The Dot1 Histone Methyltransferase and the Rad9 Checkpoint Adaptor Contribute to Cohesin-Dependent Double-Strand Break Repair by Sister Chromatid Recombination in Saccharomyces cerevisiae

Genomic integrity is threatened by multiple sources of DNA damage. DNA double-strand breaks (DSBs) are among the most dangerous types of DNA lesions and can be generated by endogenous or exogenous agents, but they can arise also during DNA replication. Sister chromatid recombination (SCR) is a key mechanism for the repair of DSBs generated during replication and it is fundamental for maintaining genomic stability. Proper repair relies on several factors, among which histone modifications play important roles in the response to DSBs. Here, we study the role of the histone H3K79 methyltransferase Dot1 in the repair by SCR of replication-dependent HO-induced DSBs, as a way to assess its function in homologous recombination. We show that Dot1, the Rad9 DNA damage checkpoint adaptor, and phosphorylation of histone H2A (γH2A) are required for efficient SCR. Moreover, we show that Dot1 and Rad9 promote DSB-induced loading of cohesin onto chromatin. We propose that recruitment of Rad9 to DSB sites mediated by γH2A and H3K79 methylation contributes to DSB repair via SCR by regulating cohesin binding to damage sites. Therefore, our results contribute to an understanding of how different chromatin modifications impinge on DNA repair mechanisms, which are fundamental for maintaining genomic stability.IN eukaryotic cells, genomic integrity is ensured by the action of the DNA damage checkpoint. This checkpoint coordinates the cellular response to DNA damage, triggering cell cycle arrest and activating DNA repair mechanisms, thus providing time for the cell to repair the damage before resuming cell cycle progression (Harrison and Haber 2006). DNA double-strand breaks (DSBs) are among the most dangerous genomic lesions and, if they are not properly repaired, they can lead to fatal consequences. DSBs can be repaired either by homologous recombination (HR) or by nonhomologous end joining (NHEJ), but only HR with the sister chromatid ensures that the fidelity of genetic information is mantained. Thus, sister chromatid recombination (SCR) is the preferred mechanism of DSB repair in mitotic cells (Kadyk and Hartwell 1992; Johnson and Jasin 2000; González-Barrera et al. 2003). Since SCR occurs between identical DNA molecules, its analysis by genetic or physical methods is difficult but, recently, a physical assay to monitor the repair by SCR of a single DSB generated during replication has been developed in budding yeast (González-Barrera et al. 2003; Cortes-Ledesma and Aguilera 2006). This SCR assay is based on a circular minichromosome harboring an internal mini-HO site, which is cleaved mainly in one strand producing ∼10% DSBs during replication, in contrast to the direct and efficient DSB induction at the full-length HO site. In this way, upon HO induction, the DSB occurs only in one chromatid and the other one remains intact and available for repair (see Figure 1A). Although this assay has been used mainly to monitor unequal SCR events, it has been demonstrated that it is an accurate indicator of the proficiency in total SCR (González-Barrera et al. 2003; Cortes-Ledesma and Aguilera 2006). Using this physical assay, it has been established that Rad52, Rad59, Rad51, and Rad54, but not Rdh54/Tid1, are involved in SCR (Cortes-Ledesma et al. 2007b). Also, SMC (structural maintenance of chromosomes) proteins including the cohesin complex and the Smc5/6 complex are required for efficient SCR (Cortes-Ledesma and Aguilera 2006; De Piccoli et al. 2006; Cortes-Ledesma et al. 2007a).Open in a separate windowFigure 1.—Dot1 is required for efficient SCR. (A) Schematic of the physical assay used to monitor repair by SCR of an HO-induced DSB in the centromeric plasmid pRS316-TINV. Fragments generated after XhoI–SpeI digestion, detected by the LEU2 probe (line with asterisks) are indicated with their corresponding sizes. Since other recombination events can also lead to the 2.9-kb fragment, only the 4.7-kb band is used to measure SCR. (B) Kinetics of HO-induced DSB formation and its repair in wild-type (MKOS-3C) and dot1 (YP764) cells incubated in galactose for the indicated times. A representative Southern blot is presented showing the different fragments detected. The 3.8-kb band corresponds to the intact plasmid and equal SCR events, the 1.4-kb and 2.4-kb fragments arise after HO cut, the 2.9-kb band results from unequal SCR and IC-BIR and the 4.7-kb band is specific for unequal SCR. (C) Quantification of DSBs (1.4-kb plus 2.4-kb bands) and SCR (4.7-kb band) relative to the total DNA. Averages and standard deviations are shown. In some cases, such as the dot1 SCR values, the error bars are hidden by the graph symbols.Detection, signaling and repair of genomic lesions occur in the context of chromatin; therefore, factors regulating chromatin structure, such as histone modifications and chromatin remodelers, play important roles in the DNA damage response (Peterson and Cote 2004; Lydall and Whitehall 2005; van Attikum and Gasser 2005; Downs et al. 2007). Mec1- and Tel1-dependent phosphorylation of histone H2A at serine 129 (hereafter referred to as γH2A) is required for DSB repair by NHEJ and likely HR (Downs et al. 2000) and also mediates recruitment of cohesin to DSB sites (Unal et al. 2004). Another chromatin modification involved in the DNA damage response is the methylation of lysine 79 of histone H3 (H3K79) mediated by Dot1 (van Leeuwen et al. 2002). During meiosis, Dot1 is required for the meiotic recombination checkpoint (San-Segundo and Roeder 2000) and, in mitotic cells, Dot1-dependent H3K79 methylation is involved in the Rad9-mediated activation of the Rad53 checkpoint kinase (Giannattasio et al. 2005; Wysocki et al. 2005). Moreover, genetic analyses of the response to different DNA damaging agents, such as ionizing radiation (IR), methyl methanesulfonate (MMS), and UV, have suggested that Dot1 modulates multiple DNA repair pathways (Game et al. 2006; Toh et al. 2006; Bostelman et al. 2007; Conde and San-Segundo 2008) and also controls DSB resection (Lazzaro et al. 2008). To gain further insight in the molecular mechanisms of DNA repair impacted by Dot1 function we have used a physical assay to monitor DSB repair by SCR as a manifestation of HR repair. We provide molecular and genetic evidence indicating that Dot1, together with γH2A, promotes SCR by Rad9-mediated recruitment of cohesin to DSB sites.     

Glycolytic oscillations in single ischemic cardiomyocytes at near anoxia

Previous studies have shown that oscillations of the metabolism can occur in cardiomyocytes under conditions simulating ischemia/reperfusion. It is not known whether they can also occur during real ischemia with near-anoxic oxygen tension. Here, using oxygen clamp in on-chip picochambers, we exposed single resting cardiomyocytes to near anoxia (pO₂ < 0.1 mm Hg). We show that at near anoxia, the mitochondrial membrane potential (ΔΨ) was kept by the F₁F₀-ATPase reversal, using glycolytic adenosine triphosphate (ATP). In many cells, activation of current through sarcolemmal K_ATP channels (I_KATP) started after a delay with one or several oscillations (frequency of 0.044 ± 0.002 Hz). These oscillations were time correlated with oscillations of ΔΨ. Metabolic oscillations at near anoxia are driven by glycolysis because (a) they were inhibited when glycolysis was blocked, (b) they persisted in cells treated with cytoplasmic reactive oxygen species scavengers, and (c) the highest rate of ATP synthesis during an oscillation cycle was associated with the generation of reducing equivalents. Glycolytic oscillations could be initiated upon rapid, but not slow, transition to near anoxia, indicating that the speed of ATP/ADP ratio drop is a determinant of their occurrence. At enhanced oxidative stress, the rate of ATP consumption was increased as indicated by rapid I_KATP activation with large-scale oscillations. These results show that metabolic oscillations occur in cardiomyocytes at near anoxia and are driven by glycolysis and modulated by mitochondria through the rate of ATP hydrolysis, which, in turn, can be accelerated by oxidative stress.     

Catalytic effect of sulfhydryl oxidase on the formation of three-dimensional structure in chymotrypsinogen A

The effect of sulfhydryl oxidase on the rate of disulfide bond formation and polypeptide chain folding in reductively denatured chymotrypsinogen A has been investigated using an immobilized zymogen preparation and a cylindrical quartz flow-through fluorescence cell. Enzymatic oxidation of the 10 sulfhydryl groups in reduced chymotrypsinogen followed first order kinetics at pH 7.0 with an apparent first order rate constant governing sulfhydryl group disappearance of 4.2 × 10^?2 min^?1. This provides a $\text{t}\text{1}\text{2}$ of 16.3 min for the sulfhydryl oxidase-catalyzed oxidation, whereas 165 min are required for nonenzymatic aerobic oxidation of one-half the sulfhydryl groups. Refolding of the reductively denatured polypeptide chains, monitored by changes in protein fluorescence, did not follow first order kinetics characteristic of a simple two-state mechanism, nor did the return of trypsin activatability. It appears that at least one intermediate must exist in such refolding, in both the uncatalyzed and sulfhydryl oxidase-catalyzed processes. Estimation of the rate constants governing refolding, assuming a single intermediate between the denatured and native states, provided values of 3 × 10^?2 min^?1 and 7 × 10^?3 min^?1 for uncatalyzed autoxidation and 4 × 10^?2 min^?1 and 1.1 × 10^?2 min^?1 for the sulfhydryl oxidase-catalyzed transition. Thus, enzymic catalysis of disulfide bond formation can lead to apparent catalysis of protein refolding as monitored both by fluorescence and by acquisition of biological function.     

Cell Survival during Complete Nutrient Deprivation Depends on Lipid Droplet-fueled β-Oxidation of Fatty Acids

Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A₂ (cPLA₂α) or down-regulation of ceramide kinase. Nutrient deprivation also induced β-oxidation of fatty acids that was sensitive to cPLA₂α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β-oxidation of fatty acids that requires biogenesis and mobilization of LD.     

Targeting lysine specific demethylase 4A (KDM4A) tandem TUDOR domain – A fragment based approach

The tandem TUDOR domains present in the non-catalytic C-terminal half of the KDM4A, 4B and 4C enzymes play important roles in regulating their chromatin localizations and substrate specificities. They achieve this regulatory role by binding to different tri-methylated lysine residues on histone H3 (H3-K4me3, H3-K23me3) and histone H4 (H4-K20me3) depending upon the specific chromatin environment. In this work, we have used a 2D-NMR based fragment screening approach to identify a novel fragment (1a), which binds to the KDM4A-TUDOR domain and shows modest competition with H3-K4me3 binding in biochemical as well as in vitro cell based assays. A co-crystal structure of KDM4A TUDOR domain in complex with 1a shows that the fragment binds stereo-specifically to the methyl lysine binding pocket forming a network of strong hydrogen bonds and hydrophobic interactions. We anticipate that the fragment 1a can be further developed into a novel allosteric inhibitor of the KDM4 family of enzymes through targeting their C-terminal tandem TUDOR domain.     

Health Related Behaviours in Normal Weight and Overweight Preschoolers of a Large Pan-European Sample: The ToyBox-Study

The aim of this study was to investigate the associations of health related behaviours (HRB) with Body Mass Index (BMI) in preschoolers, and to study the likelihood of being overweight/obese in relation to compliance with recommended HRB. The sample consisted of 3301 normal weight and overweight/obese preschoolers (mean age: 4.7 years; 52% boys, 85% normal weight) from six European countries (Belgium, Bulgaria, Germany, Greece, Poland, Spain). Height and weight were measured, total daily step counts were registered during six days, and HRB were assessed with validated parental surveys in 2012. Multiple linear and logistic regression analyses were performed. Only few HRB were significantly associated with BMI. In boys, higher water intake and higher soft drink and higher fruit consumption were significantly associated with higher BMI. Boys drinking less water than recommended were less likely to be overweight/obese (OR = 0.60), while boys who consume soft drinks were more likely to be overweight/obese (OR = 1.52). In girls, higher water intake, higher vegetable consumption, and more TV time on weekend days were significantly associated with higher BMI. Girls eating less vegetables than recommended were less likely to be overweight/obese (OR = 0.62), and girls who engaged in quiet play for more than 90 minutes on weekend days were more likely to be overweight/obese (OR = 1.64). In general, the associations between HRB and BMI or being overweight/obese were limited and mainly related to dietary intake. Awareness campaigns for caregivers should stress that HRB of young children are important and independent of children’s weight status.     

Cyclic ADP-ribose requires CD38 to regulate the release of ATP in visceral smooth muscle

It is well established that the intracellular second messenger cADP-ribose (cADPR) activates Ca(2+) release from the sarcoplasmic reticulum through ryanodine receptors. CD38 is a multifunctional enzyme involved in the formation of cADPR in mammals. CD38 has also been reported to transport cADPR in several cell lines. Here, we demonstrate a role for extracellular cADPR and CD38 in modulating the spontaneous, but not the electrical field stimulation-evoked, release of ATP in visceral smooth muscle. Using a small-volume superfusion assay and an HPLC technique with fluorescence detection, we measured the spontaneous and evoked release of ATP in bladder detrusor smooth muscles isolated from CD38(+/+) and CD38(-/-) mice. cADPR (1 nM) enhanced the spontaneous overflow of ATP in bladders isolated from CD38(+/+) mice. This effect was abolished by the inhibitor of cADPR receptors on sarcoplasmic reticulum 8-bromo-cADPR (80 μM) and by ryanodine (50?μm), but not by the nonselective P2 purinergic receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulfonate (30 μM). cADPR failed to facilitate the spontaneous ATP overflow in bladders isolated from CD38(-/-) mice, indicating that CD38 is crucial for the enhancing effects of extracellular cADPR on spontaneous ATP release. Contractile responses to ATP were potentiated by cADPR, suggesting that the two adenine nucleotides may work in synergy to maintain the resting tone of the bladder. In conclusion, extracellular cADPR enhances the spontaneous release of ATP in the bladder by influx via CD38 and subsequent activation of intracellular cADPR receptors, probably causing an increase in intracellular Ca(2+) in neuronal cells.