title: 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)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.
authors.affiliation: Division of Virology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kudoh
authors.forename: Satoko
authors.initials: S
authors.lastname: Iwahori
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Sato
authors.forename: Sanae
authors.initials: S
authors.lastname: Nakayama
authors.forename: Hiroki
authors.initials: H
authors.lastname: Isomura
authors.forename: Takayuki
authors.initials: T
authors.lastname: Murata
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Tsurumi
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D016679:Genome, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D000076228:MRE11 Homologue Protein; D010766:Phosphorylation; D051135:Rad51 Recombinase / Q000378:metabolism*; D051721:Rad52 DNA Repair and Recombination Protein / Q000378:metabolism; D011995:Recombination, Genetic; D051716:Replication Protein A / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C497194:RAD52 protein, human; D051721:Rad52 DNA Repair and Recombination Protein; D051716:Replication Protein A; C495153:RAD51 protein, human; D051135:Rad51 Recombinase; D000076228:MRE11 Homologue Protein