Analysis of DNA topology of EBV minichromosomes in HEK 293 cells.
Simian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2017-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC5706722?pdf=render |
| _version_ | 1818036132616601600 |
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| author | Alicia Castán Vanessa Fernández-Calleja Pablo Hernández Dora B Krimer Jorge B Schvartzman María-José Fernández-Nestosa |
| author_facet | Alicia Castán Vanessa Fernández-Calleja Pablo Hernández Dora B Krimer Jorge B Schvartzman María-José Fernández-Nestosa |
| author_sort | Alicia Castán |
| collection | DOAJ |
| description | Simian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen for SV40 and the Epstein-Barr Nuclear Antigen 1 (EBNA-1) for EBV. Despite the abundant information available on the genetics and biochemistry of the replication process in these systems, little is known about the changes in DNA topology that take place as molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. Here we used high-resolution two-dimensional agarose gel electrophoresis to demonstrate that in Human Embryonic Kidney (HEK) 293 cells, minichromosomes of almost the same mass carrying either the SV40 or the EBV replication origin showed similar topological features. The patterns were very similar regardless of the initiator proteins. We also showed that in a hybrid minichromosome, pEco3'Δ, that initiates replication from the SV40 origin, the presence of EBNA-1 and its putative binding to the EBV "family of repeats" induces no significant topological change. These observations challenge the idea that binding of EBNA-1 to oriP could induce negative supercoiling and favor a model suggesting that it binds to oriP in a two-step process where only the second step causes structural changes in a transient cell cycle specific manner. |
| first_indexed | 2024-12-10T07:06:05Z |
| format | Article |
| id | doaj.art-63507214f71b461a94390b87d4a593e7 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-10T07:06:05Z |
| publishDate | 2017-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-63507214f71b461a94390b87d4a593e72022-12-22T01:58:11ZengPublic Library of Science (PLoS)PLoS ONE1932-62032017-01-011211e018817210.1371/journal.pone.0188172Analysis of DNA topology of EBV minichromosomes in HEK 293 cells.Alicia CastánVanessa Fernández-CallejaPablo HernándezDora B KrimerJorge B SchvartzmanMaría-José Fernández-NestosaSimian Virus 40 (SV40) and Epstein-Barr Virus (EBV) are frequently used as model systems to study DNA replication. Their genomes are both circular duplex DNAs organized in a single replicon where replication initiates at a precise site upon binding of a specific protein: the large tumor (T) antigen for SV40 and the Epstein-Barr Nuclear Antigen 1 (EBNA-1) for EBV. Despite the abundant information available on the genetics and biochemistry of the replication process in these systems, little is known about the changes in DNA topology that take place as molecules are transfected into eukaryotic cells, assembled into chromatin and bind initiator proteins to start replication. Here we used high-resolution two-dimensional agarose gel electrophoresis to demonstrate that in Human Embryonic Kidney (HEK) 293 cells, minichromosomes of almost the same mass carrying either the SV40 or the EBV replication origin showed similar topological features. The patterns were very similar regardless of the initiator proteins. We also showed that in a hybrid minichromosome, pEco3'Δ, that initiates replication from the SV40 origin, the presence of EBNA-1 and its putative binding to the EBV "family of repeats" induces no significant topological change. These observations challenge the idea that binding of EBNA-1 to oriP could induce negative supercoiling and favor a model suggesting that it binds to oriP in a two-step process where only the second step causes structural changes in a transient cell cycle specific manner.http://europepmc.org/articles/PMC5706722?pdf=render |
| spellingShingle | Alicia Castán Vanessa Fernández-Calleja Pablo Hernández Dora B Krimer Jorge B Schvartzman María-José Fernández-Nestosa Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. PLoS ONE |
| title | Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. |
| title_full | Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. |
| title_fullStr | Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. |
| title_full_unstemmed | Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. |
| title_short | Analysis of DNA topology of EBV minichromosomes in HEK 293 cells. |
| title_sort | analysis of dna topology of ebv minichromosomes in hek 293 cells |
| url | http://europepmc.org/articles/PMC5706722?pdf=render |
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