Non-homologous end joining plays a key role in transgene concatemer formation in transgenic zebrafish embryos

This study focused on concatemer formation and integration pattern of transgenes in zebrafish embryos. A reporter plasmid based on enhanced green fluorescent protein (eGFP) driven by Cytomegalovirus (CMV) promoter, pCMV-pax6in-eGFP, was constructed to reflect transgene behavior in the host environment. After removal of the insertion fragment by double digestion with various combinations of restriction enzymes, linearized pCMV-pax6in-eGFP vectors were generated with different combinations of 5'-protruding, 3'-protruding, and blunt ends that were microinjected into zebrafish embryos. Repair of double-strand breaks (DSBs) was monitored by GFP expression following religation of the reporter gene. One-hundred-and-ninety-seven DNA fragments were amplified from GFP-positive embryos and sequenced to analyze the repair characteristics of different DSB end combinations. DSBs involving blunt and asymmetric protruding ends were repaired efficiently by direct ligation of blunt ends, ligation after blunting and fill-in, or removed by cutting. Repair of DSBs with symmetric 3'-3' protrusions was less efficient and utilized template-directed repair. The results suggest that non-homologous end joining (NHEJ) was the principal mechanism of exogenous gene concatemer formation and integration of transgenes into the genome of transgenic zebrafish.     

The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants

There are two general pathways by which multicellular eukaryotes repair double-strand DNA breaks (DSB): homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). All mammalian mutants in the NHEJ pathway demonstrate a lack of B and T lymphocytes and ionizing radiation sensitivity. Among these NHEJ mutants, the DNA-PK(cs) and Artemis mutants are the least severe, having no obvious phenotype other than the general defects described above. Ku mutants have an intermediate severity with accelerated senescence. The XRCC4 and DNA ligase IV mutants are the most severe, resulting in embryonic lethality. Here we show that the lethality of DNA ligase IV-deficiency in the mouse can be rescued when Ku86 is also absent. To explain the fact that simultaneous gene mutations in the NHEJ pathway can lead to viability when a single mutant is not viable, we propose a nuclease/ligase model. In this model, disrupted NHEJ is more severe if the Artemis:DNA-PK(cs) nuclease is present in the absence of a ligase, and Ku mutants are of intermediate severity, because the nuclease is less efficient. This model is also consistent with the order of severity in organismal phenotypes; consistent with chromosomal breakage observations reported here; and consistent with the NHEJ mutation identified in radiation sensitive human SCID patients.     

聚腺苷二磷酸-核糖聚合酶1与DNA双链断裂修复的相关性

DNA双链断裂(double strand breaks,DSBs)是细胞最严重的DNA损伤形式。细胞通过同源重组(homologous recombination,HR)和非同源末端连接(non-homologous end joining,NHEJ)途径修复DNA双链断裂损伤。聚腺苷二磷酸核糖基化(poly(ADP-ribosyl)ation,PARylation)是蛋白质翻译后修饰过程,这个过程由聚腺苷二磷酸 核糖聚合酶家族(poly(ADP-ribose)polymerases,PARPs)催化完成。PARP1作为PARPs家族最重要的成员,其在DNA损伤应答方面发挥重要作用。研究显示,PARP1在DSBs修复过程中发挥关键作用,参与DSBs的早期应答反应及其具体修复途径,可依据KU蛋白的存在与否发挥不同的特定作用。本文较全面地综述了PARP1在DNA双链断裂修复方面的潜在作用,将为临床疾病的诊治提供新的思路。     

组蛋白去乙酰化酶抑制剂对DNA双链断裂修复路径的作用

DNA双链断裂(double strand breaks, DSBs)对细胞生存是致命的.细胞内非同源末端连接(NHEJ)、重组修复(HDR)、单链退火修复(SSA)和微同源序列末端连接(MMEJ)等通路可竞争性修复DNA双链断裂损伤.在肿瘤细胞DNA中制造难以修复的基因损伤,诱导肿瘤细胞周期中止、坏死和凋亡是临床放、化疗的主要策略.组蛋白去乙酰化酶（histone deacetylase）作为抗肿瘤治疗的新靶标,其抑制剂(histonedeacetylase inhibitors, HDACi)可显著降低肿瘤细胞DSBs修复能力,增强肿瘤细胞的放、化疗敏感性.研究显示,HDACi抑制了肿瘤细胞中具有正确修复倾向的HDR和经典NHEJ通路,具有错误修复倾向的SSA和MMEJ路径也可能牵涉其中.目前,HDACi作用于DSBs修复通路的分子机制已取得较大进展,但仍有许多问题有待阐明.     

原核生物的NHEJ修复途径

DNA双链断裂(DSBs)是严重的DNA损伤形式之一,生物体对DSBs的修复可通过同源重组(HR)或非同源末端连接途径(NHEJ)进行。长期以来,人们普遍认为HR是细菌DSBs修复的惟一途径,但在分支杆菌和其它原核生物体内NHEJ途径的发现,使这一观念得以颠覆。最近的研究表明,细菌NHEJ修复系统是一个双组分系统,包含一个多功能的DNA连接酶(LigD)和DNA末端结合蛋白Ku,具有DSBs修复所需的断裂末段识别、末端加工和连接活性。重点综述细菌NHEJ修复系统的组成、结构以及生理功能。     

Non-homologous DNA end joining in the mature rat brain

Recent evidence suggests that DNA double strand breaks (DSBs) are introduced in neurons during the course of normal development, and that repair of such DSBs is essential for neuronal survival. Here we describe a non-homologous DNA end joining (NHEJ) system in the adult rat brain that may be used to repair DNA DSBs. In the brain NHEJ system, blunt DNA ends are joined with lower efficiency than cohesive or non-matching protruding ends. Moreover, brain NHEJ is blocked by DNA ligase inhibitors or by dATP and can occur in the presence or absence of exogenously added ATP. Comparison of NHEJ activities in several tissues showed that brain and testis share similar mechanisms for DNA end joining, whereas the activity in thymus seems to utilize different mechanisms than in the nervous system. The developmental profile of brain NHEJ showed increasing levels of activity after birth, peaking at postnatal day 12 and then gradually decreasing along with age. Brain distribution analysis in adult animals showed that NHEJ activity is differentially distributed among different regions. We suggest that the DNA NHEJ system may be utilized in the postnatal brain for the repair of DNA double strand breaks introduced within the genome in the postnatal brain.     

RNA干扰猪NHEJ通路修复因子对HR效率的影响

非同源末端连接(nonhomologous end joining, NHEJ)是动物基因组DNA双链断裂(double-strand break, DSB)修复的优选途径,通过与同源重组(homologous recombination, HR)竞争DSB靶点,进而抑制HR的效率。为提高HR效率,本研究针对猪NHEJ通路修复关键因子PNKP、LIG4和NHEJ1的编码序列,设计并合成相应的靶向小干扰RNA (small interfering RNA, siRNA),组成若干对RNAi (RNA interference)系统,将RNAi系统与报告质粒SSA-GFP reporter、HDR -GFP system和ssODN-GFP system共转染至猪胎儿成纤维细胞(porcine fetal fibroblasts, PFFs),检测敲低上述NHEJ关键修复因子后对HR的影响。RNAi结果显示,针对PNKP、LIG4和NHEJ1设计的siRNA均可显著敲低PNKP、LIG4和NHEJ1基因的表达(P<0.05)。选择干扰效果最好的siRNA与报告载体共转染PFFs,结果表明干扰PNKP基因表达后可显著提高单链退火(single strand annealing, SSA)修复效率、双链或单链DNA介导的同源重组定向修复(homology-directed repair, HDR)效率分别为55.7%、37.4%和73.1% (P<0.05),而干扰LIG4和NHEJ1分别提高双链和单链介导的HDR效率为37.5% 和 76.9% (P<0.05)。     

Cryo-EM of NHEJ supercomplexes provides insights into DNA repair

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Xrcc4 physically links DNA end processing by polynucleotide kinase to DNA ligation by DNA ligase IV

Nonhomologous end joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammalian cells. A critical step in this process is DNA ligation, involving the Xrcc4-DNA ligase IV complex. DNA end processing is often a prerequisite for ligation, but the coordination of these events is poorly understood. We show that polynucleotide kinase (PNK), with its ability to process ionizing radiation-induced 5'-OH and 3'-phosphate DNA termini, functions in NHEJ via an FHA-dependent interaction with CK2-phosphorylated Xrcc4. Analysis of the PNK FHA-Xrcc4 interaction revealed that the PNK FHA domain binds phosphopeptides with a unique selectivity among FHA domains. Disruption of the Xrcc4-PNK interaction in vivo is associated with increased radiosensitivity and slower repair kinetics of DSBs, in conjunction with a diminished efficiency of DNA end joining in vitro. Therefore, these results suggest a new role for Xrcc4 in the coordination of DNA end processing with DNA ligation.     

Dynamics of the Artemis and DNA-PKcs Complex in the Repair of Double-Strand Breaks

Pathologic chromosome breaks occur in human dividing cells ～10 times per day, and physiologic breaks occur in each lymphoid cell many additional times per day. Nonhomologous DNA end joining (NHEJ) is the major pathway for the repair of all of these double-strand breaks (DSBs) during most of the cell cycle. Nearly all broken DNA ends require trimming before they can be suitable for joining by ligation. Artemis is the major nuclease for this purpose. Artemis is tightly regulated by one of the largest protein kinases, which tethers Artemis to its surface. This kinase is called DNA-dependent protein kinase catalytic subunit (or DNA-PKcs) because it is only active when it encounters a broken DNA end. With this activation, DNA-PKcs permits the Artemis catalytic domain to enter a large cavity in the center of DNA-PKcs. Given this remarkably tight supervision of Artemis by DNA-PKcs, it is an appropriate time to ask what we know about the Artemis:DNA-PKcs complex, as we integrate recent structural information with the biochemistry of the complex and how this relates to other NHEJ proteins and to V(D)J recombination in the immune system.     

Cryo-EM structure of a DNA-PK trimer: higher order oligomerisation in NHEJ

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Role for Artemis nuclease in the repair of radiation-induced DNA double strand breaks by alternative end joining

Exposure of cells to ionizing radiation or radiomimetic drugs generates DNA double-strand breaks that are processed either by homologous recombination repair (HRR), or by canonical, DNA-PKcs-dependent non-homologous end-joining (C-NHEJ). Chemical or genetic inactivation of factors involved in C-NHEJ or HRR, but also their local failure in repair proficient cells, promotes an alternative, error-prone end-joining pathway that serves as backup (A-EJ). There is evidence for the involvement of Artemis endonuclease, a protein deficient in a human radiosensitivity syndrome associated with severe immunodeficiency (RS-SCID), in the processing of subsets of DSBs by HRR or C-NHEJ. It is thought that within HRR or C-NHEJ Artemis processes DNA termini at complex DSBs. Whether Artemis has a role in A-EJ remains unknown. Here, we analyze using pulsed-field gel electrophoresis (PFGE) and specialized reporter assays, DSB repair in wild-type pre-B NALM-6 lymphocytes, as well as in their Artemis^−/−, DNA ligase 4^−/− (LIG4^−/−), and LIG4^−/−/Artemis^−/− double mutant counterparts, under conditions allowing evaluation of A-EJ. Our results substantiate the suggested roles of Artemis in C-NHEJ and HRR, but also demonstrate a role for the protein in A-EJ that is confirmed in Artemis deficient normal human fibroblasts. We conclude that Artemis is a nuclease participating in DSB repair by all major repair pathways.     

Contrasting effects of Krüppel‐like factor 4 on X‐ray‐induced double‐strand and single‐strand DNA breaks in mouse astrocytes

Astrocytes, the most common cell type in the brain, play a principal role in the repair of damaged brain tissues during external radiotherapy of brain tumours. As a downstream gene of p53, the effects of Krüppel‐like factor 4 (KLF4) in response to X‐ray‐induced DNA damage in astrocytes are unclear. In the present study, KLF4 expression was upregulated after the exposure of astrocytes isolated from the murine brain. Inhibition of KLF4 expression using lentiviral transduction produced less double‐strand DNA breaks (DSB) determined by a neutral comet assay and flow cytometric analysis of phosphorylated histone family 2A variant and more single‐strand DNA breaks (SSB) determined by a basic comet assay when the astrocytes were exposed to 4 Gy of X‐ray radiation. These data suggest that radiation exposure of the tissues around brain tumour during radiation therapy causes KLF4 overexpression in astrocytes, which induces more DSB and reduces SSB. This causes the adverse effects of radiation therapy in the treatment of brain tumours. Copyright © 2013 John Wiley & Sons, Ltd.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

ATM and Artemis promote homologous recombination of radiation‐induced DNA double‐strand breaks in G2

Homologous recombination (HR) and non‐homologous end joining (NHEJ) represent distinct pathways for repairing DNA double‐strand breaks (DSBs). Previous work implicated Artemis and ATM in an NHEJ‐dependent process, which repairs a defined subset of radiation‐induced DSBs in G1‐phase. Here, we show that in G2, as in G1, NHEJ represents the major DSB‐repair pathway whereas HR is only essential for repair of ～15% of X‐ or γ‐ray‐induced DSBs. In addition to requiring the known HR proteins, Brca2, Rad51 and Rad54, repair of radiation‐induced DSBs by HR in G2 also involves Artemis and ATM suggesting that they promote NHEJ during G1 but HR during G2. The dependency for ATM for repair is relieved by depleting KAP‐1, providing evidence that HR in G2 repairs heterochromatin‐associated DSBs. Although not core HR proteins, ATM and Artemis are required for efficient formation of single‐stranded DNA and Rad51 foci at radiation‐induced DSBs in G2 with Artemis function requiring its endonuclease activity. We suggest that Artemis endonuclease removes lesions or secondary structures, which inhibit end resection and preclude the completion of HR or NHEJ.     

Genistein-induced DNA damage is repaired by nonhomologous end joining and homologous recombination in TK6 cells

Genistein (GES), a phytoestrogen, has potential chemopreventive and chemotherapeutic effects on cancer. The anticancer mechanism of GES may be related with topoisomerase II associated DNA double-strand breaks (DSBs). However, the precise molecular mechanism remains elusive. Here, we performed genetic analyses using human lymphoblastoid TK6 cell lines to investigate whether non-homologous DNA end joining (NHEJ) and homologous recombination (HR), the two major repair pathways of DSBs, were involved in repairing GES-induced DNA damage. Our results showed that GES induced DSBs in TK6 cells. Cells lacking Ligase4, an NHEJ enzyme, are hypersensitive to GES. Furthermore, the sensitivity of Ligase4^−/− cells was associated with enhanced DNA damage when comparing the accumulation of γ-H2AX foci and number of chromosomal aberrations (CAs) with WT cells. In addition, cells lacking Rad54, a HR enzyme, also presented hypersensitivity and increased DNA damages in response to GES. Meanwhile, Treatment of GES-lacking enhanced the accumulation of Rad51, an HR factor, in TK6 cells, especially in Ligase4^−/−. These results provided direct evidence that GES induced DSBs in TK6 cells and clarified that both NHEJ and HR were involved in the repair of GES-induced DNA damage, suggesting that GES in combination with inhibition of NHEJ or HR would provide a potential anticancer strategy.     

Distinct DNA repair pathways involving RecA and nonhomologous end joining in Mycobacterium smegmatis

Mycobacterium smegmatis was used to study the relationship between DNA repair processes involving RecA and nonhomologous end joining (NHEJ). The effect of gene deletions in recA and/or in two genes involved in NHEJ (ku and ligD) was tested on the ability of bacteria to join breaks in plasmids transformed into them and in their response to chemicals that damage DNA. The results provide in vivo evidence that only NHEJ is required for the repair of noncompatible DNA ends. By contrast, the response of mycobacteria to mitomycin C preferentially involved a RecA-dependent pathway.     

DNA双链断裂损伤修复系统研究进展

多种内源或外源因素都能造成细胞基因组DNA损伤,细胞内建立了复杂的修复系统来应对不同形式的损伤。其中DNA双链断裂(DNA double-strand breaks,DSBs)作为最严重的损伤形式,主要激活同源重组修复(Homologous recombination repair)和非同源末端连接(Non-homologous end joining)通路。这两条通路都是由多个修复元件参与、经过多步反应的复杂过程。两者各具特点、协同作用,共同维护细胞基因组的稳定性。对其分子机制的阐明为肿瘤放化疗的辅助治疗提供了潜在的作用靶点。