Replication Problem Research Articles

Prokaryotic and Mitochondrial Linear Genomes: Their Genesis, Evolutionary Significance, and the Problem of Replicating Chromosome Ends

Bacterial chromosomes are widely thought of as circular DNA molecules. However, linear bacterial chromosomes, as well as linear mitochondrial and plastid chromosomes, are fairly common. The most frequent causes of linearization are reparation system defects, incorporation of plasmids in the genome, and recombination compromising the circular topology of chromosomes. Genomes of some bacterial species had undergone frequent linearization-circularization events, which resulted in an increased variability of gene content at linear chromosome ends. Similarly to eukaryotes, bacteria that have linear genomes face the problem of end replication, which different species solve in a variety of ways. A theoretically important issue is the adaptive value of chromosome linearization. This review discusses theories concerning the evolution of linear genomes and supporting experiments. The most common mechanisms of linear bacterial genomes replication and possible ways of their emergence are also considered.

Tolomeres and Cancer

Telomere protects the chromosomes in normal cells, and their shortening due to cell divisions and oxidative stress induces telomere shortening causing chromosomal instability. Telomerase is an enzyme that adds TTAGG telomeric repeats at chromosomal ends. The activity of telomerase enzyme plays a significant role in initiation and progression of cancer cells. In cancer cells the telomere length is maintained by telomerase enzyme. Cancer cells survive due to the activity of telomerase enzyme due to which the length of telomere is maintained and cell evades cell death mechanisms. In cancer cells telomere shortening or dysfunctional telomeres suppress cancer progression and development due to the activation of cellular senescence pathway. In this review we summarize telomere structure, function and the role telomere plays in cancer development and progression. Hermen J. Muller and Barbara McClintock identified telomere as a structure present at the ends of the chromosomes. The word telomere is derived from the Greek word “telos” which means ends and “meres” means part. Shorter telomere length or the complete absence of telomere induces end to end fusion of the chromosomes and ultimately cause cellular senescence or cell death. James D Watson in 1970s termed end replication problem in which during DNA replication, the DNA dependant polymerase does not replicates completely at the 5’ terminal end leaving small regions of the telomere uncopied. In 1960 Leonard Hayflick and his colleagues identified that the human diploid cell can undergo limited number of cell divisions in culture. The maximum number of divisions that a cell can achieve in-vitro is known as Hayflick limit which was termed after leonard Hayflick. When the cells reaches to a limit where they can no longer divide will eventually go under biochemical and morphological changes that eventually leads to cell cycle arrest, a process known as “cellular senescence. The telomerase is an enzyme that functions to add telomere repeats to the ends of the chromosomes and was identified in 1984 by Elizbeth and her collague. The presence of telomerase enzyme activity was also identified in human cancer cell lines by Gregg in 1989. Another study conducted by Greider and associates showed the absence of telomerase enzyme in normal somatic cell. Shay and Harley in 1990s detected the presence of telomerase activity in 90 out of 101 human tumor cell samples isolated from 12 different tumor types, whereas they have found no activity in normal somatic samples (n=50) isolated from 4 different tissue types. Since then various studies on 2600 human tumor samples have shown the telomerase activity in around 90% of different tumor cells. The existence of telomerase activity in cancer cells clearly demonstrates a major role of this enzyme in cancer pathogenesis. Telomeres plays a critical role in cancer, aging, Progeria (premature aging) and various other age related disorders due to which telomere and telomerase enzyme are recently an active area of research.

Minimal Resection Takes Place during Break-Induced Replication Repair of Collapsed Replication Forks and Is Controlled by Strand Invasion

A natural and frequently occurring replication problem is generated by the action of topoisomerase I (Top1). Trapping of Top1 in a cleavage complex on the DNA generates a protein-linked DNA nick (PDN), which upon DNA replication can be transformed into a one-ended double-strand break (DSB). Break-induced replication (BIR) has been recognized as a critical repair mechanism of one-ended DSBs. Here, we have investigated resection at a one-ended DSB formed exclusively during replication due to Top1-mimicking damage. We show that resection is minimal, and only when strand invasion is abolished is extensive resection detected. When DNA synthesis is slowed by hydroxyurea treatment, extended resection is not observed, which suggests that strand invasion and/or heteroduplex formation restrains resection. Our results demonstrate that the BIR pathway acting during S phase is tailored to prevent hazardous effects of naturally and frequently occurring DNA breaks such as Top1-generated PDNs.

Ginger Extract Promotes Telomere Shortening and Cellular Senescence in A549 Lung Cancer Cells.

Replicative senescence, which is caused by telomere shortening from the end replication problem, is considered one of the tumor-suppressor mechanisms in eukaryotes. However, most cancers escape this replicative senescence by reactivating telomerase, an enzyme that extends the 3′-ends of the telomeres. Previously, we reported the telomerase inhibitory effect of a crude Zingiber officinale extract (ZOE), which suppressed hTERT expression, leading to a reduction in hTERT protein and telomerase activity in A549 lung cancer cells. In the present study, we found that ZOE-induced telomere shortening and cellular senescence during the period of 60 days when these A549 cells were treated with subcytotoxic doses of ZOE. Using assay-guided fractionation and gas chromatography/mass spectrometry analysis, we found that the major compounds in the active subfractions were paradols and shogaols of various chain lengths. The results from studies of pure 6-paradol and 6-shogaol confirmed that these two compounds could suppress hTERT expression as well as telomerase activity in A549 cells. These results suggest that these paradols and shogaols are likely the active compounds in ZOE that suppress hTERT expression and telomerase activity in these cells. Furthermore, ZOE was found to be nontoxic and had an anticlastogenic effect against diethylnitrosamine-induced liver micronucleus formation in rats. These findings suggest that ginger extract can potentially be useful in dietary cancer prevention.

Deducing Immortality

Telomere defined as a region of repetitive sequence at each end of eukaryotic chromosome which is shortened as a result of incomplete replication of linear chromosome, the so called “end replication problem”.

Telomeres Increasingly Develop Aberrant Structures in Aging Humans.

Telomeres progressively shorten with age, and it has been proposed that critically short and dysfunctional telomeres contribute to aging and aging-associated diseases in humans. For many years it was thought that telomere erosion was strictly a consequence of the "end replication problem," or the inability of replicative polymerases to completely duplicate linear DNA ends. It is becoming increasingly evident, however, that telomere shortening of cultured human cells is also caused because of other replication defects in telomeric repeats, those that cause fragile telomeres and other aberrant telomeric structures that can be detected on metaphase chromosomes. Whether these replication defects contribute to telomere erosion also in human tissues is currently unknown. By analyzing peripheral blood mononuclear cells from a total of 35 healthy subjects ranging in age from 23 to 101 years, we demonstrated that telomeres increasingly display aberrant structures with advancing donor age. Although the percentages of fragile telomeres increased only until adulthood, the percentages of chromosomes displaying sister telomere loss and sister telomere chromatid fusions increased consistently throughout the entire human life span. Our data, therefore, suggest that telomeric replication defects other than the end replication problem contribute to aging-associated telomere erosion in humans.

Content Delivery in Cache-Enabled Wireless Evolving Social Networks

Cache-enabled wireless networks have gained increasing popularity in recent years for facilitating the delivery of content. Most existing works assumed that the network is static without the change of users and content, which is not the case in the real world. In this paper, we study the content delivery in cache-enabled wireless networks, where the network evolves in terms of users and content. We adopt the affiliation network model to reveal the network evolution process, where new users and content are added into the network randomly and connected with existing nodes via the preferential attachment. Users with strong social relations tend to request the same content objects, thus content popularity is sharply concentrated. Therefore, caching technique is more effective. To maximize the content delivery rate, we formulate the optimization problem of caching replication jointly with routing strategy. We show that network evolution can greatly improve the content delivery rate. In particular, when per-user cache capacity is in the order of $\Theta (1)$ , per-user delivery rate can achieve constant, i.e., the network can scale. Finally, theoretical results are validated based on a real-world data set from Facebook.

Optimal Information Centric Caching in 5G Device-to-Device Communications

Device-to-Device (D2D) communications are a prominent feature of 5G systems, introduced to provide a native support to distributed services in mobile environments. D2D technologies enable straight interactions between mobile terminals without a compulsory involvement of base stations. In this manuscript, we study and propose an optimized caching strategy to content distribution on top of D2D technology, based on Information Centric Networking (ICN) principles. The rationale is that ICN architectures can provide seamless support to mobile services and decouple contents from node identifiers, thus providing a promising match with D2D requirements. To this end, a novel fluid-based model in proposed hereby that catches the interplay between ICN functionalities, D2D requirements, and 5G specifications. Then, based on this model, an optimal content replication problem is formulated, encompassing caching overhead and system load. Additionally, this problem is thoroughly analyzed to prove that it has an optimal solution with time threshold form. A practical algorithm $\varsigma ^*$ -OCP is further proposed in order to implement the optimal caching control in realistic environments. Finally, a massive simulation campaign is carried out to test the proposed algorithm in comparison to state-of-the-art solutions.

Fine tuning the level of the Cdc13 telomere-capping protein for maximal chromosome stability performance.

Chromosome stability relies on an adequate length and complete replication of telomeres, the physical ends of chromosomes. Telomeres are composed of short direct repeat DNA and the associated nucleoprotein complex is essential for providing end-stability. In addition, the so-called end-replication problem of the conventional replication requires that telomeres be elongated by a special mechanism which, in virtually all organisms, is based by a reverse transcriptase, called telomerase. Although, at the conceptual level, telomere functions are highly similar in most organisms, the telomeric nucleoprotein composition appears to diverge significantly, in particular if it is compared between mammalian and budding yeast cells. However, over the last years, the CST complex has emerged as a central hub for telomere replication in most systems. Composed of three proteins, it is related to the highly conserved replication protein A complex, and in all systems studied, it coordinates telomerase-based telomere elongation with lagging-strand DNA synthesis. In budding yeast, the Cdc13 protein of this complex also is essential for telomerase recruitment and this specialisation is accompanied by additional regulatory adaptations. Based on recent results obtained in yeast, here, we review these issues and present an updated telomere replication hypothesis. We speculate that the similarities between systems far outweigh the differences, once we detach ourselves from the historic descriptions of the mechanisms in the various organisms.

Abstract 1466: DAXX localizes ATRX to suppress alternative lengthening of telomeres in osteosarcoma

Abstract To maintain genome stability, proliferating cells must add telomere sequence to counteract the chromosome end replication problem. In normal cells, telomeres are lengthened through the action of the enzyme telomerase. In about 10-15% of tumors, however, telomeres are lengthened through a telomerase-independent mechanism known as Alternative Lengthening of Telomeres or ALT. Many tumors that use ALT have poor prognoses, so ALT represents an appealing therapeutic target. It has been previously observed that ALT tumors frequently carry mutations in ATRX, which partners with the protein DAXX in a chromatin remodeling complex that deposits histone variant H3.3. How these mutations facilitate the ALT pathway is not well understood. Previous work in our lab identified an ALT-positive osteosarcoma cell line, G292, in which ATRX is wild-type but DAXX has undergone a fusion event with the non-canonical kinesin KIFC3. The DAXX-KIFC3 fusion leads to a loss of DAXX function, and inducible restoration of wild-type DAXX reversibly abrogates ALT in this cell line. We observe that expression of wild-type DAXX results in localization of ATRX to PML bodies, increased occupancy of ATRX at telomeric chromatin, and higher levels of histone H3.3 at telomeres. We conclude that full-length DAXX is required for the functional localization of ATRX to telomeres. Leveraging this our inducible system, we continue to probe the role of the ATRX/DAXX complex in suppressing ALT. Citation Format: Sarah Faith Clatterbuck Soper, Kathryn E. Yost, Robert L. Walker, Marbin A. Pineda, Yuelin J. Zhu, Joshua J. Waterfall, Paul S. Meltzer. DAXX localizes ATRX to suppress alternative lengthening of telomeres in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1466.

EFFECT OF CHEMOTHERAPEUTIC DRUGS ON TELOMERE LENGTH AND TELOMERASE ACTIVITY

Fil: Bolzan, Alejandro Daniel. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - La Plata. Instituto Multidisciplinario de Biologia Celular. Provincia de Buenos Aires. Gobernacion. Comision de Investigaciones Cientificas. Instituto Multidisciplinario de Biologia Celular. Universidad Nacional de La Plata. Instituto Multidisciplinario de Biologia Celular; Argentina

Flap endonuclease overexpression drives genome instability and DNA damage hypersensitivity in a PCNA-dependent manner.

Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.

Approximation algorithms and heuristics for task scheduling in data‐intensive distributed systems

AbstractIn this work, we are interested in the problem of task scheduling on large‐scale data‐intensive computing systems. In order to achieve good performance, one must construct not only good task schedules but also good data allocation across nodes on the system, since before a task can be executed, it must have access to data distributed on the system. In this article, we present a general formulation of a static problem that combines both scheduling and replication problems in data‐intensive distributed systems. We show that this problem does not admit an approximation algorithm. However, considering a restricted version of the problem that considers some practical constraints, an approximation algorithm can be designed. From a practical perspective, we introduce a novel heuristic for the problem that is based on nodes clustering. We compare the heuristic with two adapted approaches from other works in the literature by computational simulations using an extensive set of instances based on real computer grids. We show that our heuristic often obtains the best solutions and also runs faster than other approaches.

LARP7-like protein Pof8 regulates telomerase assembly and poly(A)+TERRA expression in fission yeast

Telomerase is a reverse transcriptase complex that ensures stable maintenance of linear eukaryotic chromosome ends by overcoming the end replication problem, posed by the inability of replicative DNA polymerases to fully replicate linear DNA. The catalytic subunit TERT must be assembled properly with its telomerase RNA for telomerase to function, and studies in Tetrahymena have established that p65, a La-related protein 7 (LARP7) family protein, utilizes its C-terminal xRRM domain to promote assembly of the telomerase ribonucleoprotein (RNP) complex. However, LARP7-dependent telomerase complex assembly has been considered as unique to ciliates that utilize RNA polymerase III to transcribe telomerase RNA. Here we show evidence that fission yeast Schizosaccharomyces pombe utilizes the p65-related protein Pof8 and its xRRM domain to promote assembly of RNA polymerase II-encoded telomerase RNA with TERT. Furthermore, we show that Pof8 contributes to repression of the transcription of noncoding RNAs at telomeres.

Telomere Length Dynamics and the Evolution of Cancer Genome Architecture.

Telomeres are progressively eroded during repeated rounds of cell division due to the end replication problem but also undergo additional more substantial stochastic shortening events. In most cases, shortened telomeres induce a cell-cycle arrest or trigger apoptosis, although for those cells that bypass such signals during tumour progression, a critical length threshold is reached at which telomere dysfunction may ensue. Dysfunction of the telomere nucleoprotein complex can expose free chromosome ends to the DNA double-strand break (DSB) repair machinery, leading to telomere fusion with both telomeric and non-telomeric loci. The consequences of telomere fusions in promoting genome instability have long been appreciated through the breakage–fusion–bridge (BFB) cycle mechanism, although recent studies using high-throughput sequencing technologies have uncovered evidence of involvement in a wider spectrum of genomic rearrangements including chromothripsis. A critical step in cancer progression is the transition of a clone to immortality, through the stabilisation of the telomere repeat array. This can be achieved via the reactivation of telomerase, or the induction of the alternative lengthening of telomeres (ALT) pathway. Whilst telomere dysfunction may promote genome instability and tumour progression, by limiting the replicative potential of a cell and enforcing senescence, telomere shortening can act as a tumour suppressor mechanism. However, the burden of senescent cells has also been implicated as a driver of ageing and age-related pathology, and in the promotion of cancer through inflammatory signalling. Considering the critical role of telomere length in governing cancer biology, we review questions related to the prognostic value of studying the dynamics of telomere shortening and fusion, and discuss mechanisms and consequences of telomere-induced genome rearrangements.

Telomeres, Telomerase and Ageing.

Telomeres are specialised structures at the end of linear chromosomes. They consist of tandem repeats of the hexanucleotide sequence TTAGGG, as well as a protein complex called shelterin. Together, they form a protective loop structure against chromosome fusion and degradation. Shortening or damage to telomeres and opening of the loop induce an uncapped state that triggers a DNA damage response resulting in senescence or apoptosis.Average telomere length, usually measured in human blood lymphocytes, was thought to be a biomarker for ageing, survival and mortality. However, it becomes obvious that regulation of telomere length is very complex and involves multiple processes. For example, the "end replication problem" during DNA replication as well as oxidative stress are responsible for the shortening of telomeres. In contrast, telomerase activity can potentially counteract telomere shortening when it is able to access and interact with telomeres. However, while highly active during development and in cancer cells, the enzyme is down-regulated in most human somatic cells with a few exceptions such as human lymphocytes. In addition, telomeres can be transcribed, and the transcription products called TERRA are involved in telomere length regulation.Thus, telomere length and their integrity are regulated at many different levels, and we only start to understand this process under conditions of increased oxidative stress, inflammation and during diseases as well as the ageing process.This chapter aims to describe our current state of knowledge on telomeres and telomerase and their regulation in order to better understand their role for the ageing process.

Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe

SummaryTelomere maintenance critically depends on the distinct activities of telomerase, which adds telomeric repeats to solve the end replication problem, and RTEL1, which dismantles DNA secondary structures at telomeres to facilitate replisome progression. Here, we establish that reversed replication forks are a pathological substrate for telomerase and the source of telomere catastrophe in Rtel1−/− cells. Inhibiting telomerase recruitment to telomeres, but not its activity, or blocking replication fork reversal through PARP1 inhibition or depleting UBC13 or ZRANB3 prevents the rapid accumulation of dysfunctional telomeres in RTEL1-deficient cells. In this context, we establish that telomerase binding to reversed replication forks inhibits telomere replication, which can be mimicked by preventing replication fork restart through depletion of RECQ1 or PARG. Our results lead us to propose that telomerase inappropriately binds to and inhibits restart of reversed replication forks within telomeres, which compromises replication and leads to critically short telomeres.

Contributions of recombination and repair proteins to telomere maintenance in telomerase-positive and negative Ustilago maydis.

Homologous recombination and repair factors are known to promote both telomere replication and recombination-based telomere extension. Herein, we address the diverse contributions of several recombination/repair proteins to telomere maintenance inUstilago maydis, a fungus that bears strong resemblance to mammals with respect to telomere regulation and recombination mechanisms. In telomerase-positive U. maydis, deletion of rad51 and blm separately caused shortened but stably maintained telomeres, whereas deletion of both engendered similar telomere loss, suggesting that the repair proteins help to resolve similar problems in telomere replication. In telomerase-negative cells, the loss of Rad51 or Brh2 caused accelerated senescence and failure to generate survivors on semi-solid medium. However, slow growing survivors can be isolated through continuous liquid culturing, and these survivors exhibit type II-like as well as ALT-like telomere features. In contrast, the trt1Δ blmΔ double mutant gives rise to survivors as readily as the trt1Δ single mutant, and like the single mutant survivors, exhibit almost exclusively type I-like telomere features. In addition, we observed direct physical interactions between Blm and two telomere-binding proteins, which may thus recruit or regulate Blm at telomeres. Our findings provide the basis for further analyzing the interplays between telomerase, telomere replication, and telomere recombination.

Mathematical foundation of the replicating portfolio approach

In the last few years, the first theoretical foundations for replicating portfolios – probably the most prevailing technique for risk capital calculation in life insurance – have been given in a series of papers by Beutner, Pelsser and Schweizer. In these papers, the asymptotic behaviour of replicating portfolios concerning the approximation of the terminal value (TVL) and the fair value distribution of the liabilities (FVL) has been investigated in detail. We complement this line of research by providing results on approximations based on a finite number of replicating instruments. We do so by providing the link between the approximation error of the TVL distribution, the FVL distribution and the error in the resulting risk capital figure, either value at risk or some coherent risk measure. We further allow for a variety of practically relevant formulations of the replication problem, including cash flow matching approaches. In contrast to the existing literature, all our results apply to approaches both under the risk-neutral and the real-world measure. Our strongest bounds are due to the observation that in discrete time, the measure change from the real-world to the risk-neutral measure can be both bounded below and above by a suitable constant in the first period.

Dynamic DNA binding, junction recognition and G4 melting activity underlie the telomeric and genome-wide roles of human CST

Human CST (CTC1–STN1–TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress.