Checkpoint Mechanisms Research Articles

Studying Mitotic Checkpoint by Illustrating Dynamic Kinetochore Protein Behavior and Chromosome Motion in Living Drosophila Syncytial Embryos

The spindle assembly checkpoint (SAC) mechanism is an active signal, which monitors the interaction between chromosome kinetochores and spindle microtubules to prevent anaphase onset until the chromosomes are properly connected. Cells use this mechanism to prevent aneuploidy or genomic instability, and hence cancers and other human diseases like birth defects and Alzheimer's1. A number of the SAC components such as Mad1, Mad2, Bub1, BubR1, Bub3, Mps1, Zw10, Rod and Aurora B kinase have been identified and they are all kinetochore dynamic proteins2. Evidence suggests that the kinetochore is where the SAC signal is initiated. The SAC prime regulatory target is Cdc20. Cdc20 is one of the essential APC/C (Anaphase Promoting Complex or Cyclosome) activators3 and is also a kinetochore dynamic protein4-6. When activated, the SAC inhibits the activity of the APC/C to prevent the destruction of two key substrates, cyclin B and securin, thereby preventing the metaphase to anaphase transition7,8. Exactly how the SAC signal is initiated and assembled on the kinetochores and relayed onto the APC/C to inhibit its function still remains elusive.Drosophila is an extremely tractable experimental system; a much simpler and better-understood organism compared to the human but one that shares fundamental processes in common. It is, perhaps, one of the best organisms to use for bio-imaging studies in living cells, especially for visualization of the mitotic events in space and time, as the early embryo goes through 13 rapid nuclear division cycles synchronously (8-10 minutes for each cycle at 25 °C) and gradually organizes the nuclei in a single monolayer just underneath the cortex9.Here, I present a bio-imaging method using transgenic Drosophila expressing GFP (Green Fluorescent Protein) or its variant-targeted proteins of interest and a Leica TCS SP2 confocal laser scanning microscope system to study the SAC function in flies, by showing images of GFP fusion proteins of some of the SAC components, Cdc20 and Mad2, as the example.

A UVR-Induced G2-Phase Checkpoint Response to ssDNA Gaps Produced by Replication Fork Bypass of Unrepaired Lesions Is Defective in Melanoma

UVR is a major environmental risk factor for the development of melanoma. Here we describe a coupled DNA-damage tolerance (DDT) mechanism and G2-phase cell cycle checkpoint induced in response to suberythemal doses of UVR that is commonly defective in melanomas. This coupled response is triggered by a small number of UVR-induced DNA lesions incurred during G1 phase that are not repaired by nucleotide excision repair (NER). These lesions are detected during S phase, but rather than stalling replication, they trigger the DDT-dependent formation of single-stranded DNA (ssDNA) gaps. The ssDNA attracts replication protein A (RPA), which initiates ATR-Chk1 (ataxia telangiectasia and Rad3-related/checkpoint kinase 1) G2-phase checkpoint signaling, and colocalizes with components of the RAD18 and RAD51 postreplication repair pathways. We demonstrate that depletion of RAD18 delays both the resolution of RPA foci and exit from the G2-phase arrest, indicating the involvement of RAD18-dependent postreplication repair in ssDNA gap repair during G2 phase. Moreover, the presence of RAD51 and BRCA1 suggests that an error-free mechanism may also contribute to repair. Loss of the UVR-induced G2-phase checkpoint results in increased UVR signature mutations after exposure to suberythemal UVR. We propose that defects in the UVR-induced G2-phase checkpoint and repair mechanism are likely to contribute to melanoma development.

RAxML-Light: a tool for computing terabyte phylogenies

Motivation: Due to advances in molecular sequencing and the increasingly rapid collection of molecular data, the field of phyloinformatics is transforming into a computational science. Therefore, new tools are required that can be deployed in supercomputing environments and that scale to hundreds or thousands of cores.Results: We describe RAxML-Light, a tool for large-scale phylogenetic inference on supercomputers under maximum likelihood. It implements a light-weight checkpointing mechanism, deploys 128-bit (SSE3) and 256-bit (AVX) vector intrinsics, offers two orthogonal memory saving techniques and provides a fine-grain production-level message passing interface parallelization of the likelihood function. To demonstrate scalability and robustness of the code, we inferred a phylogeny on a simulated DNA alignment (1481 taxa, 20 000 000 bp) using 672 cores. This dataset requires one terabyte of RAM to compute the likelihood score on a single tree.Code Availability: https://github.com/stamatak/RAxML-Light-1.0.5Data Availability: http://www.exelixis-lab.org/onLineMaterial.tar.bz2Contact: alexandros.stamatakis@h-its.orgSupplementary Information: Supplementary data are available at Bioinformatics online.

Quantitative Proteomics Reveals that miR-155 Regulates the PI3K-AKT Pathway in Diffuse Large B-Cell Lymphoma

The aberrant expression of microRNA-155 (miR-155), which has emerged as having a significant impact on the biological characteristics of lymphocytes, plays important roles in B-cell malignancies, such as diffuse large B-cell lymphoma (DLBCL). DLBCL is the most common non-Hodgkin's lymphoma in the adult population, accounting for approximately 40% of newly diagnosed non-Hodgkin's lymphoma cases globally. To determine the specific function of miR-155, a quantitative proteomics approach was applied to examine the inhibitory effects of miR-155 on protein synthesis in DLBCL cells. PIK3R1 (p85α), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, was identified as a direct target of miR-155. A luciferase reporter was repressed through the direct interaction of miR-155 and the p85α 3'-untranslated region, and overexpression of miR-155 down-regulated both the transcription and translation of p85α. The PI3K-AKT signaling pathway was highly activated by the sustained overexpression of miR-155 in DHL16 cells, whereas knockdown of miR-155 in OCI-Ly3 cells diminished AKT activity. Taken together, our results reveal a novel target involved in miR-155 biological characteristics and provide a molecular link between the overexpression of miR-155 and the activation of PI3K-AKT in DLBCL.

Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint

Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that Ir-induced G2/M arrest contributed to the increase in the mitochondrial ROS level by accumulating cells in the G2/M phase.

VMckpt: lightweight and live virtual machine checkpointing

Recent advance of virtualization technology provides a new approach to check-point/restart at the virtual machine (VM) level. In contrast to traditional process-level checkpointing, checkpointing at the virtualization layer brings up several advantages, such as compatibility, transparence, flexibility and simplicity. However, because the virtualization layer has little semantic knowledge about the operation system and the applications running atop, VM-layer checkpointing requires saving the entire operating system state rather than a single process. The overhead may render the approach impractical. To reduce the size of VM checkpoint, in this paper we propose a page eviction scheme and an incremental checkpointing mechanism to avoid saving unnecessary VM pages in the checkpoint. To keep the system online transparently, we propose a live checkpointing mechanism by saving the memory image in a copy-on-write (COW) manner. We implement the performance optimization mechanisms in a prototype system, called VMckpt. Experimental results with a group of representative applications show that our page eviction scheme and incremental checkpointing can significantly reduce the checkpoint file size by up to 87% and shorten the total checkpointing/restart time by a factor of up to 71%, in comparison with the Xens default checkpointing mechanism. The observed application downtimes due to checkpointing can be reduced to as small as 300 ms.

Central Role of Ubiquitination in Genome Maintenance: DNA Replication and Damage Repair.

Faithful transmission of genetic information through generations ensures genomic stability and integrity. However, genetic alterations occur every now and then during the course of genome duplication. In order to repair these genetic defects and lesions, nature has devised several repair pathways which function promptly to prevent the cell from accumulating permanent mutations. These repair mechanisms seem to be significantly impacted by posttranslational modifications of proteins like phosphorylation and ubiquitination. Protein ubiquitination is emerging as a critical regulatory mechanism of DNA damage response. Non-proteolytic, proteasome-independent functions of ubiquitin involving monoubiquitination and polyubiquitination of DNA repair proteins contribute significantly to the signaling of DNA repair pathways. In this paper, we will particularly highlight the work on ubiquitin-mediated signaling in the repair processes involving the Fanconi anemia pathway, translesional synthesis, nucleotide excision repair, and repair of double-strand breaks. We will also discuss the role of ubiquitin ligases in regulating checkpoint mechanisms, the role of deubiquitinating enzymes, and the growing possibilities of therapeutic intervention in this ubiquitin-conjugation system.

BRCA1 tumor suppressor network: focusing on its tail

Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.

BRCA1 tumor suppressor network: focusing on its tail

Germline mutations of the BRCA1 tumor suppressor gene are a major cause of familial breast and ovarian cancer. BRCA1 plays critical roles in the DNA damage response that regulates activities of multiple repair and checkpoint pathways for maintaining genome stability. The BRCT domains of BRCA1 constitute a phospho-peptide binding domain recognizing a phospho-SPxF motif (S, serine; P, proline; × varies; F, phenylalanine). The BRCT domains are frequently targeted by clinically important mutations and most of these mutations disrupt the binding surface of the BRCT domains to phosphorylated peptides. The BRCT domain and its capability to bind phosphorylated protein is required for the tumor suppressor function of BRCA1. Through its BRCT phospho-binding ability BRCA1 forms at least three mutually exclusive complexes by binding to phosphorylated proteins Abraxas, Bach1 and CTIP. The A, B and C complexes, at lease partially undertake BRCA1's role in mechanisms of cell cycle checkpoint and DNA repair that maintain genome stability, thus may play important roles in BRCA1's tumor suppressor function.

Nucleosome assembly and genome integrity

Maintaining the stability of the replication forks is one of the main tasks of the DNA damage response. Specifically, checkpoint mechanisms detect stressed forks and prevent their collapse. In the published report reviewed here we have shown that defective chromatin assembly in cells lacking either H3K56 acetylation or the chromatin assembly factors CAF1 and Rtt106 affects the integrity of advancing replication forks, despite the presence of functional checkpoints. This loss of replication intermediates is exacerbated in the absence of Rad52, suggesting that collapsed forks are rescued by homologous recombination and providing an explanation for the accumulation of recombinogenic DNA damage displayed by these mutants. These phenotypes mimic those obtained by a partial reduction in the pool of available histones and are consistent with a model in which defective histone deposition uncouples DNA synthesis and nucleosome assembly, thus making the fork more susceptible to collapse. Here, we review these findings and discuss the possibility that defects in the lagging strand represent a major source of fork instability in chromatin assembly mutants.

Abstract A65: Targeting the defective decatenation checkpoint in melanoma.

Abstract In Australia melanomas account for 10% of all reported cancers and 3% of cancer mortalities. Current treatments offer <25% response rate and <8 months improved survival. Cancers frequently demonstrate defects in the cell cycle, particularly the checkpoint mechanisms. Loss of these protective mechanisms may provide opportunities for selectively targeting cancer cells. One G2 phase checkpoint, the decatenation checkpoint, ensures that entangled sister chromatids can be segregated with high fidelity during cellular replication. Topoisomerase II is responsible for resolving these tangles. Using ICRF-193, an inhibitor of Topoisomerase II, a panel of melanoma cell lines were investigated for decatenation checkpoint function. This analysis revealed that 17/25 melanoma cell lines possessed a defective decatenation checkpoint, suggesting it to be a common defect. Treatment with ICRF-193 caused checkpoint defective cells to undergo an aberrant mitosis, resulting in accumulation of cells exhibiting abnormal nuclear morphologies, while checkpoint functional cells remained relatively unaffected. Comparative gene expression analysis revealed differential expression of cell-division and cell-death associated genes, suggesting adaptive changes compensating for checkpoint failure. A strong gene-expression signature for the checkpoint defect was also observed in this analysis and potential bio-markers are being investigated for use identifying this defect in tumors. A synthetic lethality siRNA screen of the kinome (∼700 genes) was carried out to identify genes/pathways crucial to the viability of cell lines with a defective decatentation checkpoint. This screen identified 10 potential hits, many involved in PI3Kinase signaling. We have validated this pathway as a selective target. This research has shown that the defective decatenation checkpoint is a viable target in melanoma. Using targets identified by the synthetic lethality screens and bio-markers to the defect in tumors, it should be possible to identify tumors most likely to be sensitive to drugs targeting the checkpoint defect, providing effective and selective therapeutics for the treatment of metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A65.

Athanasia: A User-Transparent and Fault-Tolerant System for Parallel Applications

This article presents Athanasia, a user-transparent and fault-tolerant system, for parallel applications running on large-scale cluster systems. Cluster systems have been regarded as a de facto standard to achieve multitera-flop computing power. These cluster systems, as we know, have an inherent failure factor that can cause computation failure. The reliability issue in parallel computing systems, therefore, has been studied for a relatively long time in the literature, and we have seen many theoretical promises arise from the extensive research. However, despite the rigorous studies, practical and easily deployable fault-tolerant systems have not been successfully adopted commercially. Athanasia is a user-transparent checkpointing system for a fault-tolerant Message Passing Interface (MPI) implementation that is primarily based on the sync-and-stop protocol. Athanasia supports three critical functionalities that are necessary for fault tolerance: a light-weight failure detection mechanism, dynamic process management that includes process migration, and a consistent checkpoint and recovery mechanism. The main features of Athanasia are that it does not require any modifications to the application code and that it preserves many of the high performance characteristics of high-speed networks. Experimental results show that Athanasia can be a good candidate for practically deployable fault-tolerant systems in very-large and high-performance clusters and that its protocol can be applied to a variety of parallel communication libraries easily.

Fault Tolerant Architecture to Cloud Computing Using Adaptive Checkpoint

Cloud computing refers to both the applications delivered as services over the Internet and the hardware and systems software in the datacenters that provide those services. Failures of any type are common in current datacenters, partly due to the number of nodes. Fault tolerance has become a major task for computer engineers and software developers because the occurrence of faults increases the cost of using resources and to meet the user expectations, the most fundamental user expectation is, of course, that his or her application correctly finishes independent of faults in the node. This paper proposes a fault tolerant architecture to Cloud Computing that uses an adaptive Checkpoint mechanism to assure that a task running can correctly finish in spite of faults in the nodes in which it is running. The proposed fault tolerant architecture is simultaneously transparent and scalable.

Modular rollback through control logging

We present a technique, based on the use of first-class control operators, enabling programs to maintain and invoke rollback logs for sequences of reversible effects. Our technique is modular, in that it provides complete separation between some library of effectful operations, and a client, "driver" program which invokes and rolls back sequences of these operations. In particular, the checkpoint mechanism, which is entirely encapsulated within the effect library, logs not only the library's effects, but also the client's control state. Thus, logging and rollback can be almost completely transparent to the client code. This separation of concerns manifests itself nicely when we must implement software with sophisticated error handling. We illustrate with two examples that exploit the architecture to disentangle some core parsing task from its error management. The parser code is completely separate from the error-correction code, although the two components are deeply intertwined at run time.

Fault Recovery Based on Transaction Rollback in Transactional Memory

针对事务存储系统机制下的容错问题,提出一种基于事务回退的事务存储系统的故障恢复方法.该方法利用事务存储系统自身的版本管理机制,避免了额外的检查点数据保存开销,从而实现了事务存储系统高效的故障恢复.通过对容错事务存储系统的隔离性证明了该方法的正确性.最后,使用包括4 个SPLASH-2 典型用例在内的5 个测试程序对该方法进行了性能测试.实验结果表明,与经典的Checkpointing 机制相比,该方法在避免了额外的检查点数据保存开销的同时,还具有较低的故障恢复开销.

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

Deletions and duplications identified by 23 chromosome single nucleotide polymorphism (SNP) microarray are associated with aneuploidy

The Clinical Utility of the Protamine 1/Protamine 2 Ratio in Sperm

During spermiogenesis, human sperm undergo a dramatic reorganization of the chromatin in which canonical histones are replaced by two types of protamines, protamine 1 (P1) and protamine (P2). P1 and P2 are expressed approximately at a 1:1 ratio in healthy men. Alteration of this ratio is associated with male infertility. Patients with an abnormal P1/P2 ratio generally exhibit diminished semen quality, lower fertilization ability, and lower pregnancy rates when undergoing in vitro fertilization. Many studies have reported an elevated incidence of abnormal P1/P2 ratios in infertile men compared to fertile controls, and have evaluated the relationship between infertility and abnormal protamination; however, no prospective study has investigated the normal range of the P1/P2 ratio in men from the general population. Here, we report a P1/P2 reference range of 0.54 to 1.43 in a fertile, normozoospermic population. This rather wide normal range of P1/P2 led us to the conclusion that abnormal protamination is more likely indicative of other perturbations during spermatogenesis than the underlying mechanism to cause infertility. Alternatively, protamine expression may act as a checkpoint mechanism and thus be indirectly related to semen quality.

Fault Tolerance and Recovery for Grid Application Reliability using Check Pointing Mechanism

The check pointing mechanism and rollback recovery is a well-known method to achieve fault tolerance in grid computing systems. If any resource or process is tending to be faulty in run time that will be detected by check pointing mechanism through the Task Dependency Graph (TDG) and their respective worst case execution time and deadline parameters are used to decide the schedulability. The common approach is to use rollback-dependent graph or check point graph. The scheduling of concurrent tasks can be done using the proposed Concurrent Task Scheduling Algorithm (CTSA) algorithm to recover from the faulty states using replication or rollback techniques. The earlier fault detection methods are not scalable with the diversity of user applications and the frequency of faults varies dynamically making the faults hard to detect and recover. The check pointing and replication mechanisms have been used in high performance grid computing where the synchronization between communicating processes is needed to enhance the efficiency of check pointing mechanism. The performance improvements over the faulty conditions can be obtained with or without data and process replication. The experimental results show that the CTSA can lead to significant performance gain for a variety of scenarios.

Characterization of p53 expression in rainbow trout

The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells.

Scheduling of Computing Services on Intranet Networks

Nowadays, enterprises can provide computing services through their intranet networks by letting their available resources be used as virtual clusters for scientific computation during idle periods such as nights, weekends, and holidays. Generally, these idle periods do not permit to carry out the computations completely. It is therefore necessary to save the context of uncompleted applications for possible restart. This checkpointing mechanism is subject to resource constraints: the network bandwidth, the disk bandwidth, and the delay T imposed for releasing the workstations. We first introduce a function bw that gives the bandwidth bw(m,V) of a system during the checkpointing of m applications with aggregated memory requirement V. Assuming that this bandwidth is shared equitably among the applications, the scheduling problem becomes a sequence of knapsack problems with nonlinear constraints for which we propose approximate solutions. Experiments carried out on Grid5000 show that the running time of this algorithm is negligible compared to the delay T which is of the order of few minutes. This means that the proposed scheduling algorithm does not induce a significant overhead on the checkpointing process. As a consequence, our mechanism can be incorporated in a batch scheduler.