G-Quadruplexes Formation at the Upstream Region of Replication Origin (OriL) of the Pseudorabies Virus: Implications for Antiviral Targets.

Pseudorabies virus (PRV) is the causative agent of Aujeszky's disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses' genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV's attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.

Contribution of reactive oxygen species to thymineless death in Escherichia coli.

Nutrient starvation usually halts cell growth rather than causing death. Thymine starvation is exceptional, because it kills cells rapidly. This phenomenon, called thymineless death (TLD), underlies the action of several antibacterial, antimalarial, anticancer, and immunomodulatory agents. Many explanations for TLD have been advanced, with recent efforts focused on recombination proteins and replication origin (oriC) degradation. Because current proposals account for only part of TLD and because reactive oxygen species (ROS) are implicated in bacterial death due to other forms of harsh stress, we investigated the possible involvement of ROS in TLD. Here, we show that thymine starvation leads to accumulation of both single-stranded DNA regions and intracellular ROS, and interference with either event protects bacteria from double-stranded DNA breakage and TLD. Elevated levels of single-stranded DNA were necessary but insufficient for TLD, whereas reduction of ROS to background levels largely abolished TLD. We conclude that ROS contribute to TLD by converting single-stranded DNA lesions into double-stranded DNA breaks. Participation of ROS in the terminal phases of TLD provides a specific example of how ROS contribute to stress-mediated bacterial self-destruction.

Bromodomain and extraterminal inhibitors block the Epstein-Barr virus lytic cycle at two distinct steps.

Lytic infection by the Epstein-Barr virus (EBV) poses numerous health risks, such as infectious mononucleosis and lymphoproliferative disorder. Proteins in the bromodomain and extraterminal (BET) family regulate multiple stages of viral life cycles and provide promising intervention targets. Synthetic small molecules can bind to the bromodomains and disrupt function by preventing recognition of acetylated lysine substrates. We demonstrate that JQ1 and other BET inhibitors block two different steps in the sequential cascade of the EBV lytic cycle. BET inhibitors prevent expression of the viral immediate-early protein BZLF1. JQ1 alters transcription of genes controlled by the host protein BACH1, and BACH1 knockdown reduces BZLF1 expression. BET proteins also localize to the lytic origin of replication (OriLyt) genetic elements, and BET inhibitors prevent viral late gene expression. There JQ1 reduces BRD4 recruitment during reactivation to preclude replication initiation. This represents a rarely observed dual mode of action for drugs.

Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic.

Checkpoint-Independent Regulation of Origin Firing by Mrc1 through Interaction with Hsk1 Kinase.

Mrc1 is a conserved checkpoint mediator protein that transduces the replication stress signal to the downstream effector kinase. The loss of mrc1 checkpoint activity results in the aberrant activation of late/dormant origins in the presence of hydroxyurea. Mrc1 was also suggested to regulate orders of early origin firing in a checkpoint-independent manner, but its mechanism was unknown. Here we identify HBS (Hsk1 bypass segment) on Mrc1. An ΔHBS mutant does not activate late/dormant origin firing in the presence of hydroxyurea but causes the precocious and enhanced activation of weak early-firing origins during normal S-phase progression and bypasses the requirement for Hsk1 for growth. This may be caused by the disruption of intramolecular binding between HBS and NTHBS (N-terminal target of HBS). Hsk1 binds to Mrc1 through HBS and phosphorylates a segment adjacent to NTHBS, disrupting the intramolecular interaction. We propose that Mrc1 exerts a "brake" on initiation (through intramolecular interactions) and that this brake can be released (upon the loss of intramolecular interactions) by either the Hsk1-mediated phosphorylation of Mrc1 or the deletion of HBS (or a phosphomimic mutation of putative Hsk1 target serine/threonine), which can bypass the function of Hsk1 for growth. The brake mechanism may explain the checkpoint-independent regulation of early origin firing in fission yeast.

DNA-protein interaction dynamics at the Lamin B2 replication origin.

To date, a complete understanding of the molecular events leading to DNA replication origin activation in mammalian cells still remains elusive. In this work, we report the results of a high resolution chromatin immunoprecipitation study to detect proteins interacting with the human Lamin B2 replication origin. In addition to the pre-RC component ORC4 and to the transcription factors USF and HOXC13, we found that 2 components of the AP-1 transcription factor, c-Fos and c-Jun, are also associated with the origin DNA during the late G1 phase of the cell cycle and that these factors interact with ORC4. Both DNA replication and AP-1 factor binding to the origin region were perturbed by cell treatment with merbarone, a topoisomerase II inhibitor, suggesting that DNA topology is essential for determining origin function.

ATR inhibitors VE-821 and VX-970 sensitize cancer cells to topoisomerase i inhibitors by disabling DNA replication initiation and fork elongation responses.

Camptothecin and its derivatives, topotecan and irinotecan, are specific topoisomerase I (Top1) inhibitors and potent anticancer drugs killing cancer cells by producing replication-associated DNA double-strand breaks, and the indenoisoquinoline LMP-400 (indotecan) is a novel Top1 inhibitor in clinical trial. To develop novel drug combinations, we conducted a synthetic lethal siRNA screen using a library that targets nearly 7,000 human genes. Depletion of ATR, the main transducer of replication stress, came as a top candidate gene for camptothecin synthetic lethality. Validation studies using ATR siRNA and the ATR inhibitor VE-821 confirmed marked antiproliferative synergy with camptothecin and even greater synergy with LMP-400. Single-cell analyses and DNA fiber combing assays showed that VE-821 abrogates the S-phase replication elongation checkpoint and the replication origin-firing checkpoint induced by camptothecin and LMP-400. As expected, the combination of Top1 inhibitors with VE-821 inhibited the phosphorylation of ATR and Chk1; however, it strongly induced γH2AX. In cells treated with the combination, the γH2AX pattern changed over time from the well-defined Top1-induced damage foci to an intense peripheral and diffuse nuclear staining, which could be used as response biomarker. Finally, the clinical derivative of VE-821, VX-970, enhanced the in vivo tumor response to irinotecan without additional toxicity. A key implication of our work is the mechanistic rationale and proof of principle it provides to evaluate the combination of Top1 inhibitors with ATR inhibitors in clinical trials.

Rifampicin suppresses thymineless death by blocking the transcription-dependent step of chromosome initiation.

Thymineless death (TLD), a phenomenon in which thymine auxotrophy becomes lethal when cells are starved of thymine, can be prevented by the presence of rifampicin, an RNA polymerase inhibitor. Several lines of evidence link TLD to chromosome initiation events. This suggests that rifampicin-mediated TLD suppression could be due to the inhibition of RNA synthesis required for DNA chromosomal initiation at oriC, although other mechanisms cannot be discarded. In this work, we show that the addition of different rifampicin concentrations to thymine-starved cells modulates TLD and chromosomal initiation capacity (ChIC). Time-lapse experiments find increasing levels of ChIC during thymine starvation correlated with the accumulation of simple-Y, double-Y and bubble arc replication intermediates at the oriC region as visualized by two-dimensional DNA agarose gel electrophoresis. None of these structures were observed following rifampicin addition or under genetic-physiological conditions that suppress TLD, indicating that abortive chromosome replication initiations under thymine starvation are crucial for this lethality. Significantly, the introduction of mioC and gid mutations which alter transcription levels around oriC, reduces ChIC and alleviates TLD. These results show that the impairment of transcription-dependent initiation caused by rifampicin addition, is responsible for TLD suppression. Our findings here may provide new avenues for the development of improved antibacterial treatments and chemotherapies based on thymine starvation-induced cell death.

The human oncoprotein MDM2 induces replication stress eliciting early intra-S-phase checkpoint response and inhibition of DNA replication origin firing.

Conventional paradigm ascribes the cell proliferative function of the human oncoprotein mouse double minute2 (MDM2) primarily to its ability to degrade p53. Here we report that in the absence of p53, MDM2 induces replication stress eliciting an early S-phase checkpoint response to inhibit further firing of DNA replication origins. Partially synchronized lung cells cultured from p53-/-:MDM2 transgenic mice enter S phase and induce S-phase checkpoint response earlier than lung cells from p53-/- mice and inhibit firing of DNA replication origins. MDM2 activates chk1 phosphorylation, elevates mixed lineage lymphoma histone methyl transferase levels and promotes checkpoint-dependent tri-methylation of histone H3 at lysine 4, known to prevent firing of late replication origins at the early S phase. In the absence of p53, a condition that disables inhibition of cyclin A expression by MDM2, MDM2 increases expression of cyclin D2 and A and hastens S-phase entry of cells. Consistently, inhibition of cyclin-dependent kinases, known to activate DNA replication origins during firing, inhibits MDM2-mediated induction of chk1 phosphorylation indicating the requirement of this activity in MDM2-mediated chk1 phosphorylation. Our data reveal a novel pathway, defended by the intra-S-phase checkpoint, by which MDM2 induces unscheduled origin firing and accelerates S-phase entry of cells in the absence of p53.

Deletion of BRCA2 exon 27 causes defects in response to both stalled and collapsed replication forks.

BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to, respectively, facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3' overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2(lex1/lex2)) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.

Deletion of BRCA2 exon 27 causes defects in response to both stalled and collapsed replication forks.

BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to, respectively, facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3' overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2(lex1/lex2)) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.

Disintegration of nascent replication bubbles during thymine starvation triggers RecA- and RecBCD-dependent replication origin destruction.

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA(+) cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction.

EGCG debilitates the persistence of EBV latency by reducing the DNA binding potency of nuclear antigen 1.

Because the expression of EBNA1 is prevalent in all EBV-associated tumors, it has become one of the most attractive drug targets for the discovery of anti-EBV compounds. In a cell-based reporter system, EBNA1 consistently upregulated the transcription of an oriP-Luc mini-EBV episome by 6- to 8-fold. The treatment of cells with 50 µM EGCG effectively blocked the binding of EBNA1 to oriP-DNA both in vivo and in vitro, which led to the abrogation of EBNA1-dependent episome maintenance and transcriptional enhancement. Importantly, the anti-EBNA1 effects caused by EGCG ultimately impaired the persistence of EBV latent infection. Our data suggest that the inhibition of EBNA1 activity by EGCG could be a promising starting point for the development of new protocols for anti-EBV therapy.

DNA ligand designed to antagonize EBNA1 represses Epstein-Barr virus-induced immortalization.

Epstein-Barr virus (EBV) transforms human B lymphocytes into immortalized cells in vitro and is associated with various malignancies in vivo. EBNA1, which is expressed in the majority of EBV-infected cells, recognizes specific DNA sequences at the cis-acting latent origin of plasmid replication (oriP) element of the EBV genome. EBNA1 plays a critical role in the viral episome maintenance and transactivates viral transforming genes in latently infected cells. Therefore, DNA-targeting agents that can disrupt the EBNA1-oriP interaction will offer novel functional inhibitors of EBNA1. Pyrrole-imidazole polyamides, sequence-specific DNA ligands, can be designed to interfere with the binding of various transcriptional factors. Here, we synthesized pyrrole-imidazole polyamides targeting EBNA1-bound DNA sequences and developed an inhibitor for the EBNA1-oriP interaction. A pyrrole-imidazole polyamide, designated as DSE-3, bound adjacent to the EBNA1 recognition sequences located in the dyad symmetry element of oriP, and selectively inhibited EBNA1-oriP binding both in vitro and in vivo. DSE-3 also inhibited the proliferation of established lymphoblastoid cell lines by eradicating EBV episomes from the cells. In addition, DSE-3 repressed the expression of viral transforming genes after infecting human peripheral blood mononuclear cells with EBV and, as a consequence, inhibited EBV-mediated B-cell immortalization. These results suggest that EBNA1 functions will be an attractive pharmacological target for EBV-associated diseases.

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1.

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.

Initiation of DNA replication: functional and evolutionary aspects.

BACKGROUND: The initiation of DNA replication is a very important and highly regulated step in the cell division cycle. It is of interest to compare different groups of eukaryotic organisms (a) to identify the essential molecular events that occur in all eukaryotes, (b) to start to identify higher-level regulatory mechanisms that are specific to particular groups and (c) to gain insights into the evolution of initiation mechanisms. SCOPE: This review features a wide-ranging literature survey covering replication origins, origin recognition and usage, modification of origin usage (especially in response to plant hormones), assembly of the pre-replication complex, loading of the replisome, genomics, and the likely origin of these mechanisms and proteins in Archaea. CONCLUSIONS: In all eukaryotes, chromatin is organized for DNA replication as multiple replicons. In each replicon, replication is initiated at an origin. With the exception of those in budding yeast, replication origins, including the only one to be isolated so far from a plant, do not appear to embody a specific sequence; rather, they are AT-rich, with short tracts of locally bent DNA. The proteins involved in initiation are remarkably similar across the range of eukaryotes. Nevertheless, their activity may be modified by plant-specific mechanisms, including regulation by plant hormones. The molecular features of initiation are seen in a much simpler form in the Archaea. In particular, where eukaryotes possess a number of closely related proteins that form 'hetero-complexes' (such as the origin recognition complex and the MCM complex), archaeans typically possess one type of protein (e.g. one MCM) that forms a homo-complex. This suggests that several eukaryotic initiation proteins have evolved from archaeal ancestors by gene duplication and divergence.

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories.

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

The inhibitory action of P56 on select functions of E1 mediates interferon's effect on human papillomavirus DNA replication.

The interferon (IFN)-induced protein P56 inhibits human papillomavirus (HPV) DNA replication by binding to HPV E1, which has several distinct functions in initiating viral DNA replication. Here, we determined that P56 inhibited HPV type 18 (HPV18) E1's DNA helicase activity, E2 binding, and HPV Ori sequence-specific DNA binding but not nonspecific DNA binding. We observed that deletion of a single amino acid, F399, produced an E1 mutant that could not bind P56. This E1 mutant retained its ability to support Ori DNA replication, but this activity was not inhibited by IFN, demonstrating that P56 is the principal executor of the anti-HPV action of IFN.

Damage-induced phosphorylation of Sld3 is important to block late origin firing.

Origins of replication are activated throughout the S phase of the cell cycle such that some origins fire early and others fire late to ensure that each chromosome is completely replicated in a timely fashion. However, in response to DNA damage or replication fork stalling, eukaryotic cells block activation of unfired origins. Human cells derived from patients with ataxia telangiectasia are deficient in this process due to the lack of a functional ataxia telangiectasia mutated (ATM) kinase and elicit radioresistant DNA synthesis after γ-irradiation(2). This effect is conserved in budding yeast, as yeast cells lacking the related kinase Mec1 (ATM and Rad3-related (ATR in humans)) also fail to inhibit DNA synthesis in the presence of DNA damage. This intra-S-phase checkpoint actively regulates DNA synthesis by inhibiting the firing of late replicating origins, and this inhibition requires both Mec1 and the downstream checkpoint kinase Rad53 (Chk2 in humans). However, the Rad53 substrate(s) whose phosphorylation is required to mediate this function has remained unknown. Here we show that the replication initiation protein Sld3 is phosphorylated by Rad53, and that this phosphorylation, along with phosphorylation of the Cdc7 kinase regulatory subunit Dbf4, blocks late origin firing in Saccharomyces cerevisiae. Upon exposure to DNA-damaging agents, cells expressing non-phosphorylatable alleles of SLD3 and DBF4 (SLD3-m25 and dbf4-m25, respectively) proceed through the S phase faster than wild-type cells by inappropriately firing late origins of replication. SLD3-m25 dbf4-m25 cells grow poorly in the presence of the replication inhibitor hydroxyurea and accumulate multiple Rad52 foci. Moreover, SLD3-m25 dbf4-m25 cells are delayed in recovering from transient blocks to replication and subsequently arrest at the DNA damage checkpoint. These data indicate that the intra-S-phase checkpoint functions to block late origin firing in adverse conditions to prevent genomic instability and maximize cell survival.

Genome-wide replication profiles indicate an expansive role for Rpd3L in regulating replication initiation timing or efficiency, and reveal genomic loci of Rpd3 function in Saccharomyces cerevisiae.

In higher eukaryotes, heritable gene silencing is associated with histone deacetylation and late replication timing. In Saccharomyces cerevisiae, the histone deacetylase Rpd3 regulates gene expression and also modulates replication timing; however, these mechanisms have been suggested to be independent, and no global association has been found between replication timing and gene expression levels. Using 5-Bromo-2'-deoxyuridine (BrdU) incorporation to generate genome-wide replication profiles, we identified >100 late-firing replication origins that are regulated by Rpd3L, which is specifically targeted to promoters to silence transcription. Rpd3S, which recompacts chromatin after transcription, plays a primary role at only a handful of origins, but subtly influences initiation timing globally. The ability of these functionally distinct Rpd3 complexes to affect replication initiation timing supports the idea that histone deacetylation directly influences initiation timing. Accordingly, loss of Rpd3 function results in higher levels of histone H3 and H4 acetylation surrounding Rpd3-regulated origins, and these origins show a significant association with Rpd3 chromatin binding and gene regulation, supporting a general link between histone acetylation, replication timing, and control of gene expression in budding yeast. Our results also reveal a novel and complementary genomic map of Rpd3L- and Rpd3S-regulated chromosomal loci.