Regulation of Epstein-Barr Virus Origin of Plasmid Replication (OriP) by the S-Phase Checkpoint Kinase Chk2

Abstract

The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G 1 /S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.Epstein-Barr virus (EBV) is a human herpesvirus that can persist as a multicopy episome in latently infected B lymphocytes (16,30). During latent infection, the viral genomes replicate once per cell cycle and utilize the cellular machinery required for chromosomal replication (6,10,22,31,32,41,43). One virally encoded protein, EBNA1, is required for OriPdependent DNA replication and plasmid maintenance (21, 44; reviewed in references 22 and 38). EBNA1 binds to a family of repeats (FR) and a dyad symmetry element (DS) within OriP (29). The FR confers plasmid maintenance and enhances replication initiation from the DS. The DS consists of two pairs of EBNA1 binding sites flanked by telomere repeat factor (TRF) binding sites (2,9,19,42). The DS has been shown to function similarly to cellular origins that replicate once per cell cycle and assemble a prereplication complex that includes the cellular origin recognition complex (ORC) and minichromosome maintenance (MCM) subunits (6,10,31,32).ORC recruitment to the DS is thought to play a key role in determining its function as an origin of DNA replication (3,14). TRF2, along with EBNA1, was shown previously to interact with ORC subunits and to recruit the ORC to the DS (1, 23, 27, 39). The ORC interaction domains in both TRF2 and EBNA1 appear to share an amino acid composition of basic residues, often referred to as arginine-glycine-glycine (RGG) motifs (17). The basic stretches have been implicated in the intermolecular linking activity of EBNA1 and are variably termed linking region 1 (LR1) and LR2 (25). LR1 and LR2 have AT-hook DNA binding activity and confer a metaphase chromosome attachment function to EBNA1, which are t...

Ebna1

“…EBNA1 may also facilitate the recruitment of telomere repeat binding factor 2 (TRF2) to site in the DS (Deng et al 2002(Deng et al , 2003Moriyama et al 2012). TRF2 then appears to contribute to ORC recruitment to the DS in conjunction with EBNA1 (Julien et al 2004;Atanasiu et al 2006) and also affects the timing of replication in S phase through recruitment of additional proteins (Zhou et al 2009(Zhou et al , 2010.…”

TRF1 phosphorylation on T271 modulates telomerase-dependent telomere length maintenance as well as the formation of ALT-associated PML bodies

“…Among numerous potential hits, T271 (RTRT 217 ) is found to match the consensus phosphorylation site (RXXS/T) of Chk1 and a related kinase Chk2, which has been reported to phosphorylate sheltein protein TRF220. While Chk1 and Chk2 can phosphorylate T271 in vitro (K. Jeyanthan and X.-D.Zhu, unpublished data), depletion or inhibition of Chk1 or a related kinase Chk2 fails to affect T271 phosphorylation in vivo (J.R. Walker and X.-D. Zhu, unpublished data), arguing against the possibility that Chk1 and Chk2 are the kinases responsible for phosphorylating T271 in vivo .…”

Epstein-Barr Nuclear Antigen 1 (EBNA1)-dependent Recruitment of Origin Recognition Complex (Orc) on oriP of Epstein-Barr Virus with Purified Proteins

“…Besides four EBNA1 binding sites, DS carries three nonamer repeats, each resembling the telomere repeat unit, and they also contribute to replication and mitotic persistence of oriP-episome (19,22). One of the major telomere factors, TRF2 was reported to associate with DS in a manner dependent on the nonamers, and it modulates efficiency and timing of oriP-replication (23)(24)(25)(26)(27). TRF2 was actually present in an EB90mbp323-bound fraction only in the presence of DS (Fig.…”

“…Besides four EBNA1 binding sites, DS carries three nonamer repeats, each resembling the telomere repeat unit, and they also contribute to replication and mitotic persistence of oriP-episome (19,22). TRF2 (telomere repeat-binding factor 2) and several other telomere factors associate with DS in a manner dependent on the nonamers and modulate the efficiency and timing of oriP-replication (23)(24)(25)(26)(27). Because EBNA1 was reported to associate with Orc in cell extracts (13,25,28), it would be interesting to examine the possibility of in vitro sitespecific assembly of Orc and other pre-RC components on DNA bearing EBNA1 binding sites.…”