Optimal Oct-2 Affinity for an Extended DNA Site and the Effect of GST Fusion on Site Preference

Rhee, Jerry M.; Trieu, May; Turner, Eric E.

doi:10.1006/abbi.2000.2181

Cited by 10 publications

(7 citation statements)

References 41 publications

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“…19,34 Further, the GST-tag can induce unrelated, non-specific aggregation of DNA, as it is known to alter DNA-binding properties of several enzymes. 35,36 Previous work using GST-Rad54 demonstrated a sevenfold stimulation in DNA networks relative to the Rad51 only reaction with approximately 70% of the input DNA being found in coaggregates. 13 However, in the GSTRad54 only reactions, 50% of the input DNA was recovered in the pellet fraction, versus 10% in the Rad51-only reactions.…”

Section: Resultsmentioning

confidence: 98%

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Bianco

Bradfield

Castanza

et al. 2007

Journal of Molecular Biology

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Rad54 is a key component of the eukaryotic recombination machinery. Its presence in DNA strand exchange reactions in vitro results in a significant stimulation in the overall reaction rate. Using untagged Rad54, we show that this stimulation can be attributed to enhancement of the formation of a key reaction intermediate known as DNA networks. Using a novel, single DNA molecule, dualoptical tweezers approach we show how Rad54 stimulates DNA network formation. We discovered that Rad54 oligomers possess a unique ability to cross-bridge or bind dsDNA molecules positioned in close proximity. Further, Rad54 oligomers rapidly translocate dsDNA while simultaneously inducing topological loops in the DNA at the locus of the oligomer. The combination of the crossbridging and dsDNA translocation activities of Rad54 stimulates the formation of DNA networks, leading to rapid and efficient DNA strand exchange by Rad51.

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Section: Resultsmentioning

confidence: 98%

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Bianco

Bradfield

Castanza

et al. 2007

Journal of Molecular Biology

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“…pGEX-KG and pGST-Oct-2 were a gift from Eric Turner (University of California-San Diego) [87]. pGST-BZLF1 was constructed by inserting BZLF1 amino acids (aa) 1-245 into pGEX-KG using SalI and SacI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

2012

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The Epstein-Barr virus (EBV) latent-lytic switch is mediated by the BZLF1 immediate-early protein. EBV is normally latent in memory B cells, but cellular factors which promote viral latency specifically in B cells have not been identified. In this report, we demonstrate that the B-cell specific transcription factor, Oct-2, inhibits the function of the viral immediate-early protein, BZLF1, and prevents lytic viral reactivation. Co-transfected Oct-2 reduces the ability of BZLF1 to activate lytic gene expression in two different latently infected nasopharyngeal carcinoma cell lines. Furthermore, Oct-2 inhibits BZLF1 activation of lytic EBV promoters in reporter gene assays, and attenuates BZLF1 binding to lytic viral promoters in vivo. Oct-2 interacts directly with BZLF1, and this interaction requires the DNA-binding/dimerization domain of BZLF1 and the POU domain of Oct-2. An Oct-2 mutant (Δ262–302) deficient for interaction with BZLF1 is unable to inhibit BZLF1-mediated lytic reactivation. However, an Oct-2 mutant defective for DNA-binding (Q221A) retains the ability to inhibit BZLF1 transcriptional effects and DNA-binding. Importantly, shRNA-mediated knockdown of endogenous Oct-2 expression in several EBV-positive Burkitt lymphoma and lymphoblastoid cell lines increases the level of lytic EBV gene expression, while decreasing EBNA1 expression. Moreover, treatments which induce EBV lytic reactivation, such as anti-IgG cross-linking and chemical inducers, also decrease the level of Oct-2 protein expression at the transcriptional level. We conclude that Oct-2 potentiates establishment of EBV latency in B cells.

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“…Hone-Akata cells (a gift from Lawrence Young, University of Birmingham) and CNE-2 Akata cells (a gift from K. W. Lo, The Chinese University of Hong Kong [received via Diane Hayward]) are NPC epithelial cell lines superinfected with the Akata strain of EBV and were maintained in RPMI 1640 supplemented with 10% FBS,(5Ј-CCCCTCGTGGCCATTTGTAGGAACTGACCACAACACTAGAGTCC-3Ј) and reverse (5Ј-GGACTCTAGTGTTGTGGTCAGTTCCTACAAATGGC CACGAGGGG-3Ј); and Oct-1(S335D) forward (5Ј-GAAGCCTTGAACCTCG ACTTTAAGAACATGTGCAAGTTGAAGCC-3Ј) and reverse (5Ј-GGCTTCA ACTTGCACATGTTCTTAAAGTCGAGGTTCAAGGCTTC-3Ј). pGEX-KG was a gift from Eric Turner (University of California-San Diego) (65). GSTOct-1 was cloned by PCR amplifying the POU domain of Oct-1 from the cDNAOct-1 plasmid and inserting it into pGEX-KG by using HindIII and SalI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

Cellular Transcription Factor Oct-1 Interacts with the Epstein-Barr Virus BRLF1 Protein To Promote Disruption of Viral Latency

Robinson

Kwek

Hagemeier

et al. 2011

J Virol

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The Epstein-Barr virus (EBV) latent-to-lytic switch is an essential part of the viral life cycle, but the cellular factors that promote viral reactivation are not well defined. In this report, we demonstrate that the cellular transcription factor Oct-1 cooperates with the EBV immediate-early protein BRLF1 (R, Rta) to induce lytic viral reactivation. We show that cotransfected Oct-1 enhances the ability of BRLF1 to activate lytic gene expression in 293 cells stably infected with a BRLF1-defective EBV mutant (BRLF1-stop) and that Oct-1 increases BRLF1-mediated activation of lytic EBV promoters in reporter gene assays. We find that Oct-1 interacts directly with BRLF1 in vitro and that a mutant BRLF1 protein (the M140A mutant) attenuated for the ability to interact with Oct-1 in vitro is also resistant to Oct-1-mediated transcriptional enhancement in 293 BRLF1-stop cells. Furthermore, we show that cotransfected Oct-1 augments BRLF1 binding to a variety of lytic EBV promoters in chromatin immunoprecipitation (ChIP) assays (including the BZLF1, BMRF1, and SM promoters) and that BRLF1 tethers Oct-1 to lytic EBV promoters. In addition, we demonstrate that an Oct-1 mutant defective in DNA binding (the S335D mutant) still retains the ability to enhance BRLF1 transcriptional effects. Finally, we show that knockdown of endogenous Oct-1 expression reduces the level of constitutive lytic EBV gene expression in both EBV-positive B-cell and EBV-positive epithelial cell lines. These results suggest that Oct-1 acts as a positive regulator of EBV lytic gene expression and that this effect is at least partially mediated through its interaction with the viral protein BRLF1. Epstein-Barr virus (EBV), or human herpesvirus 4 (HHV4), is a double-stranded DNA gammaherpesvirus which infects approximately 90% of the world's population (66). It causes a relatively mild primary disease if acquired early in life and infectious mononucleosis if acquired after adolescence. EBV is also associated with several B-cell and epithelial cell cancers, including Burkitt lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma (NPC), and gastric carcinoma (66, 101).EBV infection persists for the life of the host as a chronic asymptomatic infection by establishing latency in memory B cells (77). However, to be transmitted from host to host, and from cell to cell, the virus periodically reactivates from latency and converts to the lytic form of replication (66). Reactivation of lytic EBV infection in host cells is controlled at the level of the BZLF1 (Z, Zta, ZEBRA, EB1) and BRLF1 (R, Rta) viral promoters, as well as the activity of the BZLF1 and BRLF1 gene products (24,42,95). The BZLF1 and BRLF1 genes encode transcription factors that cooperatively turn on the expression of the early lytic viral genes involved in lytic viral replication (2,9,16,17,23,24,26,29,31,41,50,62,68), and the overexpression of either BZLF1 or BRLF1 is sufficient to induce lytic gene expression in many latently infected cell lines (13-15, 63, 83, 96). Since the combination of both BZL...

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Optimal Oct-2 Affinity for an Extended DNA Site and the Effect of GST Fusion on Site Preference

Cited by 10 publications

References 41 publications

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

The B-Cell Specific Transcription Factor, Oct-2, Promotes Epstein-Barr Virus Latency by Inhibiting the Viral Immediate-Early Protein, BZLF1

Cellular Transcription Factor Oct-1 Interacts with the Epstein-Barr Virus BRLF1 Protein To Promote Disruption of Viral Latency

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