Limits...
Epstein-Barr Nuclear Antigen 1 modulates replication of oriP-plasmids by impeding replication and transcription fork migration through the family of repeats.

Aiyar A, Aras S, Washington A, Singh G, Luftig RB - Virol. J. (2009)

Bottom Line: We conclude that EBNA1 bound to FR regulates the replication of oriP-plasmids by impeding the migration of replication forks.Upon binding FR, EBNA1 also blocks the migration of transcription forks.Thus, in addition to regulating oriP replication, EBNA1 bound to FR also decreases the probability of detrimental collisions between two opposing replication forks, or between a transcription fork and a replication fork.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stanley S, Scott Cancer Center, LSU Health Sciences Center, New Orleans, LA 70112, USA. aaiyar@lsuhsc.edu

ABSTRACT

Background: Epstein-Barr virus is replicated once per cell-cycle, and partitioned equally in latently infected cells. Both these processes require a single viral cis-element, termed oriP, and a single viral protein, EBNA1. EBNA1 binds two clusters of binding sites in oriP, termed the dyad symmetry element (DS) and the family of repeats (FR), which function as a replication element and partitioning element respectively. Wild-type FR contains 20 binding sites for EBNA1.

Results: We, and others, have determined previously that decreasing the number of EBNA1-binding sites in FR increases the efficiency with which oriP-plasmids are replicated. Here we demonstrate that the wild-type number of binding sites in FR impedes the migration of replication and transcription forks. Further, splitting FR into two widely separated sets of ten binding sites causes a ten-fold increase in the efficiency with which oriP-plasmids are established in cells expressing EBNA1. We have also determined that EBNA1 bound to FR impairs the migration of transcription forks in a manner dependent on the number of EBNA1-binding sites in FR.

Conclusion: We conclude that EBNA1 bound to FR regulates the replication of oriP-plasmids by impeding the migration of replication forks. Upon binding FR, EBNA1 also blocks the migration of transcription forks. Thus, in addition to regulating oriP replication, EBNA1 bound to FR also decreases the probability of detrimental collisions between two opposing replication forks, or between a transcription fork and a replication fork.

Show MeSH

Related in: MedlinePlus

EBNA1 bound to FR impedes transcription fork progression. (A) Representation of transcription reporter plasmids used here. In pRSVL, the RSV LTR drives transcription of the luciferase gene, and the SV40 late polyadenylation signal is used for polyadenylation. Derivatives of pRSVL with ten, 20 or 40 EBNA1-binding sites (filled black circles) between the luciferase gene and the polyadenylation signal were constructed. Primary transcripts, and prematurely terminated transcripts present in total RNA preparations are indicated, as are mature luciferase mRNAs present in total and polyA+ RNA preparations. Primers used for RT-PCR are indicated as arrows. (B) Luciferase expression from the reporter plasmids described above. Plasmids were co-transfected with either pcDNA3 (stippled bars), or a EBNA1 DNA binding domain expression plasmid (black bars) into 293 cells. The number of EBNA1 binding sites in the reporter plasmid is indicated below each pair of bars. Luciferase activity is reported relative to the activity observed when pRSVL was co-transfected with pcDNA3. (C) RT-PCRs to detect luciferase and GAPDH transcripts in total or polyadenylated RNAs recovered from the transfected cells described in B. PCR products were visualized with ethidium bromide and the identity of the transfected plasmid is indicated above each lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654434&req=5

Figure 5: EBNA1 bound to FR impedes transcription fork progression. (A) Representation of transcription reporter plasmids used here. In pRSVL, the RSV LTR drives transcription of the luciferase gene, and the SV40 late polyadenylation signal is used for polyadenylation. Derivatives of pRSVL with ten, 20 or 40 EBNA1-binding sites (filled black circles) between the luciferase gene and the polyadenylation signal were constructed. Primary transcripts, and prematurely terminated transcripts present in total RNA preparations are indicated, as are mature luciferase mRNAs present in total and polyA+ RNA preparations. Primers used for RT-PCR are indicated as arrows. (B) Luciferase expression from the reporter plasmids described above. Plasmids were co-transfected with either pcDNA3 (stippled bars), or a EBNA1 DNA binding domain expression plasmid (black bars) into 293 cells. The number of EBNA1 binding sites in the reporter plasmid is indicated below each pair of bars. Luciferase activity is reported relative to the activity observed when pRSVL was co-transfected with pcDNA3. (C) RT-PCRs to detect luciferase and GAPDH transcripts in total or polyadenylated RNAs recovered from the transfected cells described in B. PCR products were visualized with ethidium bromide and the identity of the transfected plasmid is indicated above each lane.

Mentions: The transcription reporter plasmid pRSVL [33] was modified to introduce ten, 20, or 40 contiguous EBNA1 binding sites between the end of the luciferase open reading frame and the SV40 polyadenylation sequence in that plasmid. The structure of these reporter plasmids is shown in Figure 5A. These reporter plasmids were then co-transfected into 293 cells with a control expression plasmid (pcDNA3), or plasmid 1160 that expresses the DNA binding domain of EBNA1 (DBD). Cells were harvested two days post-transfection, FACS profiled to normalize for live transfected cells, following which luciferase levels were measured. This analysis is shown in Figure 5B. In the absence of EBNA1 binding sites on the reporter plasmid, the co-transfected DBD expression plasmid had no effect on luciferase expression. Similarly, pcDNA3 had no effect on luciferase expression from reporter plasmids that contained ten, 20 or 40 EBNA1 binding sites. However when the luciferase reporter plasmids had 20 or 40 EBNA1 binding sites, and were co-transfected with the DBD expression plasmid, there was a sharp decrease in the expression of luciferase dependent upon the number of number binding sites placed 5' to the polyadenylation signal.


Epstein-Barr Nuclear Antigen 1 modulates replication of oriP-plasmids by impeding replication and transcription fork migration through the family of repeats.

Aiyar A, Aras S, Washington A, Singh G, Luftig RB - Virol. J. (2009)

EBNA1 bound to FR impedes transcription fork progression. (A) Representation of transcription reporter plasmids used here. In pRSVL, the RSV LTR drives transcription of the luciferase gene, and the SV40 late polyadenylation signal is used for polyadenylation. Derivatives of pRSVL with ten, 20 or 40 EBNA1-binding sites (filled black circles) between the luciferase gene and the polyadenylation signal were constructed. Primary transcripts, and prematurely terminated transcripts present in total RNA preparations are indicated, as are mature luciferase mRNAs present in total and polyA+ RNA preparations. Primers used for RT-PCR are indicated as arrows. (B) Luciferase expression from the reporter plasmids described above. Plasmids were co-transfected with either pcDNA3 (stippled bars), or a EBNA1 DNA binding domain expression plasmid (black bars) into 293 cells. The number of EBNA1 binding sites in the reporter plasmid is indicated below each pair of bars. Luciferase activity is reported relative to the activity observed when pRSVL was co-transfected with pcDNA3. (C) RT-PCRs to detect luciferase and GAPDH transcripts in total or polyadenylated RNAs recovered from the transfected cells described in B. PCR products were visualized with ethidium bromide and the identity of the transfected plasmid is indicated above each lane.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654434&req=5

Figure 5: EBNA1 bound to FR impedes transcription fork progression. (A) Representation of transcription reporter plasmids used here. In pRSVL, the RSV LTR drives transcription of the luciferase gene, and the SV40 late polyadenylation signal is used for polyadenylation. Derivatives of pRSVL with ten, 20 or 40 EBNA1-binding sites (filled black circles) between the luciferase gene and the polyadenylation signal were constructed. Primary transcripts, and prematurely terminated transcripts present in total RNA preparations are indicated, as are mature luciferase mRNAs present in total and polyA+ RNA preparations. Primers used for RT-PCR are indicated as arrows. (B) Luciferase expression from the reporter plasmids described above. Plasmids were co-transfected with either pcDNA3 (stippled bars), or a EBNA1 DNA binding domain expression plasmid (black bars) into 293 cells. The number of EBNA1 binding sites in the reporter plasmid is indicated below each pair of bars. Luciferase activity is reported relative to the activity observed when pRSVL was co-transfected with pcDNA3. (C) RT-PCRs to detect luciferase and GAPDH transcripts in total or polyadenylated RNAs recovered from the transfected cells described in B. PCR products were visualized with ethidium bromide and the identity of the transfected plasmid is indicated above each lane.
Mentions: The transcription reporter plasmid pRSVL [33] was modified to introduce ten, 20, or 40 contiguous EBNA1 binding sites between the end of the luciferase open reading frame and the SV40 polyadenylation sequence in that plasmid. The structure of these reporter plasmids is shown in Figure 5A. These reporter plasmids were then co-transfected into 293 cells with a control expression plasmid (pcDNA3), or plasmid 1160 that expresses the DNA binding domain of EBNA1 (DBD). Cells were harvested two days post-transfection, FACS profiled to normalize for live transfected cells, following which luciferase levels were measured. This analysis is shown in Figure 5B. In the absence of EBNA1 binding sites on the reporter plasmid, the co-transfected DBD expression plasmid had no effect on luciferase expression. Similarly, pcDNA3 had no effect on luciferase expression from reporter plasmids that contained ten, 20 or 40 EBNA1 binding sites. However when the luciferase reporter plasmids had 20 or 40 EBNA1 binding sites, and were co-transfected with the DBD expression plasmid, there was a sharp decrease in the expression of luciferase dependent upon the number of number binding sites placed 5' to the polyadenylation signal.

Bottom Line: We conclude that EBNA1 bound to FR regulates the replication of oriP-plasmids by impeding the migration of replication forks.Upon binding FR, EBNA1 also blocks the migration of transcription forks.Thus, in addition to regulating oriP replication, EBNA1 bound to FR also decreases the probability of detrimental collisions between two opposing replication forks, or between a transcription fork and a replication fork.

View Article: PubMed Central - HTML - PubMed

Affiliation: Stanley S, Scott Cancer Center, LSU Health Sciences Center, New Orleans, LA 70112, USA. aaiyar@lsuhsc.edu

ABSTRACT

Background: Epstein-Barr virus is replicated once per cell-cycle, and partitioned equally in latently infected cells. Both these processes require a single viral cis-element, termed oriP, and a single viral protein, EBNA1. EBNA1 binds two clusters of binding sites in oriP, termed the dyad symmetry element (DS) and the family of repeats (FR), which function as a replication element and partitioning element respectively. Wild-type FR contains 20 binding sites for EBNA1.

Results: We, and others, have determined previously that decreasing the number of EBNA1-binding sites in FR increases the efficiency with which oriP-plasmids are replicated. Here we demonstrate that the wild-type number of binding sites in FR impedes the migration of replication and transcription forks. Further, splitting FR into two widely separated sets of ten binding sites causes a ten-fold increase in the efficiency with which oriP-plasmids are established in cells expressing EBNA1. We have also determined that EBNA1 bound to FR impairs the migration of transcription forks in a manner dependent on the number of EBNA1-binding sites in FR.

Conclusion: We conclude that EBNA1 bound to FR regulates the replication of oriP-plasmids by impeding the migration of replication forks. Upon binding FR, EBNA1 also blocks the migration of transcription forks. Thus, in addition to regulating oriP replication, EBNA1 bound to FR also decreases the probability of detrimental collisions between two opposing replication forks, or between a transcription fork and a replication fork.

Show MeSH
Related in: MedlinePlus