Cytometric analysis of DNA replication inhibited by emetime and cyclosporin A

Summary DNA staining methods based on aspecific interactions with dye molecules have been replaced by an immunofluorescent approach to measure DNA replication. Biotin-11-dUTP was incorporated into permeable thymocytes isolated after emetine or cyclosporin A treatment of mice. Active sites of DNA replication were amplified based on biotin-avidin interaction and verified under fluorescent microscope. Cytometry of fluorescent images allow the direct measurement of replicating DNA without aspecific detection of total cellular DNA. Cytometric analysis of replication revealed that emetine acts at the early S phase, while cyclosporin A blocks in vivo DNA synthesis at mid S phase.     

Specific inhibition of hepatitis C virus replication by cyclosporin A

The difficulty in eradicating hepatitis C virus (HCV) infection is attributable to the limited treatment options against the virus. Recently, cyclosporin A (CsA), a widely used immunosuppressive drug, has been reported to be effective against HCV infection [J. Gastroenterol. 38 (2003) 567], although little is understood about the mechanism of its action against HCV. In this study, we investigated the anti-viral effects of CsA using an HCV replicon system. Human hepatoma Huh7 cells were transfected with an HCV replicon expressing a chimeric gene encoding a luciferase reporter and neomycin phosphotransferase (Huh7/Rep-Feo). Treatment of the Huh7/Rep-Feo cells with CsA resulted in suppression of the replication of the HCV replicon in a dose-dependent manner, with an IC50 of approximately 0.5 microg/ml. There were no changes in the rate of cell growth or viability, suggesting that the effect of CsA against HCV is specific and not due to cytotoxicity. In contrast, FK506, another immunosuppressive drug, did not suppress HCV replication. CsA did not activate interferon-stimulated gene responses, suggesting that its action is independent of that of interferon. In conclusion, CsA inhibits HCV replication in vitro specifically at clinical concentrations. Further defining its mode of action against HCV replication potentially may be important for identifying novel molecular targets to treat HCV infection.     

The giant channel of the inner mitochondrial membrane is inhibited by cyclosporin A.

In patch-clamp experiments on rat liver mitoplasts, cyclosporin A inhibited the activity of the recently described (Petronilli, V., Szabó, I., and Zoratti, M. (1989) FEBS Lett. 259, 137-143) 1.3-nanosiemens channel of the inner mitochondrial membrane at concentrations in the 10(-8)-10(-7) M range. The inhibitor acts when present on the matrix side of membrane. The Ca2(+)-dependent "permeability transition channel" of mitochondria is inhibited by cyclosporin A in the same concentration range. The results suggest therefore that the same pore is responsible for the permeabilization of the inner mitochondrial membrane and for the conduction of the high currents observed in electrophysiological experiments.     

Structural analysis of DNA replication fork reversal by RecG

The stalling of DNA replication forks that occurs as a consequence of encountering DNA damage is a critical problem for cells. RecG protein is involved in the processing of stalled replication forks, and acts by reversing the fork past the damage to create a four-way junction that allows template switching and lesion bypass. We have determined the crystal structure of RecG bound to a DNA substrate that mimics a stalled replication fork. The structure not only reveals the elegant mechanism used by the protein to recognize junctions but has also trapped the protein in the initial stage of fork reversal. We propose a mechanism for how forks are processed by RecG to facilitate replication fork restart. In addition, this structure suggests that the mechanism and function of the two largest helicase superfamilies are distinct.     

Agiotensin II induced cardiac hypertrophy in vivo is inhibited by cyclosporin A in adult rats

Recently, the calciumcalmodulindependent calcineurin pathway has been defined as a central pathway for the induction of cardiac hypertrophy. The purpose of this study was to determine if cardiac hypertrophy in animals chronically treated with angiotensin II (AngII), could be prevented by blocking this pathway with cyclosporin A (CsA). Female Wistar rats were treated with AngII by subcutaneous infusion and injected twice a day with CsA (25 mg/kg) for 7 days. In the AngII treated group there was a 30% increase in the heart/body weight ratio (p < 0.05 vs. control). The increase in heart weight was blocked with CsA. Substantial increases in ANF and MHC gene expression were detected in the AngII treated animals, which were either attenuated or blocked with CsA treatment. Thus, this study demonstrates that CsA does prevent the development of cardiac hypertrophy in Ang II treated rats, suggesting that the calciumcalmodulindependent calcineurin pathway is associated with angiotensin II induced hypertrophy in vivo.     

Single molecule analysis of DNA replication

We describe here a novel approach for the study of DNA replication. The approach is based on a process called molecular combing and allows for the genome wide analysis of the spatial and temporal organization of replication units and replication origins in a sample of genomic DNA. Molecular combing is a process whereby molecules of DNA are stretched and aligned on a glass surface by the force exerted by a receding air/water interface. Since the stretching occurs in the immediate vicinity of the meniscus, all molecules are identically stretched in a size and sequence independent manner. The application of fluorescence hybridization to combed DNA results in a high resolution (1 to 4 kb) optical mapping that is simple, controlled and reproducible. The ability to comb up to several hundred haploid genomes on a single coverslip allows for a statistically significant number of measurements to be made. Direct labeling of replicating DNA sequences in turn enables origins of DNA replication to be visualized and mapped. These features therefore make molecular combing an attractive tool for genomic studies of DNA replication. In the following, we discuss the application of molecular combing to the study of DNA replication and genome stability.     

Translation termination is involved in histone mRNA degradation when DNA replication is inhibited

The levels of replication-dependent histone mRNAs are coordinately regulated with DNA synthesis. A major regulatory step in histone mRNA metabolism is regulation of the half-life of histone mRNAs. Replication-dependent histone mRNAs are the only metazoan mRNAs that are not polyadenylated. Instead, they end with a conserved stem-loop structure, which is recognized by the stem-loop binding protein (SLBP). SLBP is required for histone mRNA processing, as well as translation. We show here, using histone mRNAs whose translation can be regulated by the iron response element, that histone mRNAs need to be actively translated for their rapid degradation following the inhibition of DNA synthesis. We also demonstrate the requirement for translation using a mutant SLBP which is inactive in translation. Histone mRNAs are not rapidly degraded when DNA synthesis is inhibited or at the end of S phase in cells expressing this mutant SLBP. Replication-dependent histone mRNAs have very short 3' untranslated regions, with the stem-loop located 30 to 70 nucleotides downstream of the translation termination codon. We show here that the stability of histone mRNAs can be modified by altering the position of the stem-loop, thereby changing the distance from the translation termination codon.     

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry.     

Genome-wide analysis of DNA replication and DNA double-strand breaks using TrAEL-seq

Faithful replication of the entire genome requires replication forks to copy large contiguous tracts of DNA, and sites of persistent replication fork stalling present a major threat to genome stability. Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods that profile replication fork position and the formation of recombinogenic DNA ends. Here, we describe Transferase-Activated End Ligation sequencing (TrAEL-seq), a method that captures single-stranded DNA 3′ ends genome-wide and with base pair resolution. TrAEL-seq labels both DNA breaks and replication forks, providing genome-wide maps of replication fork progression and fork stalling sites in yeast and mammalian cells. Replication maps are similar to those obtained by Okazaki fragment sequencing; however, TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq far simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3′ ends also allows accurate detection of double-strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double-strand break hotspots in a dmc1Δ mutant that is competent for end resection but not strand invasion. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

TrAEL-seq provides genome-wide base pair resolution maps of exposed DNA 3’ ends; this reveals replication fork stalling and normal replication profiles in asynchronous, unlabelled wildtype cell populations, along with the sites of resected DNA breaks.     

A quantitative, non-radioactive single-nucleotide insertion assay for analysis of DNA replication fidelity by using an automated DNA sequencer

A method to determine the steady-state kinetic parameters of single-nucleotide insertion in replication was developed using an automated DNA sequencer. The insertion of nucleoside 5'-triphosphates into a 6-carboxyfluorescein-labeled primer by DNA polymerase was quantified from the band pattern on a gel using GeneScan software. The parameters determined by this method were consistent with those obtained by the conventional radioisotope-labeling method. This non-radioactive, fluorescent-based method is rapid and can handle a large number of samples to assess cognate or non-cognate base pair formation between natural or unnatural bases in replication.     

Lymphocyte commitment to DNA replication induced by concanavalin A

Commitment of lymphocytes to DNA replication induced by ConA was investigated by adding α-MM (a saccharide that competes with the lectin's lymphocyte binding sites) at various times to lymphocyte cultures that had been treated with a wide range of ConA concentrations. After 24 h of culture, α-MM addition has essentially no effect on cells treated with optimal concentrations of ConA, results in a marked blastogenic response of cells treated with supra-optimal (non-mitogenic) concentrations of ConA, and severely inhibits blastogenesis of cells treated with suboptimal concentrations of the lectin. Furthermore, the time of commitment is progressively shortened as the concentration of mitogen is increased. Thus, cells were committed to DNA replication as early as 5 h after incubation with supra-optimal concentrations of ConA. Induction of commitment does not occur when cells are incubated with ConA at 4 °C. Extensive crosslinking of membrane sites appears to be associated with the induction of early commitment by ConA, since the bivalent succinyl ConA failed to exhibit this effect. These findings indicate the need for re-evaluation of previous studies utilizing competing saccharides to determine kinetics of cell commitment to DNA replication.     

Control of DNA replication and chromosome ploidy by geminin and cyclin A

Alteration of the control of DNA replication and mitosis is considered to be a major cause of genome instability. To investigate the mechanism that controls DNA replication and genome stability, we used the RNA silencing-interference technique (RNAi) to eliminate the Drosophila geminin homologue from Schneider D2 (SD2) cells. Silencing of geminin by RNAi in SD2 cells leads to the cessation of mitosis and asynchronous overreplication of the genome, with cells containing single giant nuclei and partial ploidy between 4N and 8N DNA content. The effect of geminin deficiency is completely suppressed by cosilencing of Double parked (Dup), the Drosophila homologue of Cdt1, a replication factor to which geminin binds. The geminin deficiency-induced phenotype is also partially suppressed by coablation of Chk1/Grapes, indicating the involvement of Chk1/Grapes in the checkpoint control in response to overreplication. We found that the silencing of cyclin A, but not of cyclin B, also promotes the formation of a giant nucleus and overreplication. However, in contrast to the effect of geminin knockout, cyclin A deficiency leads to the complete duplication of the genome from 4N to 8N. We observed that the silencing of geminin causes rapid downregulation of Cdt1/Dup, which may contribute to the observed partial overreplication in geminin-deficient cells. Analysis of cyclin A and geminin double knockout suggests that the effect of cyclin A deficiency is dominant over that of geminin deficiency for cell cycle arrest and overreplication. Together, our studies indicate that both cyclin A and geminin are required for the suppression of overreplication and for genome stability in Drosophila cells.     

Alloantigen-specific suppressor T cells are not inhibited by cyclosporin A, but do require IL 2 for activation

Alloantigen-specific suppressor T cells are activated from normal murine spleen cells in mixed lymphocyte reactions (MLR). These T cells are radioresistant and suppress the activation of cytotoxic T lymphocytes (CTL) in second primary MLR cultures. This report demonstrates that cyclosporin A (CsA) blocks the activation of these suppressor cells at a dose of 1 microgram/ml. However, reconstitution of CsA blocked cultures with IL 2 restores the activation of the suppressor T cells, but fails to significantly restore the activation of CTL in these same cultures. This differential activation requirement was used to establish T cell lines that demonstrate enriched suppressor cell activity but depletion of CTL activity. These findings are discussed in terms of the mechanism of action of CsA in these distinct T cell subsets and the relevance to models of allograft unresponsiveness.     

Functional analysis of plastid DNA replication origins in tobacco by targeted inactivation

Sequences described as chloroplast DNA replication origins were analysed in vivo by creating deletion and insertion mutants via plastid transformation in tobacco. Deletion of the described oriA sequence, which is located within the intron of the trnI gene, resulted in heteroplastomic transformants, when the selection marker was inserted within the intron. Removal of the complete intron sequence together with the oriA sequence, however, yielded homoplastomic transformants of normal phenotype, in which wild-type signals were no longer detectable through Southern analysis, thus bringing the role of the described oriA sequence for plastome replication into question. Similarly, deletion of sequence elements upstream of trnI, which have a possible ori function in Oenothera, did not show any effect in tobacco. The two copies of oriB, which are located at the very end of the plastome Inverted Repeats, were targeted with two different transformation vectors in a cotransformation approach. While in initial transformants integration of the selection marker could be detected at both sites, the transgene was found exclusively at one site or the other after additional rounds of regeneration. Whereas the copy of oriB in Inverted Repeat B could be completely deleted, targeting of the copy in Inverted Repeat A resulted in heteroplastomic lines, as the essential ycf1 gene was also affected. Due to the strong selection against cotransformants we conclude that at least one copy of the oriB sequence is essential for plastome replication, whereas replication appears possible without oriA elements.