Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments
- 1 July 2009
- journal article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 83 (13), 6641-6651
- https://doi.org/10.1128/jvi.00049-09
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.Keywords
This publication has 48 references indexed in Scilit:
- Replication blocking lesions present a unique substrate for homologous recombinationThe EMBO Journal, 2007
- Phosphorylation of MCM4 at Sites Inactivating DNA Helicase Activity of the MCM4-MCM6-MCM7 Complex during Epstein-Barr Virus Productive ReplicationJournal of Virology, 2006
- Regulation of Replication Protein A Functions in DNA Mismatch Repair by PhosphorylationPublished by Elsevier BV ,2006
- Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular EnvironmentJournal of Biological Chemistry, 2005
- The mammalian mismatch repair protein MSH2 is required for correct MRE11 and RAD51 relocalization and for efficient cell cycle arrest induced by ionizing radiation in G2 phaseOncogene, 2003
- Mre11 Protein Complex Prevents Double-Strand Break Accumulation during Chromosomal DNA ReplicationMolecular Cell, 2001
- Stability and Nuclear Distribution of Mammalian Replication Protein A Heterotrimeric ComplexExperimental Cell Research, 2000
- Role of Protein−Protein Interactions in the Function of Replication Protein A (RPA): RPA Modulates the Activity of DNA Polymerase α by Multiple MechanismsBiochemistry, 1997
- Inhibition of S-Phase Entry of Human Fibroblasts by an Antisense Oligomer against hCDC47Biochemical and Biophysical Research Communications, 1996
- Inhibition of DNA replication factor RPA by p53Nature, 1993