A novel role for Greatwall kinase in recovery from DNA damage

Activation of the DNA damage response (DDR) is critical for genomic integrity and tumor suppression. The occurrence of DNA damage quickly evokes the DDR through ATM/ATR-dependent signal transduction, which promotes DNA repair and activates the checkpoint to halt cell cycle progression (Halazonetis et al., 2008; Motoyama and Naka, 2004; Zhou and Elledge, 2000). The "turn off" process of the DDR upon satisfaction of DNA repair, also known as "checkpoint recovery", involves deactivation of DDR elements, but the mechanism is poorly understood. Greatwall kinase (Gwl) has been identified as a key element in the G2/M transition (Archambault et al., 2007; Jackson, 2006; Zhao et al., 2008; Yu et al., 2004; Yu et al., 2006; Zhao et al., 2006) and helps maintain M phase through inhibition of PP2A/B55δ (Burgess et al., 2010; Castilho et al., 2009; Goldberg, 2010; Lorca et al., 2010; Vigneron et al., 2009), the principal phosphatase for Cdk-phosphorylated substrates. Here we show that Gwl also promotes recovery from DNA damage and is itself directly inhibited by the DNA damage response (DDR). In Xenopus egg extracts, immunodepletion of Gwl increased the DDR to damaged DNA, whereas addition of wild type, but not kinase dead Gwl, inhibited the DDR. The removal of damaged DNA from egg extracts leads to recovery from checkpoint arrest and entry into mitosis, a process impaired by Gwl depletion and enhanced by Gwl over-expression. Moreover, activation of Cdk1 after the removal of damaged DNA is regulated by Gwl. Collectively, these results defines Gwl as a new regulator of the DDR, which plays an important role in recovery from DNA     

Drosophila Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis.

Chk2 is a major target of ataxia telangiectasia-mutated (ATM) and ATM- and Rad3-related (ATR). Germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, suggesting that Chk2 is a tumor suppressor gene. To investigate the role of Chk2 in multicellular organisms, a Drosophila chk2 (Dmchk2) mutant was generated. Dmchk2 mutants are viable but show defects in maintaining genome stability and are highly sensitive to ionizing radiation. Interestingly, mutating Dmchk2 completely blocks DNA damage-induced apoptosis and partially blocks DNA damage-induced cell cycle arrest. These results indicate that Chk2 protein plays a crucial role in the DNA damage response pathway mediating cell cycle arrest and apoptosis, and that the ATM-Chk2 pathway is likely conserved in Drosophila.     

Expression of various genes is controlled by DNA methylation during mammalian development

Despite thousands of articles about 5-methylcytosine (m(5)C) residues in vertebrate DNA, there is still controversy concerning the role of genomic m(5)C in normal vertebrate development. Inverse correlations between expression and methylation are seen for many gene regulatory regions [Heard et al., 1997; Attwood et al., 2002; Plass and Soloway, 2002] although much vertebrate DNA methylation is in repeated sequences [Ehrlich et al., 1982]. At the heart of this debate is whether vertebrate DNA methylation has mainly a protective role in limiting expression of foreign DNA elements and endogenous transposons [Walsh and Bestor, 1999] or also is important in the regulation of the expression of diverse vertebrate genes involved in differentiation [Attwood et al., 2002]. Enough thorough studies have now been reported to show that many tissue- or development-specific changes in methylation at vertebrate promoters, enhancers, or insulators regulate expression and are not simply inconsequential byproducts of expression differences. One line of evidence comes from mutants with inherited alterations in genes encoding DNA methyltransferases and from rodents or humans with somatically acquired changes in DNA methylation that illustrate the disease-producing effects of abnormal methylation. Another type of evidence derives from studies of in vivo correlations between tissue-specific changes in DNA methylation and gene expression coupled with experiments demonstrating cause-and-effect associations between DNA hyper- or hypomethylation and gene expression. In this review, I summarize some of the strong evidence from both types of studies. Taken together, these studies demonstrate that DNA methylation in mammals modulates expression of many genes during development, causing major changes in or important fine-tuning of expression. Also, I discuss previously established and newly hypothesized mechanisms for this epigenetic control.     

Acetylation of Werner protein at K1127 and K1117 is important for nuclear trafficking and DNA repair

Werner syndrome is a rare autosomal recessive disorder where Werner (WRN) gene is mutated. Being a nucleolar protein, during DNA damage, WRN translocates at the damage site where its catalytic function is required in DNA repair. Several studies have indicated that WRN acetylation may modulate WRN trafficking and catalytic function (Blander et al., 2002; Lozada et al., 2014). Among the six acetylation sites in WRN protein identified by mass-spectrometry analysis (Li et al., 2010) we here explore the role of acetylation sites in C-terminal of WRN (K1127, K1117, K1389, K1413) because the C- terminal domain is the hub for protein- protein interaction and DNA binding activity (Brosh et al. [4]; Muftuoglu et al., 2008; Huang et al., 2006). To explore their functional activity, we created mutations in these sites by changing the acetylation residue lysine (K) to a non-acetylation residue arginine (R) and expressed them in WRN mutant cell lines. We observed that K1127R and K1117R mutants are sensitive to the DNA damaging agents etoposide and mitomycin C and display deficient DNA repair. Importantly, deacetylation of WRN by SIRT1 (Mammalian Sir2) is necessary for restoration of WRN localization at nucleoli after completion of DNA repair. Among all putative acetylation sites, K1127R, K1117R and the double mutant K1127R/K1117R showed significantly delayed re-entry to the nucleolus after damage recovery, even when SIRT1 is overexpressed. These mutants showed partial interaction with SIRT1 compared to WT WRN. Thus, our results suggest that K1127 and K1117 are the major sites of acetylation, necessary for DNA repair. These results elucidate the mechanism by which SIRT1 regulates WRN trafficking via these acetylation sites during DNA damage.     

Intracellular free Ca2+ in the cell cycle in human fibroblasts: transitions between G1 and G0 and progression into S phase.

Intracellular free calcium ([Ca2+]i) has been proposed to play an important part in the regulation of the cell cycle. Although a number of studies have shown that stimulation of quiescent cells with growth factors causes an immediate rise in [Ca2+]i (Rabinovitch et al., 1986; Vincentini and Villereal, 1986; Hesketh et al., 1988; Tucker et al., 1989, Wahl et al., 1990), a causal relationship between the [Ca2+]i transient and the ability of the cells to reenter the cell cycle has not been firmly established. We have found that blocking the mitogen-induced elevation of [Ca2+]i with the cytoplasmic [Ca2+]i buffer dimethyl BAPTA (dmBAPTA) also blocks subsequent entry of cells into S phase. The dose response curves for inhibition of serum stimulation of [Ca2+]i and DNA synthesis by dmBAPTA are virtually identical including an anomalous stimulation observed at low levels of dmBAPTA. Reversal of the [Ca2+]i buffering effect of dmBAPTA by transient exposure of the cells to the Ca2+ ionophore ionomycin also reverses the inhibition of DNA synthesis 20-24 h later. Ionomycin by itself does not stimulate DNA synthesis. These data are consistent with the conclusion that a transient increase in [Ca2+]i occurring shortly after serum stimulation of quiescent fibroblasts is necessary but not sufficient for subsequent entry of the cells into S phase. This study is the first to show a direct relationship between early serum stimulated Cai2+ increase and subsequent DNA synthesis in human cells. It also goes beyond recent studies on BALB/3T3 cells by providing dose response data and demonstrating reversibility, which are strong indications of a cause and effect relationship.     

ATM signals miRNA biogenesis through KSRP

In this issue of Molecular Cell, Zhang and colleagues (Zhang et?al., 2011) describe a critical link between the DNA damage response and the miRNA pathway, in which DNA double-strand breaks (DSBs) induce ATM-dependent KSRP phosphorylation to facilitate pri-miRNA processing.     

Mobilization and recruitment of HP1β: A bimodal response to DNA breakage

The pathways that signal double-strand DNA breaks (DSBs) in mammalian cells are central to the maintenance of genome integrity. We have reported (Ayoub et al., Nature 2008; 453: 682-6) that the rapid mobilization of the heterochromatin protein, HP1β, within seconds from DSB sites promotes chromatin changes like H2AX phosphorylation that trigger this response. Notably, this paper and a subsequent report (Ayoub et al., Cell Cycle 2009; 8: 1494-500), demonstrate that transient HP1β mobilization is followed by its accumulation over time at DSB sites. Indeed, two recent papers (Luijsterburg et al., J Cell Biol 2009; 185:577-86 and Zarebski et al., Cytometry A May 2009) suggest that HP1 recruitment to damage sites, rather than its rapid mobilization, is the predominant behaviour exhibited by this protein. Here, we present new experimental analyses which corroborate that fluorophore-tagged HP1β exhibits two distinct behaviours at DSB sites in living cells – rapid, transient mobilization, most evident in heterochromatic regions, followed by slower recruitment. Experimental methods allowing visualization of these behaviours are described. Interestingly, chemical inhibition of the DNA-damage responsive enzyme, casein kinase 2 (CK2), suppresses HP1β mobilization while permitting recruitment. Our findings reconcile recent findings in a new model, wherein rapid HP1β mobilization from DSBs mediated by its phosphorylation on Thr51 by CK2, is followed by, and may overlap with, its accumulation at these sites via the chromoshadow domain, independent of Thr51. Our analyses provide fresh insight into the earliest events that trigger the DNA damage response in mammalian cells.     

Polyploid tissues in the nematode Caenorhabditis elegans

During larval development, the number of somatic nuclei in C. elegans hermaphrodites increases from 558 to 959 (J. E. Sulston and H. R. Horvitz, Dev. Biol. 56, 110-156, 1977; J. E. Sulston et al., Dev. Biol. 100, 64-119, 1983). At the same time, the animals increase about 60-fold in volume. We have measured the DNA contents of several classes of nuclei by quantitating the fluorescence of Hoescht 33258 stained DNA (D. G. Albertson et al., Dev. Biol. 63, 165-178, 1978). Probably all embryonic nuclei, including those of neurons, muscles, hypodermis, and intestine, are diploid at hatching. Neurons, muscles, and nondividing hypodermal nuclei remain diploid throughout larval development. The DNA content of the intestinal nuclei doubles at the end of each larval stage, reaching 32C by the adult stage. New hypodermal cells, generated by division of seam cells in the larval stages, undergo an additional round of DNA replication before fusing with the major syncytium (hyp7, Sulston et al., 1983). Thus the larval hyp7 syncytium comprises a fixed number of diploid embryonic nuclei plus an increasing number of tetraploid postembryonic nuclei. Some of the endoreduplications that occur in the intestinal and hypodermal lineages of C. elegans may correspond to nuclear or cellular divisions in another nematode Panagrellus redivivus (P. W. Sternberg and H. R. Horvitz, Dev. Biol. 93, 181-205, 1982).     

Application of the DNA adductome approach to assess the DNA-damaging capability of in vitro micronucleus test-positive compounds

The in vitro micronucleus (MN) test is widely used for screening genotoxic compounds, but it often produces false-positive results. To consider the significance of positive results, it is important to know whether DNA adducts are formed in the cells treated with the test compound. Recently, Matsuda et al. developed the DNA adductome approach to detect DNA adducts comprehensively ([4] Kanaly, et al., Antioxid. Redox Signal., 2006, 8, 993-1001). We applied this method to assess the DNA-damaging capability of in vitro MN test-positive compounds. CHL/IU cells were treated with compounds from three categories: (1) carcinogens causing DNA alkylation, ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; (2) carcinogens producing DNA bulky adducts, 2-amino-6-phenyl-1-methylimidazo[4,5-b]pyrene, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, and 4-nitroquinoline-1-oxide, and (3) non-carcinogens, caffeine, maltol, and sodium chloride, with or without metabolic activation. With the conditions in which all test compounds gave positive results in the MN tests, DNA was extracted from the cells and hydrolyzed to deoxyribonucleosides, which were subsequently subjected to LC/ESI-MS/MS analysis. All carcinogens (categories 1 and 2) produced various DNA adduct peaks, and some of the m/z peak values corresponded to known adducts. No non-carcinogens produced DNA adducts, indicating that these compounds produced MN through different mechanisms from the adduct formation. These results indicate that the adductome approach is useful to demonstrate DNA damage formation of MN test-positive compounds and to understand their mechanisms of action.     

The epithelial Na(+) channel (ENaC) is related to the hypertonicity-induced Na(+) conductance in rat hepatocytes

The epithelial Na(+) channel (ENaC) is composed of the subunits alpha, beta, and gamma [Canessa et al., Nature 367 (1994) 463-467] and typically exhibits a high affinity to amiloride [Canessa et al., Nature 361 (1993) 467-470]. When expressed in Xenopus oocytes, conflicting results were reported concerning the osmo-sensitivity of the channel [Ji et al., Am. J. Physiol. 275 (1998) C1182-C1190; Hawayda and Subramanyam, J. Gen. Physiol. 112 (1998) 97-111; Rossier, J. Gen. Physiol. 112 (1998) 95-96]. Rat hepatocytes were the first system in which amiloride-sensitive sodium currents in response to hypertonic stress were reported [Wehner et al., J. Gen. Physiol. 105 (1995) 507-535; Wehner et al., Physiologist 40 (1997) A-4]. Moreover, all three ENaC subunits are expressed in these cells [B?hmer et al., Cell. Physiol. Biochem. 10 (2000) 187-194]. Here, we injected specific antisense oligonucleotides directed against alpha-rENaC into single rat hepatocytes in confluent primary culture and found an inhibition of hypertonicity-induced Na(+) currents by 70%. This is the first direct evidence for a role of the ENaC in cell volume regulation.     

Xenopus egg lysates repair heat-generated DNA nicks with an average patch size of 36 nucleotides.

Base excision repair (BER) is an essential DNA repair pathway since it processes spontaneous (endogenous) DNA damage such as abasic sites, oxidized and alkylated bases, as well as mismatches arising from deamination of cytosine and 5-methylcytosine. Some of these lesions are repaired by the exchange of a single deoxynucleotide [Dianov, G. et al. (1992) Mol. Cell. Biol. 12, 1605-1612; Wiebauer, K. and Jiricny, J. (1990) Proc. Natl. Acad. Sci. USA, 87, 5842-5845] or a few deoxynucleotides [Matsumoto, Y. et al. (1994) Mol. Cell. Biol., 14 6187-6197]. Here we report that DNA single strand breaks induced by hyperthermic conditions are repaired with an average patch size of approximately 36 nt in Xenopus laevis egg lysates.     

Involvement of phenylalanine 272 of DNA polymerase beta in discriminating between correct and incorrect deoxynucleoside triphosphates

DNA polymerase beta is a small monomeric polymerase that participates in base excision repair and meiosis [Sobol, R., et al. (1996) Nature 379, 183-186; Plug, A., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1327-1331]. A DNA polymerase beta mutator mutant, F272L, was identified by an in vivo genetic screen [Washington, S., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1321-1326]. Residue 272 is located within the deoxynucleoside triphosphate (dNTP) binding pocket of DNA polymerase beta according to the known DNA polymerase beta crystal structures [Pelletier, H., et al. (1994) Science 264, 1891-1893; Sawaya, M., et al. (1997) Biochemistry 36, 11205-11215]. The F272L mutant produces errors at a frequency 10-fold higher than that of wild type in vivo and in the in vitro HSV-tk gap-filling assay. F272L shows an increase in the frequency of both base substitution mutations and frameshift mutations. Single-enzyme turnover studies of misincorporation by wild type and F272L DNA polymerase beta demonstrate that there is a 4-fold decrease in fidelity of the mutant as compared to that of the wild type enzyme for a G:A mismatch. The decreased fidelity is due primarily to decreased discrimination between the correct and incorrect dNTP during ground-state binding. These results suggest that the phenylalanine 272 residue is critical for maintaining fidelity during the binding of the dNTP.     

The novel surveillance mechanism of the Trp53-dependent s-phase checkpoint ensures chromosome damage repair and preimplantation-stage development of mouse embryos fertilized with x-irradiated sperm

Cell cycle checkpoints and apoptosis function as surveillance mechanisms in somatic tissues. However, some of these mechanisms are lacking or are restricted during the preimplantation stage. Previously, we reported the presence of a novel Trp53-dependent S-phase checkpoint that suppresses pronuclear DNA synthesis in mouse zygotes fertilized with X-irradiated sperm (sperm-irradiated zygotes) (Shimura et al., Mol. Cell. Biol. 22, 2220-2228, 2002). Here we studied the role of the Trp53-dependent S-phase checkpoint in the early stage of development of sperm-irradiated zygotes. In the Trp53(+/+) genetic background, all of the sperm-irradiated zygotes cleaved successfully to the two-cell stage despite the fact that half of them carried a sub-2N amount of DNA. These zygotes progressed normally to the eight-cell stage and then implanted, but the subsequent fetal development was suppressed in a dose-dependent manner. In contrast, sperm-irradiated Trp53(-/-) embryos lacking an S-phase checkpoint exhibited an abnormal segregation of chromosomes at the first cleavage, even though they carried an apparently normal 2N amount of DNA. They were morphologically abnormal with numerous micronuclei, and they degenerated before reaching the eight-cell stage. As a consequence, no implants were observed for sperm-irradiated Trp53(-/-) embryos. These results suggest that the Trp53-dependent S-phase checkpoint is a surveillance mechanism involved in the repair of chromosome damage and ensures the preimplantation-stage development of sperm-irradiated embryos.     

Parole terms for a killer

In a series of discoveries over the preceding decade, a number of laboratories have unequivocally established that apoptotic proteins and pathways are well conserved cell fate determinants, which act independent of a cell death response. Within this context, the role for apoptotic proteins in the induction of cell differentiation has been widely documented. Despite these discoveries, little information has been forthcoming regarding a conserved mechanism by which apoptotic proteins achieve this non-death outcome. In the following discussion, we will explore the premise that the penultimate step in apoptosis, genome wide DNA damage/strand breaks act as a conserved genomic reprogramming event necessary for cell differentiation (Larsen et al., Proc Natl Acad Sci USA 2010; 107 (9):4230-5). Moreover, we hypothesis that directed DNA damage, as mediated by known apoptotic proteins, may participate in numerous forms of regulated gene expression.