Differential usage of non-homologous end-joining and homologous recombination in double strand break repair

Sonoda, Eiichiro; Hochegger, Helfrid; Saberi, Alihossein; Taniguchi, Yoshihito; Takeda, Shunichi

doi:10.1016/j.dnarep.2006.05.022

Cited by 455 publications

(356 citation statements)

References 62 publications

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“…HR is an error-free repair system, but it is limited to the late S and G2 phases because the replicated DNA strand is used as a template. 37 We used a GFP-based repair assay for HR repair in DR-GFP U2OS cells as described previously. 38 U2OS cells with a single integrated copy of the HR repair substrate were co-transfected with I-SceI and pcDNA or BZLF1 plasmids, followed by FACS analysis of GFP-positive events to assay for DSB repair by HR.…”

Section: Bzlf1 Impairs Dsb Repair and Activates Atmdependent Signalinmentioning

confidence: 99%

Epstein–Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage

Yang

Deng

Hau

et al. 2015

Laboratory Investigation

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Epstein-Barr virus (EBV) infection is closely associated with several human malignancies including nasopharyngeal carcinoma (NPC). The EBV immediate-early protein BZLF1 is the key mediator that switches EBV infection from latent to lytic forms. The lytic form of EBV infection has been implicated in human carcinogenesis but its molecular mechanisms remain unclear. BZLF1 has been shown to be a binding partner of several DNA damage response (DDR) proteins. Its functions in host DDR remain unknown. Thus, we explore the effects of BZLF1 on cellular response to DNA damage in NPC cells. We found that expression of BZLF1 impaired the binding between RNF8 and MDC1 (mediator of DNA damage checkpoint 1), which in turn interfered with the localization of RNF8 and 53BP1 to the DNA damage sites. The RNF8-53BP1 pathway is important for repair of DNA double-strand breaks and DNA damage-induced G2/M checkpoint activation. Our results showed that, by impairing DNA damage repair as well as abrogating G2/M checkpoint, BZLF1 induced genomic instability and rendered cells more sensitive to ionizing radiation. Moreover, the blockage of 53BP1 and RNF8 foci formation was recapitulated in EBV-infected cells. Taken together, our study raises the possibility that, by causing mis-localization of important DDR proteins, BZLF1 may function as a link between lytic EBV infection and impaired DNA damage repair, thus contributing to the carcinogenesis of EBV-associated human epithelial malignancies. Epstein-Barr virus (EBV) is a gamma herpesvirus and infection with EBV is associated with a variety of epithelial and B-cell cancers. 1,2 EBV has a biphasic life cycle consisting of latent and lytic stages. The switch from latent to lytic infection is triggered by the EBV immediate-early transcription factor, BZLF1 (ZEBRA, Zta, Z, EB1). 3,4 A number of reports have demonstrated that lytic EBV activation is intimately linked to the pathogenesis of EBV-induced malignancies including the development of NPC. [5][6][7][8] The mammalian DNA damage response (DDR) network has pivotal roles in maintaining genome stability and tumor suppression. [9][10][11][12][13] In the presence of double-strand break (DSBs), the protein complex composed of MRE11-RAD50-NBS1 (MRN) recruits and activates the ataxia-telangiectasiamutated (ATM) kinase at the vicinity of DSBs, 10 which leads to the phosphorylation of the histone variant H2AX (γH2AX). γH2AX decorates chromatin domains flanking DSBs, and is directly engaged by the mediator of DNA damage checkpoint 1 (MDC1). MDC1 loading onto the damaged chromatin subsequently permits the productive accumulation of a cohort of DNA damage signaling and mediator proteins. 14,15 In particular, the RING finger ubiquitin ligase RNF8 is targeted to MDC1 via its phospho-binding forkheadassociated domain, where it initiates a cascade of chromatin ubiquitination events important for tethering of DNA damage mediator and repair proteins, including 53BP1 and BRCA1, [16][17][18][19] the dysregulation of which compromises genome stability and cont...

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Section: Bzlf1 Impairs Dsb Repair and Activates Atmdependent Signalinmentioning

confidence: 99%

Epstein–Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage

Yang

Deng

Hau

et al. 2015

Laboratory Investigation

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“…DNA DSBs are repaired by either the NHEJ or HR pathways (13,27,31,65,68,76). Homologous recombination requires a sister chromatid, homolog, or homologous sequence on a heterolog to be used as a template for DNA DSB repair and thus requires more significant chromatin remodeling and greater DNA accessibility to facilitate DNA unwinding, strand invasion, and DNA replication-based repair mechanisms.…”

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confidence: 99%

MOF and Histone H4 Acetylation at Lysine 16 Are Critical for DNA Damage Response and Double-Strand Break Repair

Sharma

Gupta

et al. 2010

Molecular and Cellular Biology

284

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The human MOF gene encodes a protein that specifically acetylates histone H4 at lysine 16 (H4K16ac). Here we show that reduced levels of H4K16ac correlate with a defective DNA damage response (DDR) and double-strand break (DSB) repair to ionizing radiation (IR). The defect, however, is not due to altered expression of proteins involved in DDR. Abrogation of IR-induced DDR by MOF depletion is inhibited by blocking H4K16ac deacetylation. MOF was found to be associated with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a protein involved in nonhomologous end-joining (NHEJ) repair. ATM-dependent IR-induced phosphorylation of DNA-PKcs was also abrogated in MOF-depleted cells. Our data indicate that MOF depletion greatly decreased DNA double-strand break repair by both NHEJ and homologous recombination (HR). In addition, MOF activity was associated with general chromatin upon DNA damage and colocalized with the synaptonemal complex in male meiocytes. We propose that MOF, through H4K16ac (histone code), has a critical role at multiple stages in the cellular DNA damage response and DSB repair.In eukaryotes, specifically in mammals, the mechanisms by which the DNA damage response (DDR) components gain access to broken DNA in compacted chromatin remain a mystery. The DNA damage response occurs within the context of chromatin, and its structure is altered post-DNA double-strand break (DSB) induction. Major alterations include (i) chromatin remodeling via ATP-dependent activities and covalent histone modifications and (ii) incorporation of histone variants into nucleosomes. Chromatin structure creates a natural barrier to damaged DNA sites, which suggests that histone modifications will play a primary role in DDR by facilitating repair protein access to DNA breaks (43,58,87,88). While some experimental evidence indicates that preexisting histone modifications may play an important role in DDR, the precise role of chromatin status prior to DNA damage on DDR is yet to be clearly established. For instance, biochemical and cell biology studies indicate that repair proteins (53BP1, Schizosaccharomyces pombe Crb2 [SpCrb2], and Saccharomyces cerevisiae Rad9 [ScRad9]) require methylated Lys79 of histone H3 (H3-K79) (29) or methylated Lys20 of histone H4 (H4-K20) and/or CBP/p300-mediated acetylation of histone H3 on lysine 56 (9,15,29,66,93) for focus formation at DNA-damaged sites. These modifications are normally present on chromatin, and none has been reported to change in response to ionizing radiation (IR)-induced DNA damage. However, it is yet to be established whether preexisting acetylation of specific histone residues at the time of cellular exposure to IR plays any critical role in DDR. While recent studies demonstrate that in human cells, histone H3 acetylated at K9 (H3K9ac) and H3K56ac are rapidly and reversibly reduced in response to DNA damage, most histone acetylation modifications do not change appreciably after genotoxic stress (80).The amino-terminal tail of histone H4 is a well-described target fo...

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“…The repair of double strand DNA breaks (DSB) in mammalian cells occurs via the distinct mechanisms of homology directed repair (HDR) or nonhomologous end joining (NHEJ) (8).…”

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confidence: 99%

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

Santiago

Chan²,

Liu³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

330

230

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Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR ؊/؊ cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR ؊/؊ cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.genetic engineering ͉ zinc-finger proteins T he use of gene knockouts in basic research, functional genomics, and industrial cell line engineering is severely limited by an absence of methods for rapid targeting and disruption of an investigator-specified gene. Early approaches to somatic cell gene disruption used genome-wide nontargeted methods, including ionizing radiation and chemical-induced mutagenesis (1, 2) whereas more recent methods used targeted homologous recombination (HR) (3). However, the Ͼ1,000-fold lower frequency of the targeted HR event relative to random integration in most mammalian cell lines (beyond mouse ES cells) can necessitate screening thousands of clones and take several months to identify a biallelic targeted gene knockout. Strategies including positive and negative marker selection and promoter-trap can boost efficiencies considerably, although these approaches present their own technical challenges and are not always successful in achieving high efficiency targeting (4, 5). Although advances with adeno-associated viral delivery strategies continue to improve the efficiency of knockouts (6, 7), the frequency is still very low and the time required to achieve biallelic gene knockout remains a barrier to its routine adoption. Here, we present a general solution for rapid gene knockout in mammalian cells.The repair of double strand DNA breaks (DSB) in mammalian cells occurs via the distinct mechanisms of homol...

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Differential usage of non-homologous end-joining and homologous recombination in double strand break repair

Cited by 455 publications

References 62 publications

Epstein–Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage

Epstein–Barr virus BZLF1 protein impairs accumulation of host DNA damage proteins at damage sites in response to DNA damage

MOF and Histone H4 Acetylation at Lysine 16 Are Critical for DNA Damage Response and Double-Strand Break Repair

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

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