Baculovirus F-Box Protein LEF-7 Modifies the Host DNA Damage Response To Enhance Virus Multiplication

The DNA damage response (DDR) of a host organism represents an effective antiviral defense that is frequently manipulated and exploited by viruses to promote multiplication. We report here that the large DNA baculoviruses, which require host DDR activation for optimal replication, encode a conserved replication factor, LEF-7, that manipulates the DDR via a novel mechanism. LEF-7 suppresses DDR-induced accumulation of phosphorylated host histone variant H2AX (γ-H2AX), a critical regulator of the DDR. LEF-7 was necessary and sufficient to block γ-H2AX accumulation caused by baculovirus infection or DNA damage induced by means of pharmacological agents. Deletion of LEF-7 from the baculovirus genome allowed γ-H2AX accumulation during virus DNA synthesis and impaired both very late viral gene expression and production of infectious progeny. Thus, LEF-7 is essential for efficient baculovirus replication. We determined that LEF-7 is a nuclear F-box protein that interacts with host S-phase kinase-associated protein 1 (SKP1), suggesting that LEF-7 acts as a substrate recognition component of SKP1/Cullin/F-box (SCF) complexes for targeted protein polyubiquitination. Site-directed mutagenesis demonstrated that LEF-7''s N-terminal F-box is necessary for γ-H2AX repression and Autographa californica multiple nucleopolyhedrovirus (AcMNPV) replication events. We concluded that LEF-7 expedites virus replication most likely by selective manipulation of one or more host factors regulating the DDR, including γ-H2AX. Thus, our findings indicate that baculoviruses utilize a unique strategy among viruses for hijacking the host DDR by using a newly recognized F-box protein.     

病毒感染与宿主细胞DNA损伤应答

病毒感染宿主细胞后,利用细胞内的营养物质和原料进行复制和增殖,同时能引起宿主细胞启动抗病毒免疫应答的防御机制。此外,近年来的研究还表明病毒感染能够引起宿主细胞的DNA损伤应答,该反应是细胞另一种防止病毒入侵的自我保护机制。同时发现,病毒在长期进化过程中形成了不同的机制来对抗宿主细胞的DNA损伤应答,从而消除细胞对其复制和繁殖产生的不利影响。因此,研究和阐述病毒感染后引起宿主细胞DNA损伤应答途径的机制,可使我们采取相应对策选择新的抗病毒靶点,从而有利于新型抗病毒药物的开发。     

Adeno-Associated Virus Replication Induces a DNA Damage Response Coordinated by DNA-Dependent Protein Kinase

The parvovirus adeno-associated virus (AAV) contains a small single-stranded DNA genome with inverted terminal repeats that form hairpin structures. In order to propagate, AAV relies on the cellular replication machinery together with functions supplied by coinfecting helper viruses such as adenovirus (Ad). Here, we examined the host cell response to AAV replication in the context of Ad or Ad helper proteins. We show that AAV and Ad coinfection activates a DNA damage response (DDR) that is distinct from that seen during Ad or AAV infection alone. The DDR was also triggered when AAV replicated in the presence of minimal Ad helper proteins. We detected autophosphorylation of the kinases ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and signaling to downstream targets SMC1, Chk1, Chk2, H2AX, and XRCC4 and multiple sites on RPA32. The Mre11 complex was not required for activation of the DDR to AAV infection. Additionally, we found that DNA-PKcs was the primary mediator of damage signaling in response to AAV replication. Immunofluorescence revealed that some activated damage proteins were found in a pan-nuclear pattern (phosphorylated ATM, SMC1, and H2AX), while others such as DNA-PK components (DNA-PKcs, Ku70, and Ku86) and RPA32 accumulated at AAV replication centers. Although expression of the large viral Rep proteins contributed to some damage signaling, we observed that the full response required replication of the AAV genome. Our results demonstrate that AAV replication in the presence of Ad helper functions elicits a unique damage response controlled by DNA-PK.Replication of viral genomes produces a large amount of extrachromosomal DNA that may be recognized by the cellular DNA damage machinery. This is often accompanied by activation of DNA damage response (DDR) signaling pathways and recruitment of cellular repair proteins to sites of viral replication. Viruses therefore provide good model systems to study the recognition and response to DNA damage (reviewed in reference 48). The Mre11/Rad50/Nbs1 (MRN) complex functions as a sensor of chromosomal DNA double-strand breaks (DSBs) and is involved in activation of damage signaling (reviewed in reference 41). The MRN complex also localizes to DNA DSBs and is found at viral replication compartments during infection with a number of DNA viruses (6, 40, 47, 70, 75, 77, 87, 93). The phosphatidylinositol 3-kinase-like kinases (PIKKs) ataxia telangiectasia mutated (ATM), ATM and Rad3-related kinase (ATR), and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) are involved in the signal transduction cascades activated by DNA damage (reviewed in references 43, 51, and 71). These kinases respond to distinct types of damage and regulate DSB repair during different phases of the cell cycle (5), either through nonhomologous end-joining (NHEJ) or homologous recombination pathways (reviewed in references 63, 81, and 86). The DNA-PK holoenzyme is composed of DNA-PKcs and two regulatory subunits, the Ku70 and Ku86 heterodimer. DNA-PK functions with XRCC4/DNA ligase IV to repair breaks during NHEJ, and works with Artemis to process DNA hairpin structures during VDJ recombination and during a subset of DNA DSB events (46, 50, 86). While the kinase activity of DNA-PKcs leads to phosphorylation of a large number of substrates in vitro as well as autophosphorylation of specific residues (reviewed in references 16 and 85), it is currently unclear how DNA-PKcs contributes to signaling in cells upon different types of damage.The adeno-associated virus (AAV) genome consists of a molecule of single-stranded DNA with inverted terminal repeats (ITRs) at both ends that form double-hairpin structures due to their palindromic sequences (reviewed in reference 52). The ITRs are important for replication and packaging of the viral genome and for integration into the host genome. Four viral Rep proteins (Rep78, Rep68, Rep52, and Rep40) are also required for replication and packaging of the AAV genome into virions assembled from the Cap proteins. Although the Rep and Cap genes are replaced in recombinant AAV vectors (rAAV) that retain only the ITRs flanking the gene of interest, these vectors can be replicated by providing Rep in trans (reviewed in reference 7). Productive AAV infection requires helper functions supplied by adenovirus (Ad) or other viruses such as herpes simplex virus (HSV) (reviewed in reference 27), together with components of the host cell DNA replication machinery (54, 55, 58). In the presence of helper viruses or minimal helper proteins from Ad or HSV, AAV replicates in the nucleus at centers where the viral DNA and Rep proteins accumulate (35, 76, 84, 89). Cellular and viral proteins involved in AAV replication, including replication protein A (RPA), Ad DNA-binding protein (DBP), and HSV ICP8, localize with Rep proteins at these viral centers (29, 33, 76).A number of published reports suggest associations between AAV and the cellular DNA damage machinery. For example, transduction by rAAV vectors is increased by genotoxic agents and DNA damaging treatments (1, 62, 91) although the mechanisms involved remain unclear. Additionally, the ATM kinase negatively regulates rAAV transduction (64, 92), and we have shown that the MRN complex poses a barrier to both rAAV transduction and wild-type AAV replication (11, 67). UV-inactivated AAV particles also appear to activate a DDR involving ATM and ATR kinases that perturbs cell cycle progression (39, 60, 88). It has been suggested that this response is provoked by the AAV ITRs (60) and that UV-treated particles mimic stalled replication forks in infected cells (39). In addition to AAV genome components, the viral Rep proteins have been observed to exhibit cytotoxicity and induce S-phase arrest (3, 65).The role of cellular repair proteins in AAV genome processing has also been explored by examining the molecular fate of rAAV vectors, which are converted into circular and concatemeric forms that persist episomally (18, 19, 66). Proteins shown to regulate circularization in cell culture include ATM and the MRN complex (14, 64), while in vivo experiments using mouse models have implicated ATM and DNA-PK in this process (14, 20, 72). Additionally, DNA-PKcs and Artemis have recently been shown to cleave the ITR hairpins of rAAV vectors in vivo in a tissue-dependent manner (36). Despite these studies, it is not clear how damage response factors function together and how they impact AAV transduction and replication in human cells.In this study we examined the cellular response to AAV replication in the context of Ad infection or helper proteins. We show that coinfection with AAV and Ad activates a DDR that is distinct from that seen during infection with Ad alone. The ATM and DNA-PKcs damage kinases are activated and signal to downstream substrates, but the response does not require the MRN complex and is primarily mediated by DNA-PKcs. Although expression of the large Rep proteins induced some DDR events, full signaling appeared to require AAV replication and was accompanied by accumulation of DNA-PK at viral replication compartments. Our results demonstrate that AAV replication induces a unique DNA damage signal transduction response and provides a model system for studying DNA-PK.     

DNA Damage Response in Nucleoli

Molecular Biology - Nucleoli, the largest subnuclear compartments, are formed around arrays of ribosomal gene repeats transcribed by RNA polymerase I. The primary function of nucleoli is ribosome...     

SUMO化修饰在DNA损伤响应中的作用

物理或化学等多种因素均可以引起DNA损伤。为维持机体基因组的稳定性,机体形成了精确完整的机制来修复损伤的／DNA。SUMO（smallubiquitin-relatedmodifier,SUMO）化修饰与其他蛋白翻译后修饰一样,具有多种生物学功能。近年来的研究表明,其在DNA损伤修复中也具有非常重要的作用。该文就DNA损伤修复、SUMO,96修饰系统及其二者关系的最新研究进展作了较为全面的介绍和总结。     

UV反应不一定等同于DNA损伤反应

哺乳类细胞遭受紫外线（UV）和其他DNA损伤剂作用后短时间内出现的基因转录诱导称为UV反应.过去认为这种反应是DNA损伤的结果．但是近年来的一些研究对这一观点提出了不少质疑,因而有必要在此讨论几个产生争议的主要问题,并对UV反应的触发机制及UV反应的功能作一些探讨     

c-Abl在DNA损伤反应中的功能

非受体酪氨酸激酶c-Abl在正常生理及病理条件下具有多种生物学功能。当电离辐射、顺铂、丝裂霉素C等DNA损伤诱导剂诱导DNA损伤反应后,c-Abl可参与DNA损伤反应后的细胞周期调控、基因重组修复及细胞凋亡调控等,进而决定细胞在DNA损伤反应条件下的状态。简要介绍了c-Abl在DNA损伤反应中的作用及其进展。     

Deubiquitylating Enzymes and DNA Damage Response Pathways

Covalent post-translational modification of proteins by ubiquitin and ubiquitin-like factors has emerged as a general mechanism to regulate myriad intra-cellular processes. The addition and removal of ubiquitin or ubiquitin-like proteins from factors has recently been demonstrated as a key mechanism to modulate DNA damage response (DDR) pathways. It is thus, timely to evaluate the potential for ubiquitin pathway enzymes as DDR drug targets for therapeutic intervention. The synthetic lethal approach provides exciting opportunities for the development of targeted therapies to treat cancer: most tumours have lost critical DDR pathways, and thus rely more heavily on the remaining pathways, while normal tissues are still equipped with all DDR pathways. Here, we review key deubiquitylating enzymes (DUBs) involved in DDR pathways, and describe how targeting DUBs may lead to selective therapies to treat cancer patients.     

Chlamydia Infection Promotes Host DNA Damage and Proliferation but Impairs the DNA Damage Response

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DNA Damage Response: Three Levels of DNA Repair Regulation

Genome integrity is challenged by DNA damage from both endogenous and environmental sources. This damage must be repaired to allow both RNA and DNA polymerases to accurately read and duplicate the information in the genome. Multiple repair enzymes scan the DNA for problems, remove the offending damage, and restore the DNA duplex. These repair mechanisms are regulated by DNA damage response kinases including DNA-PKcs, ATM, and ATR that are activated at DNA lesions. These kinases improve the efficiency of DNA repair by phosphorylating repair proteins to modify their activities, by initiating a complex series of changes in the local chromatin structure near the damage site, and by altering the overall cellular environment to make it more conducive to repair. In this review, we focus on these three levels of regulation to illustrate how the DNA damage kinases promote efficient repair to maintain genome integrity and prevent disease.The DNA in each of our cells accumulates thousands of lesions every day. This damaged DNA must be removed for the DNA code to be read properly. Fortunately, cells contain multiple DNA repair mechanisms including: base excision repair (BER) that removes damaged bases, mismatch repair (MMR) that recognizes base incorporation errors and base damage, nucleotide excision repair (NER) that removes bulky DNA adducts, and cross-link repair (ICL) that removes interstrand cross-links. In addition, breaks in the DNA backbone are repaired via double-strand break (DSB) repair pathways including homologous recombination (HR) and nonhomologous end joining (NHEJ). Some of these mechanisms can operate independently to repair simple lesions. However, the repair of more complex lesions involving multiple DNA processing steps is regulated by the DNA damage response (DDR). For the most difficult to repair lesions, the DDR can be essential for successful repair.The DDR consists of multiple pathways, but for the purposes of this review we will focus on the DDR kinase signaling cascades controlled by the phosphatidylinositol 3-kinase-related kinases (PIKK). These kinases include DNA-dependent protein kinase (DNA-PKcs), ataxia telangiectasia-mutated (ATM), and ATM and Rad3-related (ATR). DNA-PKcs and ATM are primarily involved in DSB repair, whereas ATR responds to a wide range of DNA lesions, especially those associated with DNA replication (Cimprich and Cortez 2008). ATR’s versatility makes it essential for the viability of replicating cells in mice and humans (Brown and Baltimore 2000; de Klein et al. 2000; Cortez et al. 2001). In the case of ATM, inherited biallelic mutations cause ataxia-telangiectasia—a disorder characterized by neurodegeneration, immunodeficiency, and cancer (Shiloh 2003; Lavin 2008). ATM mutations are also frequently found in several types of tumors (Negrini et al. 2010).The DDR kinases share several common regulatory mechanisms of activation (Lovejoy and Cortez 2009). All three DDR kinases sense damage through protein–protein interactions that serve to recruit the kinases to damage sites. Once localized, posttranslational modifications and other protein–protein interactions fully activate the kinases to initate a cascade of phosphorylation events. The best-studied substrate of DNA-PKcs is actually DNA-PKcs itself, and autophosphorylation is an important step in direct religation of the DSB via nonhomologous end joining (NHEJ) (Weterings and Chen 2007; Dobbs et al. 2010). ATM and ATR have both unique and shared substrates that participate in DNA repair, checkpoint signaling, and determining cell fate decisions such as apoptosis and sensescence.     

Hematopoietic Stem Cell Quiescence Attenuates DNA Damage Response and Permits DNA Damage Accumulation During Aging

The aging of tissue-specific stem and progenitor cells is believed to be central to the pathophysiological conditions arising in aged individuals. While the mechanisms driving stem cell aging are poorly understood, mounting evidence points to age-dependent DNA damage accrual as an important contributing factor. While it has been postulated that DNA damage may deplete stem cell numbers with age, recent studies indicate that murine hematopoietic stem cell (HSC) reserves are in fact maintained despite the accrual of genomic damage with age. Evidence suggests this to be a result of the quiescent (G0) cell cycle status of HSC, which results in an attenuation of checkpoint control and DNA damage responses for repair or apoptosis. When aged stem cells that have acquired damage are called into cycle under conditions of stress or tissue regeneration however, their functional capacity was shown to be severely impaired. These data suggest that age-dependent DNA damage accumulation may underlie the diminished capacity of aged stem cells to mediate a return to homeostasis after acute stress or injury. Moreover, the cytoprotection afforded by stem cell quiescence in stress-free, steady-state conditions suggests a mechanism through which potentially dangerous lesions can accumulate in the stem cell pool with age.     

Causes and Consequences of the DNA Damage Response

A prerequisite for maintaining genome stability in all cell types is the accurate repair and efficient signaling of DNA double strand breaks (DSBs). It is believed that DSBs are initially detected by damage sensors that trigger the activation of transducing kinases. These transducers amplify the damage signal, which is then relayed to effector proteins, which regulate the progression of the cell cycle, DNA repair and apoptosis. Errors in the execution of the repair and/or signaling of DSBs can give rise to multi-systemic disorders characterized by tissue degeneration, infertility, immune system dysfunction, age-related pathologies and cancer. This special Spotlight issue of Cell Cycle highlights recent advances in our understanding of the biology and significance of the DNA damage response. A range of issues are addressed including mechanistic ones: what is the aberrant DNA structure that triggers the activation of the checkpoint - how does chromatin structure influence the recruitment of repair and checkpoint proteins- how does chromosomal instability contribute to the evolution of cancer. In addition, questions related to the physiology of the DNA damage response in normal and abnormal cells is explored: what is the in vivo consequence of altering specific amino acids in a DNA damage sensor- does DNA damage accumulation in stem cells cause aging- how is neurodegeneration linked to deficiencies in specific DNA repair pathways, and finally, what is the biological basis for selection of aberrant DNA damage responses in cancer cells?     

脆性组氨酸三联体突变体在 DNA 损伤应答中的作用机制

目的:用建立的稳定表达脆性组氨酸三联体(Fhit)突变体的细胞株,研究 Fhit 与复制蛋白 A(RPA)之间的相互作用对细胞在 DNA 损伤后的影响.方法:用电离辐射或 DNA 损伤诱导剂喜树碱处理稳定表达 Fhit 突变体的阳性细胞株 HeLa-FhitA/D/F 后,通过流式细胞技术、MTT 比色法及克隆形成实验,检测这些细胞系的细胞周期变化情况以及对 DNA 损伤诱导剂的敏感性.结果:DNA 损伤诱导剂处理后,Fhit 突变体基的高表达可以使细胞表现出更强的G2期阻滞及对 DNA 损伤诱导剂更耐受.结论:Fhit 与 RPA 相互作用的改变影响了细胞对 DNA 损伤诱导剂的耐受性,为阐明 Fhit 在维持基组完整性方面的机理提供了线索.