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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.

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Split FRs distinguish between the replication factor titration and replication fork barrier models. (A) Schematic representation of the oriP region from plasmids designed to distinguish between the replication factor titration and the replication fork barrier models. The identity of the plasmid is indicated to the left of each schematic. DS is represented as a striped oval, and the EBNA1-binding sites in FR as filled black circles. The number of EBNA1 binding sites within each FR is indicated above each FR. FRs are separated from each other (plasmid AGP213) or from DS (plasmid AGP212) by the EBV sequences normally present between FR and DS. (B) Stable replication of oriP replication reporters under selection in 293/EBNA1 cells. 293/EBNA1 cells were transfected with the indicated plasmid, placed under puromycin selection for 18 days, at which time replicated DpnI-resistant episomal DNAs were recovered and quantified as described in the Methods. "M" indicates the migration position of standards used for quantitation, and the amounts of standards loaded are indicated above each lane. The identity of the transfected plasmid is indicated above each lane. "A" indicates the migration position of DpnI-resistant, linearized plasmid DNAs.
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Figure 3: Split FRs distinguish between the replication factor titration and replication fork barrier models. (A) Schematic representation of the oriP region from plasmids designed to distinguish between the replication factor titration and the replication fork barrier models. The identity of the plasmid is indicated to the left of each schematic. DS is represented as a striped oval, and the EBNA1-binding sites in FR as filled black circles. The number of EBNA1 binding sites within each FR is indicated above each FR. FRs are separated from each other (plasmid AGP213) or from DS (plasmid AGP212) by the EBV sequences normally present between FR and DS. (B) Stable replication of oriP replication reporters under selection in 293/EBNA1 cells. 293/EBNA1 cells were transfected with the indicated plasmid, placed under puromycin selection for 18 days, at which time replicated DpnI-resistant episomal DNAs were recovered and quantified as described in the Methods. "M" indicates the migration position of standards used for quantitation, and the amounts of standards loaded are indicated above each lane. The identity of the transfected plasmid is indicated above each lane. "A" indicates the migration position of DpnI-resistant, linearized plasmid DNAs.

Mentions: To test the models presented in Figure 2, two additional replication reporter plasmids illustrated in Figure 3A were constructed. In the first, AGP212, two FRs with ten binding sites each were placed on either side of DS, and separated from DS by the EBV sequences normally present between FR and DS. In the second, AGP 213, two FRs with ten binding sites each were placed in tandem, but separated from each other by the EBV sequences normally present between FR and DS. AGP212 and AGP213 were transfected into 293/EBNA1 cells and their ability to form puromycin resistant colonies was evaluated (Table 2). As indicated in the table, both plasmids containing 20 EBNA1-binding sites split into two sets of ten binding sites form puromycin resistant colonies far more efficiently than a plasmid containing 20 contiguous EBNA1 binding sites in FR, or a plasmid that contains wild-type FR (p-value < 0.05 by the Wilcoxon rank-sum test). Not only do AGP212 and AGP213 form colonies more efficiently than AGP74, they also give rise to puromycin-resistant colonies more efficiently than AGP73 that contains a single block of ten EBNA1-binding sites. This result favors the "replication fork barrier" model over the "replication factor titration" model.


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)

Split FRs distinguish between the replication factor titration and replication fork barrier models. (A) Schematic representation of the oriP region from plasmids designed to distinguish between the replication factor titration and the replication fork barrier models. The identity of the plasmid is indicated to the left of each schematic. DS is represented as a striped oval, and the EBNA1-binding sites in FR as filled black circles. The number of EBNA1 binding sites within each FR is indicated above each FR. FRs are separated from each other (plasmid AGP213) or from DS (plasmid AGP212) by the EBV sequences normally present between FR and DS. (B) Stable replication of oriP replication reporters under selection in 293/EBNA1 cells. 293/EBNA1 cells were transfected with the indicated plasmid, placed under puromycin selection for 18 days, at which time replicated DpnI-resistant episomal DNAs were recovered and quantified as described in the Methods. "M" indicates the migration position of standards used for quantitation, and the amounts of standards loaded are indicated above each lane. The identity of the transfected plasmid is indicated above each lane. "A" indicates the migration position of DpnI-resistant, linearized plasmid DNAs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Split FRs distinguish between the replication factor titration and replication fork barrier models. (A) Schematic representation of the oriP region from plasmids designed to distinguish between the replication factor titration and the replication fork barrier models. The identity of the plasmid is indicated to the left of each schematic. DS is represented as a striped oval, and the EBNA1-binding sites in FR as filled black circles. The number of EBNA1 binding sites within each FR is indicated above each FR. FRs are separated from each other (plasmid AGP213) or from DS (plasmid AGP212) by the EBV sequences normally present between FR and DS. (B) Stable replication of oriP replication reporters under selection in 293/EBNA1 cells. 293/EBNA1 cells were transfected with the indicated plasmid, placed under puromycin selection for 18 days, at which time replicated DpnI-resistant episomal DNAs were recovered and quantified as described in the Methods. "M" indicates the migration position of standards used for quantitation, and the amounts of standards loaded are indicated above each lane. The identity of the transfected plasmid is indicated above each lane. "A" indicates the migration position of DpnI-resistant, linearized plasmid DNAs.
Mentions: To test the models presented in Figure 2, two additional replication reporter plasmids illustrated in Figure 3A were constructed. In the first, AGP212, two FRs with ten binding sites each were placed on either side of DS, and separated from DS by the EBV sequences normally present between FR and DS. In the second, AGP 213, two FRs with ten binding sites each were placed in tandem, but separated from each other by the EBV sequences normally present between FR and DS. AGP212 and AGP213 were transfected into 293/EBNA1 cells and their ability to form puromycin resistant colonies was evaluated (Table 2). As indicated in the table, both plasmids containing 20 EBNA1-binding sites split into two sets of ten binding sites form puromycin resistant colonies far more efficiently than a plasmid containing 20 contiguous EBNA1 binding sites in FR, or a plasmid that contains wild-type FR (p-value < 0.05 by the Wilcoxon rank-sum test). Not only do AGP212 and AGP213 form colonies more efficiently than AGP74, they also give rise to puromycin-resistant colonies more efficiently than AGP73 that contains a single block of ten EBNA1-binding sites. This result favors the "replication fork barrier" model over the "replication factor titration" model.

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