Analysis of DNA topology of EBV minichromosomes in HEK 293 cells

Abstract

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.

Replication Fork Barriers and Topological Barriers: Progression of DNA Replication Relies on DNA Topology Ahead of Forks

“…Thus, the replication of EBV minichromosomes proceeds in a predominantly unidirectional manner. [ 52,55 ]…”

Closing the DNA replication cycle: from simple circular molecules to supercoiled and knotted DNA catenanes

“…In Figure 4, we show topological difficulties encountered during replication of small circular DNA molecules in bacterial cells. Nuclear DNA in eukaryotic cells, including infecting viral DNA, does not need to be constantly maintained in negatively supercoiled form, contrary to what is the case of bacterial DNA (44,45). In eukaryotic chromosomes a transient, local pulse of negative supercoiling facilitating transcription initiation can be generated when needed by the ejection of individual histone octamers in the vicinity of promoters of genes that need to be activated (46).…”