Checkpoint Adaptation Research Articles

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.

Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents

In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage.

The budding yeast polo-like kinase Cdc5 regulates the Ndt80 branch of the meiotic recombination checkpoint pathway

Defects in chromosome synapsis and/or meiotic recombination activate a surveillance mechanism that blocks meiotic cell cycle progression to prevent anomalous chromosome segregation and formation of aberrant gametes. In the budding yeast zip1 mutant, which lacks a synaptonemal complex component, the meiotic recombination checkpoint is triggered, resulting in extremely delayed meiotic progression. We report that overproduction of the polo-like kinase Cdc5 partially alleviates the meiotic prophase arrest of zip1, leading to the formation of inviable meiotic products. Unlike vegetative cells, we demonstrate that Cdc5 overproduction does not stimulate meiotic checkpoint adaptation because the Mek1 kinase remains activated in zip1 2μ-CDC5 cells. Inappropriate meiotic divisions in zip1 promoted by high levels of active Cdc5 do not result from altered function of the cyclin-dependent kinase (CDK) inhibitor Swe1. In contrast, CDC5 overexpression leads to premature induction of the Ndt80 transcription factor, which drives the expression of genes required for meiotic divisions, including CLB1. We also show that depletion of Cdc5 during meiotic prophase prevents the production of Ndt80 and that CDK activity contributes to the induction of Ndt80 in zip1 cells overexpressing CDC5. Our results reveal a role for Cdc5 in meiotic checkpoint control by regulating Ndt80 function.

Polo-like kinases and DNA damage checkpoint: beyond the traditional mitotic functions

Polo-like kinases (Plks) are a family of serine-threonine kinases that play a pivotal role in cell cycle progression and in cellular response to DNA damage. The Plks are highly conserved from yeast to mammals. There are five Plk family members (Plk1-5) in humans, of which Plk1, is the best characterized. The Plk1 isoform is being aggressively pursued as a target for cancer therapy, following observations that this protein is overexpressed in human tumors and is actively involved in malignant transformation. The roles of Plks in mitotic entry, spindle pole functions and cytokinesis are well established and have been the subject of several recent reviews. In this review, we discuss functions of Plks other than their classical roles in mitotic progression. When cells incur DNA damage, they activate checkpoint mechanisms that result in cell cycle arrest and allow time for repair. If the damage is extensive and cannot be repaired, cells will undergo cell death by apoptosis. If the damage is repaired, cells can resume cycling, as part of the process known as checkpoint recovery. If the damage is not repaired or incompletely repaired, cells can override the checkpoint and resume cycling with damaged DNA, a process called checkpoint adaptation. The Plks play a role in all three outcomes and their involvement in these processes will be the subject of this review.

Polymerase Epsilon Is Required To Maintain Replicative Senescence

Replicative senescence is a permanent cell cycle arrest in response to extensive telomere shortening. To understand the mechanisms behind a permanent arrest, we screened for factors affecting replicative senescence in budding yeast lacking telomere elongation pathways. Intriguingly, we found that DNA polymerase epsilon (Pol ε) acts synergistically with Exo1 nuclease to maintain replicative senescence. In contrast, the Pol ε-associated checkpoint and replication protein Mrc1 facilitates escape from senescence. To understand this paradox, in which DNA-synthesizing factors cooperate with DNA-degrading factors to maintain arrest, whereas a checkpoint protein opposes arrest, we analyzed the dynamics of double- and single-stranded DNA (ssDNA) at chromosome ends during senescence. We found evidence for cycles of DNA resection, followed by resynthesis. We propose that resection of the shortest telomere, activating a Rad24(Rad17)-dependent checkpoint pathway, alternates in time with an Mrc1-regulated Pol ε resynthesis of a short, double-stranded chromosome end, which in turn activates a Rad9(53BP1)-dependent checkpoint pathway. Therefore, instead of one type of DNA damage, different types (ssDNA and a double-strand break-like structure) alternate in a "vicious circle," each activating a different checkpoint sensor. Every time resection and resynthesis switches, a fresh signal initiates, thus preventing checkpoint adaptation and ensuring the permanent character of senescence.

Polo-like Kinase 1 Activated by the Hepatitis B Virus X Protein Attenuates Both the DNA Damage Checkpoint and DNA Repair Resulting in Partial Polyploidy

Hepatitis B virus X protein (pX), implicated in hepatocarcinogenesis, induces DNA damage because of re-replication and allows propagation of damaged DNA, resulting in partial polyploidy and oncogenic transformation. The mechanism by which pX allows cells with DNA damage to continue proliferating is unknown. Herein, we show pX activates Polo-like kinase 1 (Plk1) in the G(2) phase, thereby attenuating the DNA damage checkpoint. Specifically, in the G(2) phase of pX-expressing cells, the checkpoint kinase Chk1 was inactive despite DNA damage, and protein levels of claspin, an adaptor of ataxia telangiectasia-mutated and Rad3-related protein-mediated Chk1 phosphorylation, were reduced. Pharmacologic inhibition or knockdown of Plk1 restored claspin protein levels, Chk1 activation, and p53 stabilization. Also, protein levels of DNA repair protein Mre11 were decreased in the G(2) phase of pX-expressing cells but not with Plk1 knockdown. Interestingly, in pX-expressing cells, Mre11 co-immunoprecipitated with transfected Plk1 Polo-box domain, and inhibition of Plk1 increased Mre11 stability in cycloheximide-treated cells. These results suggest that pX-activated Plk1 by down-regulating Mre11 attenuates DNA repair. Importantly, concurrent inhibition of Plk1, p53, and Mre11 increased the number of pX-expressing cells with DNA damage entering mitosis, relative to Plk1 inhibition alone. By contrast, inhibition or knockdown of Plk1 reduced pX-induced polyploidy while increasing apoptosis. We conclude Plk1, activated by pX, allows propagation of DNA damage by concurrently attenuating the DNA damage checkpoint and DNA repair, resulting in polyploidy. We propose this novel Plk1 mechanism initiates pX-mediated hepatocyte transformation.

Abstract 684: Trifluoperazine modulates DNA damage-induced cell death in human lung cancer cells through inhibition of DNA repair, cell cycle delays, and augmentation of pro-apoptotic signaling

Abstract Trifluoperazine modulates DNA damage-induced cell death in human lung cancer cells through inhibition of DNA repair, cell cycle delays, and augmentation of pro-apoptotic signaling Resistance of human tumors to DNA damaging chemotherapy poses severe limitations to the efficacy of current available treatment. Small molecule compounds of the phenothiazine class, such as trifluoperazine (TFP), have documented chemosensitizing activities. We exposed human non-small cell lung cancer (NSCLC) cell lines to the DNA damaging drug bleomycin in the presence or absence of TFP, and studied potential differences with regard to DNA repair and cell death signaling. A colony formation assay showed that TFP significantly enhanced the cytotoxic potential of bleomycin, at doses achievable clinically. Bleomycin treatment resulted in a reversible arrest at the G2-M phase of the cell cycle. Notably, TFP delayed the repair of bleomycin-induced DNA double strand breaks (DSBs), causing higher residual levels of γH2AX 24 h after treatment. Despite the presence of unrepaired DNA DSBs, cells co-treated with bleomycin and TFP overcame the G2-M checkpoint arrest, as evidenced by the reappearance of the mitosis marker histone H3 phospho-Ser10 and reduction in CFSE fluorescence. This process, which is reminiscent of checkpoint adaptation, was associated with increased micronucleation and secondary arrest in G2-M phase during subsequent rounds of the cell cycle. Furthermore, the onset of secondary checkpoint arrest in cells co-treated with bleomycin and TFP coincided with the activation of pro-apoptotic Bak and Bax, and was followed by activation of caspase-3, which occurred preferentially in cells in the G2-M phase. In conclusion, we demonstrate here that the chemosensitizing effect of TFP in NSCLC cells involves interference with DNA DSBs repair, resulting in secondary G-M checkpoint arrest after initial checkpoint adaptation, and concomitant increases in pro-apoptotic signaling. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 684.

Analysis of Polo-like kinase Cdc5 in the meiosis recombination checkpoint

In meiosis, accumulation of recombination intermediates or defects in chromosome synapsis trigger checkpoint-mediated arrest in prophase I. Such 'checkpoints' are important surveillance mechanisms that ensure temporal dependence of cell cycle events. The budding yeast Polo-like kinase, Cdc5, has been identified as a key regulator of the meiosis I chromosome segregation pattern. Here we have analysed the role of Cdc5 in the recombination checkpoint and observed that Polo-like kinase is not required for checkpoint activation in yeast meiosis. Surprisingly, depletion of CDC5 in the Drad17 checkpoint-defective background resulted in nuclear fragmentation to levels even higher than that observed inDdmc1 Drad17 cells that bypass the checkpoint arrest despite accumulating DNA double-strand breaks. The spindle morphology of Cdc5-depleted cells included short, thick metaphase I spindles in mononucleate cells and disassembled spindles in binucleate and tetranucleate cells, although this phenotype does not appear to be the cause of the nuclear fragmentation. An exaggeration of chromosome synapsis defects occurred in Cdc5-depleted Drad17 cells and may contribute to the nuclear fragmentation phenotype. The analysis also uncovered a role for Cdc5 in maintaining spindle integrity in Ddmc1 Drad17 cells. Further analysis confirmed that adaptation to DNA damage does occur in meiosis and that CDC5 is required for this process. The cdc5-ad mutation that renders cells unable to adapt to DNA damage in mitosis did not affect checkpoint adaptation in meiosis, indicating that the mechanisms of checkpoint adaptation in mitosis and meiosis are not fully conserved.

CDC5 Inhibits the Hyperphosphorylation of the Checkpoint Kinase Rad53, Leading to Checkpoint Adaptation

Author SummaryCellular surveillance mechanisms, termed checkpoints, have evolved to recognize the presence of DNA damage, halt cell division, and promote repair. The purpose of these checkpoints is to prevent the next generation of cells from inheriting a damaged genome. However, after futile attempts at repair over several hours of growth arrest, yeast cells eventually adapt and continue with cell division despite the presence of persistent DNA lesions. This process of adaptation employs CDC5, a kinase that also has essential roles in promoting cell division in the absence of DNA damage. We found that increasing levels of Cdc5 promote adaptation by suppressing the hyperphosphorylation of the checkpoint kinase Rad53, which in turn suppresses the DNA damage checkpoint and relieves cell division arrest. Intriguingly, overexpression of PLK1, the human homolog of CDC5, has been reported in various tumor types and has been linked to poor prognosis. Therefore, understanding the mechanism of adaptation in yeast may provide valuable insight into the role of PLK1 overexpression in tumor progression. Two related papers, published in PLoS Biology (van Vugt et al., doi:10.1371/journal.pbio.1000287) and PLoS Genetics (Donnianni et al., doi:10.1371/journal.pgen.1000763), similarly investigate the phenomenon of checkpoint adaptation.

Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation

In budding yeast the evolutionarily conserved checkpoint response varies in its sensitivity to DNA damaging agents through the cell cycle. Specifically, higher amounts of damage are needed to activate the downstream checkpoint kinase Rad53 in S-phase cells. We examined here whether phosphorylation of Rad53 itself by cell cycle-dedicated kinases regulates Rad53 activation. We found that during unperturbed growth Rad53 exhibits a small phosphorylation-dependent electrophoretic mobility shift in G2, M and G1 phases of the cell cycle that is lost in S phase. We show that Rad53 is phosphorylated in vitro by Cdc5, a mitotic Polo-like kinase, and by the yeast cyclin-dependent kinase, Cdc28. Consistently, the cell cycle-dependent Rad53 mobility shift requires both Cdc5 and Cdc28 activities. We mapped the in vitro targeted phosphorylation sites by mass spectrometry and confirmed with mass spectroscopy that serines 774, 789 and 791 within Rad53 are phosphorylated in vivo in M-phase arrested cells. By creating nonphosphorylatable mutations in the endogenous RAD53 gene, we confirmed that the CDK and Polo kinase target sites are responsible for the observed cell cycle-dependent shift in protein mobility. The loss of phospho-acceptor sites does not interfere with Rad53 activation but accelerates checkpoint adaptation after induction of a single irreparable double-strand break. We thus demonstrate that cell cycle-dependent phosphorylation can fine-tune the response of Rad53 to DNA damage.

A role for Chk2 in DNA damage induced mitotic delays in human colorectal cancer cells

Progression into mitosis in the presence of DNA damage leads to spindle checkpoint (SAC) dependent mitotic delays and cytokinesis failure. In Drosophila embryos, DNA damage does not delay mitotic entry but triggers Chk2 kinase dependent delays in mitotic exit. It is unclear if damage associated mitotic delays in human cells result from kinase signaling or breaks in centromere DNA that disrupt kinetochore function and activate the SAC. We show that transgenic expression of Human Chk2 in a Drosophila chk2 mutant background restores damage induced mitotic delays during early embryogenesis. Parental HCT116 colorectal cancer cells that progress into mitosis following DNA damage, due to either G2 checkpoint adaptation or G2 checkpoint abrogation by caffeine or the Chk1 inhibitor UCN-01, delay in mitosis and show high rates of cytokinesis failure. Significantly, these mitotic responses are suppressed in HCT116 chk2 knockout cells, and the response is restored by transgenic expression of wild type Chk2. However, both parental and chk2-/-HCT116 cells arrested in G2 for prolonged periods by DNA damage prior to release from the G2 block do show significant mitotic delays. Chk2 thus appears to have a conserved function in control of mitotic progression following G2/M transition with DNA damage. However, prolonged G2 arrest with DNA damage can trigger Chk2-independent mitotic delays that may be secondary to kinetochore disruption.

Aberrant Polo-Like Kinase 1-Cdc25A Pathway in Metastatic Hepatocellular Carcinoma

Most studies on pathogenesis of tumor metastasis focus on cell adhesion and migration. Little is understood of how cell cycle pathways critically affect cell fate of metastatic cells and their sensitivity to anticancer drugs. In this study, we investigated cell cycle checkpoint progression and regulation in the presence of cisplatin in metastatic hepatocellular carcinoma (HCC) cells. Cisplatin-mediated cell cycle progression and Polo-like kinase 1 (Plk1)-Cdc25A pathway were compared between metastatic and nonmetastatic HCC cells by flow cytometry, Western blots, and reverse transcription-PCR. Cdc25A expression in clinical HCC samples was detected using immunohistochemistry and its association with clinical HCC metastasis was analyzed. Cisplatin induced degradation of Cdc25A in nonmetastatic HCC cells but not in metastatic HCC cells. Hence, metastatic HCC cells showed defective S-M cell cycle phase arrest and continued to enter mitosis. Tumor expression of Cdc25A was strongly associated with metastatic diseases in HCC patients, and elevated Cdc25A expression significantly correlated with HCC tumor-node-metastasis staging and venous invasion. Metastatic HCC cells did not show down-regulation of Plk1 that was normally induced by DNA damage. Blockage of Plk1 expression in metastatic HCC cells initiated Cdc25A degradation in response to DNA damage, suggesting that Plk1 could be an upstream regulator of Cdc25A. Deregulated Plk1-Cdc25A pathway in metastatic HCC cells and primary tumors did not result in drug-induced mitotic catastrophe but rather in accumulation of damaged DNA due to checkpoint adaptation. Metastatic HCC cells showed a defective S-M checkpoint following cisplatin treatment and potential aberrant checkpoint adaptation, which might result from deregulation of Plk1-Cdc25A pathway.

Regulation of Intra-S Phase Checkpoint by Ionizing Radiation (IR)-dependent and IR-independent Phosphorylation of SMC3

Structure maintenance of chromosome 1 (SMC1) is phosphorylated by ataxia telangiectasia-mutated (ATM) in response to ionizing radiation (IR) to activate intra-S phase checkpoint. A role of CK2 in DNA damage response has been implicated in many previous works, but the molecular mechanism for its activation is not clear. In the present work, we report that SMC3 is phosphorylated at Ser-1067 and Ser-1083 in vivo. Ser-1083 phosphorylation is IR-inducible, depends on ATM and Nijmegen breakage syndrome 1 (NBS1), and is required for intra-S phase checkpoint. Interestingly, Ser-1067 phosphorylation is constitutive and is not induced by IR but also affects intra-S phase checkpoint. Phosphorylation of Ser-1083 is weakened in cells expressing S1067A mutant, suggesting interplay between Ser-1067 and Ser-1083 phosphorylation in DNA damage response. Consistently, small interfering RNA knockdown of CK2 leads to attenuated phosphorylation of Ser-1067 as well as intra-S phase checkpoint defect. Our data provide evidence that phosphorylation of a core cohesin subunit SMC3 by ATM plays an important role in DNA damage response and suggest that a constitutive phosphorylation by CK2 may affect intra-S phase checkpoint by modulating SMC3 phosphorylation by ATM.

Checkpoint adaptation in human cells

Checkpoint adaptation was originally described in Saccharomyces cerevisiae as the ability to divide following a sustained checkpoint arrest in the presence of unrepairable DNA breaks. A process of checkpoint adaptation was also reported in Xenopus in response to the replication inhibitor aphidicolin. Recently, we showed that checkpoint adaptation also occurs in human cells. Although cells undergoing checkpoint adaptation will frequently die in subsequent cell cycles owing to excessive DNA damage, some of the cells may be able to survive and proliferate with damaged DNA. Thus, checkpoint adaptation in human cells may potentially promote genomic instability and lead to cancer. Here, I discuss the current evidence for checkpoint adaptation in human cells and possible mechanisms and implications of this phenomenon.

G2 checkpoint‐dependent DNA repair and its response to catalase in Down syndrome and control lymphocyte cultures

The amount of DNA lesions repaired in G2 and also G2 timing are controlled by the DNA damage-dependent checkpoint. Down syndrome (DS) lymphocytes showed twice as much constitutive DNA damage in G2 than control ones, when recording it as chromosomal aberrations in metaphase, after caffeine-induced checkpoint abrogation. During G2, DS lymphocytes repaired 1.5 times more DNA lesions than control ones. However the DS cells displayed a decreased threshold for checkpoint adaptation, as the spontaneous override of the G2 to mitosis transition block induced by the checkpoint took place in the DS cells when they had three times more DNA lesions than controls. Catalase addition to cultures scavenges hydrogen peroxide diffused from cells, resulting in subsequent intracellular depletion (Antunes and Cadenas, 2000). The intracellular H2O2 level seemed to regulate the G2 checkpoint. Thus, in controls, H2O2 depletion (induced by 3.2-50 microg/mL catalase) prevented its functioning: chromosomal damage increased while G2 shortened. Conversely, in the DS lymphocytes, 12.5 microg/mL catalase lengthened G2 and decreased chromosomal damage, in spite that the amount of DNA repaired in G2 was half of that repaired in the catalase-free DS lymphocytes.

Adaptation to the Ionizing Radiation–Induced G2 Checkpoint Occurs in Human Cells and Depends on Checkpoint Kinase 1 and Polo-like Kinase 1 Kinases

Checkpoint adaptation was originally defined in yeast as the ability to divide despite the presence of damaged DNA. An important unanswered question is whether checkpoint adaptation also occurs in human cells. Here, we show that following the ionizing radiation-induced G(2) checkpoint, human osteosarcoma cells entered mitosis with gamma-H2AX foci, a marker for unrepaired DNA double-strand breaks. Exit from the G(2) checkpoint was accelerated by inhibiting the checkpoint kinase 1 (Chk1) and delayed by overexpressing wild-type Chk1 or depleting the Polo-like kinase 1 (Plk1). Chk1 and Plk1 controlled this process, at least partly, via independent signaling pathways. Our results suggest that human cells are able to exit the checkpoint arrest and divide before the damage has been fully repaired. Such cell division in the presence of damaged DNA may be detrimental for genetic stability and could potentially contribute to cancer development.

CDK Pho85 targets CDK inhibitor Sic1 to relieve yeast G1 checkpoint arrest after DNA damage

In budding yeast, DNA damage in G1 activates a Rad9-dependent checkpoint that targets the cyclin-dependent kinase (CDK) Cdc28 to delay G1 exit. After a transient arrest, cells may enter S phase before completing DNA repair. We used genetic analysis to identify the stress-responsive CDK Pho85, the cyclin Pho80 and the targeted transcription factors Pho4 and Swi5 as determinants of G1 checkpoint adaptation. Consistent with opposing roles for the Cdc28 inhibitor Sic1 in blocking G1 exit and Pho85 in targeting Sic1 for proteolysis, mutation of Sic1 curtails G1 checkpoint delay, whereas Pho85 inhibition after DNA damage promotes Sic1 stability. G1 checkpoint delay in mutants lacking both Sic1 and Pho4 is independent of Pho85 activity. These data establish a G1 checkpoint adaptation pathway where Pho85 mediates Pho4 downregulation and Sic1 degradation to release Cdc28 activity and promote onset of S phase.

Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptationin response to DNA damage

Ino80 and Swr1 are ATP-dependent chromatin remodeling enzymes that have been implicated in DNA repair. Here we show that Ino80 is required for cell cycle checkpoint adaptation in response to a persistent DNA double-strand break (DSB). The failure of cells lacking Ino80 to escape checkpoint arrest correlates with an inability to maintain high levels of histone H2AX phosphorylation and an increased incorporation of the Htz1p histone variant into chromatin surrounding the DSB. Inactivation of Swr1 eliminates this DNA damage-induced Htz1p incorporation and restores H2AX phosphorylation and checkpoint adaptation. We propose that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, and that they regulate the incorporation of different histone H2A variants that can either promote or block cell cycle checkpoint adaptation.

CHROMATIN REMODELING: REGULATION OF CHROMATIN HIGHER‐ORDER FOLDING AND DNA REPAIR

Dynamic changes in chromosome structure are catalyzed by ATP-dependent chromatin remodeling enzymes and by histone modifying enzymes, such as histone acetyltransferases. We will present two stories that illustrate distinct roles for both types of chromatin-based enzymes. First, we will discuss our recent work on Ino80 and Swr1 which are ATP-dependent chromatin remodeling enzymes that have been implicated in DNA repair. We have found that Ino80 is required for maintanence of high levels of histone H2AX phosphorylation and for cell cycle checkpoint adaptation in response to a persistent DNA double strand break (DSB). Our data indicate that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, and that they regulate the incorporation of different histone H2A variants that can either promote or block cell cycle checkpoint adaptation. The second story will focus on histone H4 lysine 16 acetylation which is a prevalent and reversible post-translational modification in eukaryotic organisms. We have used a native chemical ligation strategy to generate histone H4 homogeneously acetylated at K16. When this histone is incorporated into nucleosomal arrays, we find that acetylation of this single lysine residue inhibits the formation of compact 30 nm-like fibers and impedes the ability of chromatin to form cross-fiber interactions. This is the first example of a single histone modification that is sufficient to modulate higher-order chromatin structure.

The G2 checkpoint activated by DNA damage does not prevent genome instability in plant cells

Root growth, G2 length, and the frequency of aberrant mitoses and apoptotic nuclei were recorded after a single X-ray irradiation, ranging from 2.5 to 40 Gy, in Allium cepa L. root meristematic cells. After 72 h of recovery, root growth was reduced in a dose-dependent manner from 10 to 40 Gy, but not at 2.5 or 5 Gy doses. Flow cytometry plus TUNEL (TdT-mediated dUTP nick end labeling) showed that activation of apoptosis occurred only after 20 and 40 Gy of X-rays. Nevertheless, irrespective of the radiation dose, conventional flow cytometry showed that cells accumulated in G2 (4C DNA content). Simultaneously, the mitotic index fell, though a mitotic wave appeared later. Cell accumulation in G2 was transient and partially reversed by caffeine, thus it was checkpoint-dependent. Strikingly, the additional G2 time provided by this checkpoint was never long enough to complete DNA repair. Then, in all cases, some G2 cells with still-unrepaired DNA underwent checkpoint adaptation, i.e., they entered into the late mitotic wave with chromatid breaks. These cells and those produced by the breakage of chromosomal bridges in anaphase will reach the G1 of the next cell cycle unrepaired, ensuring the appearance of genome instability.