Sequences Adjacent to oriP Improve the Persistence of Epstein-Barr Virus-Based Episomes in B Cells

ABSTRACT Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spansoriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriPalone.

Variant Chromatin Structure of theoriP Region of Epstein-Barr Virus and Regulation of EBER1 Expression by Upstream Sequences andoriP

ABSTRACT Most of the Epstein-Barr virus genome in latently infected cells is in a standard nucleosomal structure, but the region encompassingoriP and the Epstein-Barr virus-encoded small RNA (EBER) genes shows a distinctive pattern when digested with micrococcal nuclease. This pattern corresponds to a previously mapped nuclear matrix attachment region. Although the EBER genes are adjacent to oriP, there is only a two- to fourfold effect oforiP on EBER expression. However, sequences containing a consensus ATF site upstream of EBER1 are important for EBER1 expression.

Nucleoskeleton and initiation of DNA replication in metazoan cells

To determine whether or not initiation sites for DNA replication in mammalian cells are defined by association with nuclear structure, attachments between the nucleoskeleton and the hamster DHFR gene initiation zone were examined. Nucleoskeletons were prepared by encapsulating cells in agarose and then extracting them with a nonionic detergent in a physiological buffer. The fraction of DNA that remained following endonuclease digestion was resistant to salt, sensitive to Sarkosyl, and essentially unchanged by glutaraldehyde crosslinking. Although newly replicated DNA was preferentially attached to the nucleoskeleton, no specific sequence was preferentially attached within a 65 kb locus containing the DHFR gene, two origins of bi-directional replication and at least one nuclear matrix attachment region. Instead, the entire region went from preferentially unattached to preferentially attached as cells progressed from G1 to late S-phase. Thus, initiation sites in mammalian chromosomes are not defined by attachments to the nucleoskeleton. To further assess the relationship between the nucleoskeleton and DNA replication, plasmid DNA containing the DHFR initiation region was replicated in a Xenopus egg extract. All of the DNA associated with the nucleoskeleton prior to S-phase without preference for a particular sequence and was released upon mitosis. However, about half of this DNA was trapped rather than bound to the nucleoskeleton. Thus, attachments to the nucleoskeleton can form in the absence of either DNA replication or transcription, but if they are required for replication, they are not maintained once replication is completed.