A FOXO-dependent replication checkpoint restricts proliferation of damaged cells

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Sensing, signaling, and responding to DNA damage: organization of the checkpoint pathways in mammalian cells

The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. Unlike typical signal transduction mechanisms that respond to one or a few stimuli, checkpoints can be activated by a broad spectrum of extrinsically or intrinsically derived DNA damage or replication interference. Recent investigations have shed light on how the damage and replication checkpoints are able to respond to such diverse stimuli. The activation of checkpoints not only attenuates cell cycle progression but also facilitates DNA repair and recovery of faltered replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. Recently, more checkpoint targets from the cell cycle and DNA replication apparatus have been identified, revealing the increasing complexity of the checkpoint control of the cell cycle. In this article, we discuss current models of the DNA damage and replication checkpoints and highlight recent advances in the field.     

Mutation of the mouse Rad17 gene leads to embryonic lethality and reveals a role in DNA damage-dependent recombination

Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents.     

细胞周期检控点

细胞周期是高度有组织的时序调控过程,受到DNA损伤检控点、DNA复制检控点和纺锤体检控点等细胞周期检控点的精确调控。细胞周期检控点的作用主要是调节细胞周期的时序转换,以确保DNA复制、染色体分离等细胞重要生命活动的高度精确性,并对DNA损伤、DNA复制受阻、纺锤体组装和染色体分离异常等细胞损伤及时做出反应,以防止突变和遗传不稳定的发生。细胞周期检控点的功能缺陷,将导致细胞基因组的不稳定,与细胞癌变密切相关。因此细胞周期检控点对于维持细胞遗传信息的稳定性和完整性以及防止细胞癌变和遗传疾病的发生起着至关重要的作用。     

Flap Endonuclease 1 Limits Telomere Fragility on the Leading Strand

The existence of redundant replication and repair systems that ensure genome stability underscores the importance of faithful DNA replication. Nowhere is this complexity more evident than in challenging DNA templates, including highly repetitive or transcribed sequences. Here, we demonstrate that flap endonuclease 1 (FEN1), a canonical lagging strand DNA replication protein, is required for normal, complete leading strand replication at telomeres. We find that the loss of FEN1 nuclease activity, but not DNA repair activities, results in leading strand-specific telomere fragility. Furthermore, we show that FEN1 depletion-induced telomere fragility is increased by RNA polymerase II inhibition and is rescued by ectopic RNase H1 expression. These data suggest that FEN1 limits leading strand-specific telomere fragility by processing RNA:DNA hybrid/flap intermediates that arise from co-directional collisions occurring between the replisome and RNA polymerase. Our data reveal the first molecular mechanism for leading strand-specific telomere fragility and the first known role for FEN1 in leading strand DNA replication. Because FEN1 mutations have been identified in human cancers, our findings raise the possibility that unresolved RNA:DNA hybrid structures contribute to the genomic instability associated with cancer.     

Mitochondrial reactive oxygen species-mediated genomic instability in low-dose irradiated human cells through nuclear retention of cyclin D1

Mitochondria are associated with various radiation responses, including adaptive responses, mitophagy, the bystander effect, genomic instability, and apoptosis. We recently identified a unique radiation response in the mitochondria of human cells exposed to low-dose long-term fractionated radiation (FR). Such repeated radiation exposure inflicts chronic oxidative stresses on irradiated cells via the continuous release of mitochondrial reactive oxygen species (ROS) and decrease in cellular levels of the antioxidant glutathione. ROS-induced oxidative mitochondrial DNA (mtDNA) damage generates mutations upon DNA replication. Therefore, mtDNA mutation and dysfunction can be used as markers to assess the effects of low-dose radiation. In this study, we present an overview of the link between mitochondrial ROS and cell cycle perturbation associated with the genomic instability of low-dose irradiated cells. Excess mitochondrial ROS perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of protein phosphatase 2A after low-dose long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 induces genomic instability in low-dose irradiated cells.     

The significance of telomeric aggregates in the interphase nuclei of tumor cells

Telomeres are TTAGGG repetitive motifs found at the ends of vertebrate chromosomes. In humans, telomeres are protected by shelterin, a complex of six proteins (de Lange [2005] Genes Dev. 19: 2100-2110). Since (Müller [1938] Collecting Net. 13: 181-198; McClintock [1941] Genetics 26: 234-282), their function in maintaining chromosome stability has been intensively studied. This interest, especially in cancer biology, stems from the fact that telomere dysfunction is linked to genomic instability and tumorigenesis (Gisselsson et al. [2001] Proc. Natl. Acad. Sci. USA 98: 12683-12688; Deng et al. [2003] Genes Chromosomes Cancer 37: 92-97; DePinho and Polyak [2004] Nat. Genetics 36: 932-934; Meeker et al. [2004] Clin. Cancer Res. 10: 3317-3326). In the present overview, we will discuss the role of telomeres in genome stability, recent findings on three-dimensional (3D) changes of telomeres in tumor interphase nuclei, and outline future avenues of research.     

APOBEC3A damages the cellular genome during DNA replication

The human APOBEC3 family of DNA-cytosine deaminases comprises 7 members (A3A-A3H) that act on single-stranded DNA (ssDNA). The APOBEC3 proteins function within the innate immune system by mutating DNA of viral genomes and retroelements to restrict infection and retrotransposition. Recent evidence suggests that APOBEC3 enzymes can also cause damage to the cellular genome. Mutational patterns consistent with APOBEC3 activity have been identified by bioinformatic analysis of tumor genome sequences. These mutational signatures include clusters of base substitutions that are proposed to occur due to APOBEC3 deamination. It has been suggested that transiently exposed ssDNA segments provide substrate for APOBEC3 deamination leading to mutation signatures within the genome. However, the mechanisms that produce single-stranded substrates for APOBEC3 deamination in mammalian cells have not been demonstrated. We investigated ssDNA at replication forks as a substrate for APOBEC3 deamination. We found that APOBEC3A (A3A) expression leads to DNA damage in replicating cells but this is reduced in quiescent cells. Upon A3A expression, cycling cells activate the DNA replication checkpoint and undergo cell cycle arrest. Additionally, we find that replication stress leaves cells vulnerable to A3A-induced DNA damage. We propose a model to explain A3A-induced damage to the cellular genome in which cytosine deamination at replication forks and other ssDNA substrates results in mutations and DNA breaks. This model highlights the risk of mutagenesis by A3A expression in replicating progenitor cells, and supports the emerging hypothesis that APOBEC3 enzymes contribute to genome instability in human tumors.     

Generation and characterization of cells that can be conditionally depleted of mitochondrial SOD2

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.     

Direct imaging of DNA in living cells reveals the dynamics of chromosome formation

Individual chromosomes are not directly visible within the interphase nuclei of most somatic cells; they can only be seen during mitosis. We have developed a method that allows DNA strands to be observed directly in living cells, and we use it to analyze how mitotic chromosomes form. A fluorescent analogue (e.g., Cy5-dUTP) of the natural precursor, thymidine triphosphate, is introduced into cells, which are then grown on the heated stage of a confocal microscope. The analogue is incorporated by the endogenous enzymes into DNA. As the mechanisms for recognizing and removing the unusual residues do not prevent subsequent progress around the cell cycle, the now fluorescent DNA strands can be followed as they assemble into chromosomes, and segregate to daughters and granddaughters. Movies of such strands in living cells suggest that chromosome axes follow simple recognizable paths through their territories during G2 phase, and that late replicating regions maintain their relative positions as prophase chromosomes form. Quantitative analysis confirms that individual regions move little during this stage of chromosome condensation. As a result, the gross structure of an interphase chromosome territory is directly related to that of the prophase chromosome.     

Effects of oriented substrates on cell morphology,the cell cycle,and the cytoskeleton in Ros 17/2.8 cells

Absence of gravity or microgravity influences the cellular functions of bone forming osteoblasts. The underlying mechanism, however, of cellular sensing and responding to the gravity vector is poorly understood. This work quantified the impact of vector-directional gravity on the biological responses of Ros 17/2.8 cells grown on upward-, downward- or edge-on-oriented substrates. Cell morphology and nuclear translocation, cell proliferation and the cell cycle, and cytoskeletal reorganization were found to vary significantly in the three orientations. All of the responses were duration-dependent. These results provide a new insight into understanding how osteoblasts respond to static vector-directional gravity.     

Cell cycle effects of hypertonic stress on various human cells in culture

Long-term exposure to hypertonic (HT) culture media has been found to perturb the cell cycle and change gene expression in various animal cell types. A lower growth rate, with exit of cells from the cycling compartment has been observed previously in human transformed EUE. cells. The aim of this study was to investigate if the kinetic changes after long-term HT stress, were typical of transformed cells or could be also found in primary cultures of normal cells. Human transformed cells from normal and neoplastic tissues, and normal human cells of epithelial and connective origin have been studied. After the incorporation of bromodeoxyuridine (BrdUrd), the frequency of S-phase cells was estimated by dual-parameter flow cytometry of DNA content versus BrdUrd immuno-labelling; the total growth fraction was also estimated, after immunolabelling with an anti-PCNA antibody. We also investigated, by polyacrylamide gel electrophoresis, changes in the amount of a 35 kDa protien band, which increased in EUE cells grown in an HT medium, and which may be directly involved in cell resistance to hypertonicity. Lower BrdUrd labelling indices and higher frequencies of cells in the G_0/1 range of DNA content were common features of all the cells in HT media, irrespective of their tissue of origin; other cycle phases may also be involved, depending on the cell type considered. The mechanisms by which cells cope with the HT environment could however, differ, since only some cell types showed an increase of the 35 kDa stress protein found originally in HT EUE cells.     

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.     

Loss of Geminin induces rereplication in the presence of functional p53

Strict regulation of DNA replication is essential to ensure proper duplication and segregation of chromosomes during the cell cycle, as its deregulation can lead to genomic instability and cancer. Thus, eukaryotic organisms have evolved multiple mechanisms to restrict DNA replication to once per cell cycle. Here, we show that inactivation of Geminin, an inhibitor of origin licensing, leads to rereplication in human normal and tumor cells within the same cell cycle. We found a CHK1-dependent checkpoint to be activated in rereplicating cells accompanied by formation of gammaH2AX and RAD51 nuclear foci. Abrogation of the checkpoint leads to abortive mitosis and death of rereplicated cells. In addition, we demonstrate that the induction of rereplication is dependent on the replication initiation factors CDT1 and CDC6, and independent of the functional status of p53. These data show that Geminin is required for maintaining genomic stability in human cells.     

Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S‐phase checkpoint

The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1‐dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single‐strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1‐dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3′ ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.