein A (RPA). The Tipin-RPA interaction stabilizes Timeless-Tipin and Tipin-Claspin complexes on RPA-coated ssDNA and in doing so promotes Claspin-mediated phosphorylation of Chk1 by ATR. Our results therefore indicate that RPA-covered ssDNA not only supports recruitment and activation of ATR but also, through Tipin and Claspin, it plays an important role in the action of ATR on its critical downstream target Chk1.
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an alternative form of Replication Protein a expressed in normal human tissues supports dna repair
Journal of Biological Chemistry, 2010Co-Authors: Michael G Kemp, Aaron C. Mason, Stuart J. Haring, Aziz Sancar, Joyce T Reardon, Gloria E O Borgstahl, Aura Carreira, Stephen C Kowalczykowski, Marc S WoldAbstract:Replication Protein A (RPA) is a heterotrimeric Protein complex required for a large number of DNA metabolic processes, including DNA Replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA Replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.
Walter J. Chazin - One of the best experts on this subject based on the ideXlab platform.
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surface reengineering of rpa70n enables cocrystallization with an inhibitor of the Replication Protein a interaction motif of atr interacting Protein
Biochemistry, 2013Co-Authors: Andreas O. Frank, Alex G. Waterson, J.d. Patrone, B. Vangamudi, J.p. Kennedy, Stephen W. Fesik, Walter J. ChazinAbstract:Replication Protein A (RPA) is the primary single-stranded DNA (ssDNA) binding Protein in eukaryotes. The N-terminal domain of the RPA70 subunit (RPA70N) interacts via a basic cleft with a wide range of DNA processing Proteins, including several that regulate DNA damage response and repair. Small molecule inhibitors that disrupt these Protein–Protein interactions are therefore of interest as chemical probes of these critical DNA processing pathways and as inhibitors to counter the upregulation of DNA damage response and repair associated with treatment of cancer patients with radiation or DNA-damaging agents. Determination of three-dimensional structures of Protein–ligand complexes is an important step for elaboration of small molecule inhibitors. However, although crystal structures of free RPA70N and an RPA70N–peptide fusion construct have been reported, RPA70N–inhibitor complexes have been recalcitrant to crystallization. Analysis of the P61 lattice of RPA70N crystals led us to hypothesize that the lig...
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A new structural framework for integrating Replication Protein A into DNA processing machinery
Nucleic Acids Research, 2013Co-Authors: Chris Brosey, Chunli Yan, Susan E Tsutakawa, William T Heller, Robert P Rambo, John A. Tainer, Ivaylo Ivanov, Walter J. ChazinAbstract:By coupling the protection and organization of single-stranded DNA (ssDNA) with recruitment and alignment of DNA processing factors, Replication Protein A (RPA) lies at the heart of dynamic multi-Protein DNA processing machinery. Nevertheless, how RPA coordinates biochemical functions of its eight domains remains unknown. We examined the structural biochemistry of RPA’s DNA-binding activity, combining small-angle X-ray and neutron scattering with all-atom molecular dynamics simulations to investigate the architecture of RPA’s DNA-binding core. The scattering data reveal compaction promoted by DNA binding; DNA-free RPA exists in an ensemble of states with inter-domain mobility and becomes progressively more condensed and less dynamic on binding ssDNA. Our results contrast with previous models proposing RPA initially binds ssDNA in a condensed state and becomes more extended as it fully engages the substrate. Moreover, the consensus view that RPA engages ssDNA in initial, intermediate and final stages conflicts with our data revealing that RPA undergoes two (not three) transitions as it binds ssDNA with no evidence for a discrete intermediate state. These results form a framework for understanding how RPA integrates the ssDNA substrate into DNA processing machinery, provides substrate access to its binding partners and promotes the progression and selection of DNA processing pathways.
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human dna helicase b hdhb binds to Replication Protein a and facilitates cellular recovery from Replication stress
Journal of Biological Chemistry, 2012Co-Authors: Gulfem D Guler, Walter J. Chazin, Sivaraja Vaithiyalingam, Elisabeth Kremmer, Hanjian Liu, Diana R Arnett, Ellen FanningAbstract:Maintenance of genomic stability in proliferating cells depends on a network of Proteins that coordinate chromosomal Replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal Replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of Replication Protein A (RPA) recruited to chromatin. We show using purified Proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling Proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from Replication stress.
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a high throughput fluorescence polarization anisotropy assay for the 70n domain of Replication Protein a
Analytical Biochemistry, 2012Co-Authors: Elaine M Souzafagundes, Walter J. Chazin, Michael D. Feldkamp, Andreas O. Frank, Olivia W. Rossanese, Stephen W. Fesik, Daniel Dorset, Edward T OlejniczakAbstract:Replication Protein A (RPA) interacts with multiple checkpoint Proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint Proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical Protein–Protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z′ factor of ⩾0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.
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bid binds to Replication Protein a and stimulates atr function following replicative stress
Molecular and Cellular Biology, 2011Co-Authors: Yang Liu, Walter J. Chazin, Sivaraja Vaithiyalingam, Qiong Shi, Sandra S ZinkelAbstract:Proapoptotic BH3-interacting death domain agonist (BID) regulates apoptosis and the DNA damage response. Following replicative stress, BID associates with Proteins of the DNA damage sensor complex, including Replication Protein A (RPA), ataxia telangiectasia and Rad3 related (ATR), and ATR-interacting Protein (ATRIP), and facilitates an efficient DNA damage response. We have found that BID stimulates the association of RPA with components of the DNA damage sensor complex through interaction with the basic cleft of the N-terminal domain of the RPA70 subunit. Disruption of the BID-RPA interaction impairs the association of ATR-ATRIP with chromatin as well as ATR function, as measured by CHK1 activation and recovery of DNA Replication following hydroxyurea (HU). We further demonstrate that the association of BID with RPA stimulates the association of ATR-ATRIP to the DNA damage sensor complex. We propose a model in which BID associates with RPA and stimulates the recruitment and/or stabilization of ATR-ATRIP to the DNA damage sensor complex.
Ellen Fanning - One of the best experts on this subject based on the ideXlab platform.
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human dna helicase b hdhb binds to Replication Protein a and facilitates cellular recovery from Replication stress
Journal of Biological Chemistry, 2012Co-Authors: Gulfem D Guler, Walter J. Chazin, Sivaraja Vaithiyalingam, Elisabeth Kremmer, Hanjian Liu, Diana R Arnett, Ellen FanningAbstract:Maintenance of genomic stability in proliferating cells depends on a network of Proteins that coordinate chromosomal Replication with DNA damage responses. Human DNA helicase B (HELB or HDHB) has been implicated in chromosomal Replication, but its role in this coordinated network remains undefined. Here we report that cellular exposure to UV irradiation, camptothecin, or hydroxyurea induces accumulation of HDHB on chromatin in a dose- and time-dependent manner, preferentially in S phase cells. Replication stress-induced recruitment of HDHB to chromatin is independent of checkpoint signaling but correlates with the level of Replication Protein A (RPA) recruited to chromatin. We show using purified Proteins that HDHB physically interacts with the N-terminal domain of the RPA 70-kDa subunit (RPA70N). NMR spectroscopy and site-directed mutagenesis reveal that HDHB docks on the same RPA70N surface that recruits S phase checkpoint signaling Proteins to chromatin. Consistent with this pattern of recruitment, cells depleted of HDHB display reduced recovery from Replication stress.
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a dynamic model for Replication Protein a rpa function in dna processing pathways
Nucleic Acids Research, 2006Co-Authors: Ellen Fanning, Vitaly Klimovich, Andrew R NagerAbstract:Processing of DNA in Replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding Protein. This Protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed. Many ssDNA-binding Proteins interact physically and functionally with a variety of other DNA processing Proteins. These interactions are thought to temporally order and guide the parade of Proteins that ‘trade places’ on the ssDNA, a model known as ‘hand-off’, as the processing pathway progresses. How this hand-off mechanism works remains poorly understood. Recent studies of the conserved eukaryotic ssDNA-binding Protein Replication Protein A (RPA) suggest a novel mechanism by which Proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA. This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA Replication and other DNA processing pathways, and proposes a general model for the role of RPA in Protein-mediated hand-off.
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interaction of dna polymerase alpha primase with cellular Replication Protein a and sv40 t antigen
The EMBO Journal, 1992Co-Authors: Irene Dornreiter, Ilka Gilbert, D Winkler, L F Erdile, Thomas J Kelly, Ellen FanningAbstract:Abstract The purified human single-stranded DNA binding Protein, Replication Protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique. RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase. The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase. Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase. These results suggest that the specific Protein-Protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA Replication.
Peter D Nagy - One of the best experts on this subject based on the ideXlab platform.
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P33 Replication Protein interacts with Osh6p oxysterol-binding Protein in the vicinity of Ufe1-positive membranes.
2018Co-Authors: Zsuzsanna Sasvari, Nikolay Kovalev, Paulina Alatriste Gonzalez, Peter D NagyAbstract:(A) Interaction between of TBSV nYFP-p33 Replication Protein and the Osh6-cYFP Protein was detected by BiFC. Partial co-localization of RFP-Ufe1 with the BiFC signal (see merged image) demonstrates that the interaction between p33 Replication Protein and Osh6p Protein occurs in the vicinity of the Ufe1-positive subdomain of the ER membrane. The images were taken at the 16 h time point. (B) Co-purification of Osh6p with the p33 Replication Protein from subcellular membranes is inhibited by co-expression of the dominant-negative Ufe1ΔTM. Top panels: Western blot analysis of co-purified His6-tagged cellular Osh6p Protein (lanes 3–4) with Flag-affinity purified FLAG-p33 (lanes 1–2) from detergent-solubilized membrane fraction of wt yeast. His6-Osh6p was detected with anti-His antibody, while FLAG-p33 was detected with anti-FLAG antibody. Note that yeasts co-expressed the dominant-negative Ufe1ΔTM (lanes 2 and 4). Bottom panels: Western blot of total HA-Ufe1p or HA-Use1p in the total yeast extract using anti-HA antibody. Bottom panel: Western blot of total FLAG-p33 and His6-Osh6p in the total yeast extracts. (C) Confocal laser microscopy analysis shows the diminished interaction between the TBSV nYFP-p33 Replication Protein and the Osh6-cYFP Protein (detected by BiFC) when the dominant-negative Ufe1ΔTM is co-expressed in wt yeast cells. (D) Interaction between TBSV nYFP-p33 Replication Protein and the AtSyp81-cYFP Protein (detected by BiFC) occurs at vMCS. Co-localization of ORP3A-CFP (small OSBP-like Protein, ortholog of yeast Osh6p) with the BiFC signal (see merged image) demonstrates that the interaction between p33 Replication Protein and AtSyp81 in subdomains of the ER membrane likely facilitates vMCS formation. The Proteins were expressed via agroinfiltration in N. benthamiana, followed by inoculation with TBSV. The images were taken 3 days latter. (E) Co-expression of the dominant-negative Ufe1ΔTM interferes with TBSV p33-driven sterol enrichment at internal Replication sites. Sterols (ergosterols) were stained with filipin dye, followed by fluorescent microscopic analysis. Note that ergosterol are mostly present in the plasma membrane when dominant-negative Ufe1ΔTM was co-expressed with the viral components (right images), whereas sterols were present at internal viral Replication sites in the absence of Ufe1ΔTM (images on the left).
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tombusviruses upregulate phospholipid biosynthesis via interaction between p33 Replication Protein and yeast lipid sensor Proteins during virus Replication in yeast
Virology, 2014Co-Authors: Daniel Barajas, Monika Sharma, Peter D NagyAbstract:Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral Replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 Replication Protein, which interacts with Opi1p FFAT domain Protein and Scs2p VAP Protein. Opi1p and Scs2p are phospholipid sensor Proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation and enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV Replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its Replication in yeast cells.
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rna chaperone activity of the tombusviral p33 Replication Protein facilitates initiation of rna synthesis by the viral rdrp in vitro
Virology, 2011Co-Authors: Jozsef Stork, Nikolay Kovalev, Zsuzsanna Sasvari, Peter D NagyAbstract:Small plus-stranded RNA viruses do not code for RNA helicases that would facilitate the proper folding of viral RNAs during Replication. Instead, these viruses might use RNA chaperones as shown here for the essential p33 Replication Protein of Tomato bushy stunt virus (TBSV). In vitro experiments demonstrate that the purified recombinant p33 promotes strand separation of a DNA/RNA duplex. In addition, p33 renders dsRNA templates sensitive to single-strand specific S1 nuclease, suggesting that p33 can destabilize highly structured RNAs. We also demonstrate that the RNA chaperone activity of p33 facilitates self-cleavage by a ribozyme in vitro. In addition, purified p33 facilitates in vitro RNA synthesis on double-stranded (ds)RNA templates up to 5-fold by a viral RNA-dependent RNA polymerase. We propose that the RNA chaperone activity of p33 facilitates the initiation of plus-strand synthesis as well as affects RNA recombination. Altogether, the TBSV RNA chaperone might perform similar biological functions to the helicases of other RNA viruses with much larger coding capacity.
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cpr1 cyclophilin and ess1 parvulin prolyl isomerases interact with the tombusvirus Replication Protein and inhibit viral Replication in yeast model host
Virology, 2010Co-Authors: Venugopal Mendu, Daniel Barajas, Menghsuen Chiu, Zhenghe Li, Peter D NagyAbstract:Abstract To identify host Proteins interacting with the membrane-bound Replication Proteins of tombusviruses, we performed membrane yeast two-hybrid (MYTH) screens based on yeast cDNA libraries. The screens led to the identification of 57 yeast Proteins interacting with Replication Proteins of two tombusviruses. Results from a split ubiquitin assay with 12 full-length yeast Proteins and the viral Replication Proteins suggested that the Replication Proteins of two tombusviruses interact with a similar set of host Proteins. Follow-up experiments with the yeast Cpr1p cyclophilin, which has prolyl isomerase activity that catalyzes cis–trans isomerization of peptidyl–prolyl bonds, confirmed that Cpr1p interacted with the viral p33 Replication Protein in yeast and in vitro . Replication of Tomato bushy stunt virus replicon RNA increased in cpr1 Δ yeast, while over-expression of Cpr1p decreased viral Replication. We also show that the Ess1p parvulin prolyl isomerase partly complements Cpr1p function as an inhibitor of tombusvirus Replication.
