Double-Stranded Linear Duck Hepatitis B Virus (DHBV) Stably Integrates at a Higher Frequency than Wild-Type DHBV in LMH Chicken Hepatoma Cells

Integration of hepadnavirus DNAs into host chromosomes can have oncogenic consequences. Analysis of host-viral DNA junctions of DHBV identified the terminally duplicated r region of the viral genome as a hotspot for integration. Since the r region is present on the 5′ and 3′ ends of double-stranded linear (DSL) hepadnavirus DNAs, these molecules have been implicated as integration precursors. We have produced a LMH chicken hepatoma cell line (LMH 66-1 DSL) which replicates exclusively DSL duck hepatitis B virus (DHBV) DNA. To test whether linear DHBV DNAs integrate more frequently than the wild type open circular DHBV DNAs, we have characterized the integration frequency in LMH 66-1 DSL cells by using a subcloning approach. This approach revealed that 83% of the LMH 66-1 DSL subclones contained new integrations, compared to only 16% of subclones from LMH-D2 cells replicating wild-type open circular DHBV DNA. Also, a higher percentage of the LMH 66-1 DSL subclones contained two or more new integrations. Mathematical analysis suggests that the DSL DHBV DNAs integrated stably once every three generations during subcloning whereas wild-type DHBV integrated only once every four to five generations. Cloning and sequencing of new integrations confirmed the r region as a preferred integration site for linear DHBV DNA molecules. One DHBV integrant was associated with a small deletion of chromosomal DNA, and another DHBV integrant occurred in a telomeric repeat sequence.     

Loss and acquisition of duck hepatitis B virus integrations in lineages of LMH-D2 chicken hepatoma cells.

Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis.     

3-氨基苯酰胺对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接作用的影响

端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老和凋亡有着密切的关系.在各种DNA损伤中,单链断裂(single-strand breaks, SSBs)是最常见的类型之一,既可直接通过内源活性氧或离子化辐射产生,也可间接地在DNA代谢或碱基切除修复期间产生.已知多聚(ADP-核糖)聚合酶［poly(ADPribose) polymerase, PARP］在SSBs修复中起着极为重要的作用.本实验观察了PARP抑制剂3-氨基苯酰胺(3-aminobenzamide, 3-AB)对氧化应激诱导的HeLa细胞端粒DNA链断裂重连接的效应以及对过氧化氢(H2O2)抑制HeLa细胞增殖的影响.结果表明3-AB能够显著地抑制氧化应激诱导的HeLa细胞端粒DNA链断裂后的重连接作用,并能增强H2O2对HeLa细胞增殖的抑制作用,提示PARP参与了端粒DNA链断裂损伤的修复过程.     

Effect of DNA repair inhibitor (3-aminobenzamide) on genetic stability of loach (Misgurnus fossilis) embryos derived from cryopreserved sperm

Semen cryopreservation is widely used in clinical medicine, agriculture, aquaculture and biomedical research, but it is an inefficient technique that induces extensive cytoplasmic damage and loss of fertilising ability. Whether any genetic damage (i.e. DNA strand breakage or mutation) is also induced is still unclear. However, previous data has indicated that this is likely. The present study was designed to explore this possibility further by using inhibitors of the DNA repair system to block DNA repair in embryos derived from cryopreserved spermatozoa. If cryopreservation causes strand breaks in sperm DNA it might be expected that inhibition of a repair enzyme such as poly(ADP-ribose) polymerase (PARP) would enhance any such negative effect of cryopreservation. To check this hypothesis 3-aminobenzamide (3-AB) was used as an inhibitor of PARP. Weather loach (Misgurnus fossilis) eggs were fertilised using cryopreserved as well as fresh spermatozoa. Embryos derived from cryopreserved spermatozoa were exposed to 10 mM 3-AB for 2 h after fertilisation. The experiments were carried out using 43,544 embryos from 5 females and 10 males. Embryo survival was evaluated at different stages until the hatching stage. Sperm cryopreservation significantly decreased embryo survival (53.6+/-2.79% compared to 76.97+/-2.79% of control; P<0.01). The addition of 3-AB to the medium with embryos derived from cryopreserved sperm further decreased embryo survival from 53.6+/-2.79% to 46.1+/-2.79% (P<0.01) whereas there was no adverse effect of 3-AB exposed embryos derived from fresh sperm (76.97+/-2.79% of control compared to 74.8+/-2.79% of control+3-AB). The effect of 3-AB provides indirect evidence that cryopreservation might induce instability in sperm DNA, and that such damage can be repaired by the oocyte repair system after fertilisation.     

Effect of poly(ADP-ribosyl)ation inhibitors on the genotoxic effects of the boron neutron capture reaction

The boron neutron capture (BNC) reaction results from the interaction of 10B with low-energy thermal neutrons and gives rise to highly damaging lithium and alpha-particles. In this work the genotoxicity caused by the BNC reaction in V79 Chinese hamster cells was evaluated in the presence of poly(ADP-ribosyl)ation inhibitors. Poly(ADP-ribose) polymerase-1 (PARP-1), the most important member of the PARP enzyme family, is considered to be a constitutive factor of the DNA damage surveillance network present in eukaryotic cells, acting through a DNA break sensor function. Inhibition of poly(ADP-ribosyl)ation was achieved with the classical compound 3-aminobenzamide (3-AB), and with two novel and very potent inhibitors, 5-aminoisoquinolinone (5-AIQ) and PJ-34. Dose-response increases in the frequencies of aberrant cells excluding gaps (%ACEG) and chromosomal aberrations excluding gaps per cell (CAEG/cell) were observed for increasing exposures to the BNC reaction. The presence of 3-AB did not increase the %ACEG or CAEG/cell, nor did it change the pattern of the induced chromosomal aberrations. Results with 5-AIQ and PJ-34 were in agreement with the results obtained with 3-AB. We further studied the combined effect of a PARP inhibitor and a DNA-dependent protein kinase (DNA-PK) inhibitors (3-AB and wortmannin, respectively) on the genotoxicity of the BNC reaction, by use of the cytokinesis-block micronucleus assay. DNA-PK is also activated by DNA breaks and binds DNA ends, playing a role of utmost importance in the repair of double-strand breaks. Our results show that the inhibition of poly(ADP-ribosyl)ation does not particularly modify the genotoxicity of the BNC reaction, and that PARP inhibition together with a concomitant inhibition of DNA-PK revealed barely the same sensitizing effect as DNA-PK inhibition per se.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.     

Topoisomerase I-mediated integration of hepadnavirus DNA in vitro.

Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule.     

The role of DNA damage response proteins at telomeres--an "integrative" model

Telomeres are specialized structures at chromosome ends which play the key role in chromosomal end protection. There is increasing evidence that many DNA damage response proteins are involved in telomere maintenance. For example, cells defective in DNA double strand break repair proteins including Ku, DNA-PKcs, RAD51D and the MRN (MRE11/RAD51/NBS1) complex show loss of telomere capping function. Similarly, mouse and human cells defective in ataxia telangiectasia mutated (ATM) have defective telomeres. A total of 14 mammalian DNA damage response proteins have, so far, been implicated in telomere maintenance. Recent studies indicate that three more proteins, namely BRCA1, hRad9 and PARP1 are involved in telomere maintenance. The involvement of a wide range of DNA damage response proteins at telomeres raises an important question: do telomere maintenance mechanisms constitute an integral part of DNA damage response machinery? A model termed the "integrative" model is proposed here to argue in favour of telomere maintenance being an integral part of DNA damage response. The "integrative" model is supported by the observation that a telomeric protein, TRF2, is not confined to its local telomeric environment but it migrates to the sites of DNA breakage following exposure of cells to ionizing radiation. Furthermore, even if telomeres are maintained in a non-canonical way, as in the case of Drosophila, DNA damage response proteins are still involved in telomere maintenance suggesting integration of telomere maintenance mechanisms into the DNA damage response network.     

Beneficial Effects of Poly (ADP-ribose) Polymerase Inhibition Against the Reperfusion Injury in Heart Transplantation

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD + and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD + and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.     

Beneficial effects of poly (ADP-ribose) polymerase inhibition against the reperfusion injury in heart transplantation

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD + and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD + and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.     

Condensin I recruitment to base damage-enriched DNA lesions is modulated by PARP1

Condensin I is important for chromosome organization and segregation in mitosis. We previously showed that condensin I also interacts with PARP1 in response to DNA damage and plays a role in single-strand break repair. However, whether condensin I physically associates with DNA damage sites and how PARP1 may contribute to this process were unclear. We found that condensin I is preferentially recruited to DNA damage sites enriched for base damage. This process is dictated by PARP1 through its interaction with the chromosome-targeting domain of the hCAP-D2 subunit of condensin I.     

Human DNA topoisomerase I-mediated cleavage and recombination of duck hepatitis B virus DNA in vitro

In this study, we report that eukaryotic topoisomerase I (top1) can linearize the open circular DNA of duck hepatitis B virus (DHBV). Using synthetic oligonucleotides mimicking the three-strand flap DR1 region of the DHBV genome, we found that top1 cleaves the DNA plus strand in a suicidal manner, which mimics the linearization of the virion DNA. We also report that top1 can cleave the DNA minus strand at specific sites and can linearize the minus strand via a non-homologous recombination reaction. These results are consistent with the possibility that top1 can act as a DNA endo-nuclease and strand transferase and play a role in the circularization, linearization and possibly integration of viral replication intermediates.     

Retrovirus integration and chromatin structure: Moloney murine leukemia proviral integration sites map near DNase I-hypersensitive sites.

The chromatin conformation of mouse genome regions containing Moloney murine leukemia proviral intergration sites in two Mov mouse strains and randomly selected integration sites in virus-infected mouse 3T3 fibroblasts was analyzed. All integrations have occurred into chromosomal regions containing several DNase-hypersensitive sites, and invariably the proviral integration sites map within a few hundred base pairs of a DNase-hypersensitive site. The probability that this close association between proviral integration sites and DNase-hypersensitive sites was due to chance was calculated to be extremely low (2 X 10(-4]. Because the proviral integrations analyzed were not selected for an altered phenotype, our results suggest that DNase-hypersensitive regions are preferred targets for retrovirus integration.     

Q-FADD: A Mechanistic Approach for Modeling the Accumulation of Proteins at Sites of DNA Damage

The repair of DNA damage requires the ordered recruitment of many different proteins that are responsible for signaling and subsequent repair. A powerful and widely used tool for studying the orchestrated accumulation of these proteins at damage sites is laser microirradiation in live cells, followed by monitoring the accumulation of the fluorescently labeled protein in question. Despite the widespread use of this approach, there exists no rigorous method for characterizing the recruitment process quantitatively. Here, we introduce a diffusion model that explicitly accounts for the unique sizes and shapes of individual nuclei and uses two variables: D_eff, the effective coefficient of diffusion, and F, the fraction of mobile protein that accumulates at sites of DNA damage. Our model quantitatively describes the accumulation of three test proteins, poly-ADP-ribose polymerases 1 and 2 (PARP1/2) and histone PARylation factor 1. D_eff for PARP1, as derived by our approach, is 6× greater than for PARP2 and in agreement with previous literature reports using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Our data indicate that histone PARylation factor 1 arrives at sites of DNA damage independently of either PARP. Importantly, our model, which can be applied to existing data, allows for the direct comparison of the coefficient of diffusion for any DNA repair protein between different cell types, obtained in different laboratories and by different methods, and also allows for the interrogation of cell-to-cell variability.     

DNA ligase III acts as a DNA strand break sensor in the cellular orchestration of DNA strand break repair

In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme''s SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3.