Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Abstract

Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-Replication protein A (RPA), the eukaryotic singlestranded DNA (ssDNA)-binding protein, is a heterotrimeric complex composed of three tightly associated subunits of 70, 32, and 14 kDa (referred as to RPA70, RPA32, and RPA14, respectively) that is essential for DNA replication, recombination, and all major types of DNA repair (4). RPA participates in such diverse pathways through its ability to interact with DNA and numerous proteins involved in its processing. During DNA replication, RPA associates with ssDNA at forks and facilitates nascent-strand DNA synthesis by replicative DNA polymerases localized at replication foci during S phase. Under DNA-damaging conditions, RPA binds to ssDNA at damaged sites and interacts with repair and recombination components to process double-strand DNA breaks (DSBs) and other lesions (6,14,21,32,38,41).RPA undergoes both DNA damage-independent and -dependent phosphorylation on the N-terminal 33 residues of RPA32. Unstressed cell cycle-dependent phosphorylation occurs during the G 1 /S-phase transition and in M phase, primarily at the conserved cyclin-CDK phosphorylation sites of Ser-23 and Ser-29 in the N terminus of the RPA32 subunit (13,15). In contrast, stress-induced hyperphosphorylation of RPA is much more extensive. Nine potential phosphorylation sites within the N-terminal domain of RPA32, Ser-4, Ser-8, Ser-11/Ser-12/Ser-13, Thr-21, Ser-23, Ser-29, and Ser-33, in response to DNAdamaging agents, have been suggested (33,54). Although this region of RPA32 is not required for the ssDNA-binding activity of RPA (5, 22), a phosphorylation-induced subtle conformation change in RPA, resulting from altered intersubunit interactions, regulates the interaction of RPA with both interacting proteins and DNA (30). The hyperphosphorylated form of RPA32 is unable to localize to replication centers in normal cells, while binding to DNA damage foci is unaffected (46). Therefore, RPA phosphorylation following damage is thought to both prevent RPA from catalyzing DNA replication and potentially serve as a marker to recruit repair factors to sites of DNA damage. RPA localizes to nuclear foci where DNA repair is occurring after DNA damage and is essential for multiple DNA repair pathways, participating in damage recognition, excision, and resynthesis reactions (4, 56).Mammalian cells can repair DSBs by homologous recombination (HR) or by nonhomologous end joining. HR is an accurate repair process, the first step of which is the resection of the 5Ј ends of the DSB to generate 3Ј ssDNA overhangs. This reaction is carried out by the Mre11/Rad50/Nbs1 (MRN) complex, which not only functions as a damage sensor upstream of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) activation but also plays a role in DSB repair (4).

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

“…We also previously noted that expression of an ATPase-defective version of Rad51 acts as a transdominant inhibitor of recombination between HSV-1 tsS and tsK mutant viruses, resulting in reduced yield of virus with a wild type genotype (47). Furthermore, it was recently observed that siRNA-mediated knockdown of Rad51 caused an approximate 5-fold reduction in Epstein-Barr virus lytic replication (48). Here, we find that replication of UV-damaged HSV-1 DNA is reduced 50 -150-fold by siRNA-mediated knockdown of Rad54, Rad52, and Rad51 proteins demonstrating a direct role in HSV-1 recombination repair.…”

Coronavirus Infection Induces DNA Replication Stress Partly through Interaction of Its Nonstructural Protein 13 with the p125 Subunit of DNA Polymerase δ

“…As activation of DNA damage response can promote apoptosis, viruses have also evolved ways to tailor the damage signaling by targeting a specific sensor or a repair protein for degradation or mislocalization to suppress the downstream signaling. For example, Epstein-Barr virus lytic replication induces ATM-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11-Rad50-Nbs1 (MRN) complexes as well as homologous recombinational repair (HRR) factors such as RPA, Rad51, and Rad52 to sites of viral genome synthesis in the nucleus (72,73). The nuclear antigen 3C (EBNA3C) of Epstein-Barr virus could release G 2 /M cell cycle checkpoint by direct interaction with Chk2 (74).…”

Replication of Epstein-Barr Viral DNA

“…Lytic DNA replication involves both replication and homologous recombination of DNA, which are two interdependent processes active during the lytic phase of EBV's life cycle (Pfuller and Hammerschmidt 1996). Several cellular recombination and DNA repair factors have been recently found recruited to EBV's replication compartments (Daikoku et al 2006;Kudoh et al 2009;Sugimoto et al 2011) and induction of EBV's lytic phase induces a genuine DNA-damage response signal Sato et al 2010). It now seems that oriLyt is complex not only in mediating mechanistically distinct biphasic DNA replication but also in using a complex repertoire of viral and cellular proteins to carry out these two modes of replication.…”