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      The HSV-1 Exonuclease, UL12, Stimulates Recombination by a Single Strand Annealing Mechanism

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          Abstract

          Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.

          Author Summary

          The repair of DNA damage is essential to maintain genomic stability. Cells have at least four distinct DNA repair pathways, and defects in any of them can lead to tumor formation and cancer progression. Herpes Simplex Virus-1 (HSV-1) manipulates components of the host DNA repair pathways. In this paper we showed that DNA repair by the single strand annealing (SSA) pathway was increased during HSV infection and that other pathways were inhibited. We also show that a viral nuclease in conjunction with either a viral or cellular single strand annealing protein can stimulate the SSA pathway. We suggest that viral DNA synthesis occurs via an SSAdependent mechanism that is reminiscent of that used by bacterial viruses such as λ. Interestingly, λ has evolved an SSA-mediated repair mechanism to exchange genetic information that has also been used to enhance gene targeting in bacteria. It is thus possible that HSV proteins could be similarly used as tools to stimulate gene targeting in human cells leading to more effective strategies for gene therapy. Furthermore, the diversity of HSV reported in human populations, combined with the high rate of genetic exchange during infection, suggests that SSA may play a role in viral evolution and pathogenesis.

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          The DNA damage response: ten years after.

          The DNA damage response (DDR), through the action of sensors, transducers, and effectors, orchestrates the appropriate repair of DNA damage and resolution of DNA replication problems, coordinating these processes with ongoing cellular physiology. In the past decade, we have witnessed an explosion in understanding of DNA damage sensing, signaling, and the complex interplay between protein phosphorylation and the ubiquitin pathway employed by the DDR network to execute the response to DNA damage. These findings have important implications for aging and cancer.
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            Human CtIP promotes DNA end resection.

            In the S and G2 phases of the cell cycle, DNA double-strand breaks (DSBs) are processed into single-stranded DNA, triggering ATR-dependent checkpoint signalling and DSB repair by homologous recombination. Previous work has implicated the MRE11 complex in such DSB-processing events. Here, we show that the human CtIP (RBBP8) protein confers resistance to DSB-inducing agents and is recruited to DSBs exclusively in the S and G2 cell-cycle phases. Moreover, we reveal that CtIP is required for DSB resection, and thereby for recruitment of replication protein A (RPA) and the protein kinase ATR to DSBs, and for the ensuing ATR activation. Furthermore, we establish that CtIP physically and functionally interacts with the MRE11 complex, and that both CtIP and MRE11 are required for efficient homologous recombination. Finally, we reveal that CtIP has sequence homology with Sae2, which is involved in MRE11-dependent DSB processing in yeast. These findings establish evolutionarily conserved roles for CtIP-like proteins in controlling DSB resection, checkpoint signalling and homologous recombination.
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              XRCC3 promotes homology-directed repair of DNA damage in mammalian cells.

              Homology-directed repair of DNA damage has recently emerged as a major mechanism for the maintenance of genomic integrity in mammalian cells. The highly conserved strand transferase, Rad51, is expected to be critical for this process. XRCC3 possesses a limited sequence similarity to Rad51 and interacts with it. Using a novel fluorescence-based assay, we demonstrate here that error-free homology-directed repair of DNA double-strand breaks is decreased 25-fold in an XRCC3-deficient hamster cell line and can be restored to wild-type levels through XRCC3 expression. These results establish that XRCC3-mediated homologous recombination can reverse DNA damage that would otherwise be mutagenic or lethal.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                August 2012
                August 2012
                9 August 2012
                : 8
                : 8
                : e1002862
                Affiliations
                [1 ]Molecular, Microbial and Structural Biology Department, University of Connecticut Health Center, Farmington, Connecticut, United States of America
                [2 ]Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America
                [3 ]Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, United States of America
                University of Glasgow, United Kingdom
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AJS KNM YK EAH JMS SKW. Performed the experiments: AJS KNM YK. Analyzed the data: AJS KNM SKW. Contributed reagents/materials/analysis tools: AJS YK EAH JMS. Wrote the paper: AJS SKW.

                Article
                PPATHOGENS-D-12-01159
                10.1371/journal.ppat.1002862
                3415443
                22912580
                a9596629-54a8-44b8-a36b-62a75011d3fc
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 11 May 2012
                : 1 July 2012
                Page count
                Pages: 10
                Funding
                This work was supported by Public Health Service grants AI069136 to SKW, GM088351 to EAH and CA120954 to JMS and Connecticut stem cell research grant 09-SCA-UCHC-34 to AJS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Biochemistry
                Microbiology
                Molecular Cell Biology

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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