Characterisation of eye-lens DNases: long term persistence of activity in post apoptotic lens fibre cells

Fibre cells in the ocular lens exhibit a constitutive apoptotic process of nuclear degradation that includes chromatin breakage, generating a ladder pattern of DNA fragments. This process is intrinsic to the normal terminal differentiation program. Despite the loss of nucleus and cytoplasmic organelles, the terminal differentiated fibre cells remain in the lens during the whole life span of the individual. The lens cells thus provide a unique system in which to determine the presence and fate of endonucleases once the chromatin has been cleaved. We report here on the presence of DNase activity in nucleated and anucleated lens cells. Using a nuclease gel assay and double-stranded DNA as substrate, we found active 30 and 60 kDa DNases. The enzymatic activities were Ca(2+), Mg(2+) dependent, and active at neutral pH. The relative amount of these forms changed during development and aging of the lens fibre cells. Both forms were inhibited by Zn(2+), aurintricarboxylic acid, and G-actin. The proteins were also separated by SDS-PAGE, renatured after removing SDS and incubated in the presence of native DNA adsorbed to a membrane. Therefore it was possible to demonstrate, by means of a nick translation reaction, that the enzymes produced single strand cuts. Based on these findings we propose that these chick lens nucleases are probably related to DNase I.     

Mn2+-dependent endonuclease activity of chromatin

The endogenous endonuclease activity of chromatin in isolated rat liver nuclei in the presence of Mn2+, Mg2+ and Ca2+ + Mg2+ was studied. The existence of a Mn2+-dependent endonuclease activity not coupled with the Ca2+, Mg2+-dependent endonuclease was demonstrated, which was weaker than the former one in isolated cell nuclei but higher than in the preparation of Ca2+, Mg2+-dependent nuclease obtained by gel filtration through Toyopearl HW 60F. The Mn2+-dependent splitting of chromatin predominantly occurs at linker DNA of distal parts of chromatin loops. A split-off of purified DNA was more universal than in the presence of Ca2+, Mg2+-dependent endonuclease; the hydrolysis rate of native and denaturated DNA appeared to be the same.     

Purification and characterization of the Ca2+ plus Mg2+-dependent endodeoxyribonuclease from calf thymus chromatin

Ca2+ plus Mg2+-dependent endodeoxyribonuclease was extracted from calf thymus chromatin and purified to a state free from contamination by other DNases. This DNase required both Ca2+ and Mg2+, or Mn2+ alone for its activity and the optimum pH for activity was at 6.5-7.5. No specificity for the 5'-base was observed. The molecular weight of the DNase was estimated to be about 25,000-30,000 by glycerol gradient centrifugation. Actin and antibody for pancreatic DNase (DNase I) did not inhibit the enzyme, whereas both strongly inhibited DNase I, suggesting that these two DNases are different enzymes.     

Behavior of pericentromere heterochromatin regions under different conditions of chromosome decondensation in isolated nuclei]

In isolated mouse nuclei the chromocenters were shown to be the pericentromeric heterochromatin regions (PCHR). After the decreasing of bivalent ion concentration (0.1 mM Ca2+, 2 mM Mg2+) the main and peripheral parts of the chromatin remained on the contrary as the compact chromatin bodies. The additional ultrasound treatment of isolated nuclei in the presence of 0.1 mM Ca2+ with DNAase II and triton X-100 resulted in the species enriched by the condensed PCHR.     

Propidium iodide staining correlates with the extent of DNA degradation in isolated nuclei.

Gradual degradation of internucleosomal DNA is a hallmark of apoptosis and can be simulated by incubating isolated thymocyte nuclei in the presence of 5 mM Mg2+ and 5 mM Ca2+ at 37 degrees C. Staining of nuclei with the DNA binding fluorescent dye propidium iodide (PI) showed that intensity of fluorescence correlated with the extent of DNA degradation. PI fluorescence was increased in the presence of DNase I. Thus it seems that the cleavage of chromatin DNA by DNase 1 or by the endogenous enzyme increases the accessibility of DNA for the dye. No increase of fluorescence was observed in the presence of the known inhibitors of the endogenous endonuclease: Zn2+ and EGTA. However, the presence of Zn2+ led to decreased staining of the nuclei by PI and caused a shift in the scatter profile of the nuclei, suggesting that a conformational change of chromatin is induced by this ion. This correlation between intensity of PI staining and DNA degradation should be useful to compare endogenous nuclease levels in lymphocyte populations.     

Structure of rat liver nuclei after depletion and reassociation of histone H1

Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination.     

Effect of the cytoplasm of irradiated cells on chromatin degradation in mouse thymocytes and hepatocytes

The addition of a cytoplasmic fraction, isolated from cells 3h after irradiation of mice, to exposed or intact thymocyte nuclei causes a 2- or 3-fold acceleration of chromatin degradation in the nuclei incubated in conditions optimum for activity of Ca2+,Mg2+-dependent endonuclease to be manifest. In contrast to thymocytes, no chromatin degradation products are found in liver cells of irradiated mice. The cytoplasmic fraction isolated from hepatocytes of irradiated animals fails to activate chromatin degradation in thymocyte nuclei.     

Action of apoptotic endonuclease DNase gamma on naked DNA and chromatin substrates

The internucleosomal cleavage of genomic DNA is a biochemical hallmark of apoptosis. DNase gamma, a Mg2+/Ca2+-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases, but its biochemical characteristic has not been fully elucidated. Here, using recombinant DNase gamma, we showed that DNase gamma is a Mg2+/Ca2+-dependent single-stranded DNA nickase and has a high activity at low ionic strength. Under higher ionic strength, such as physiological buffer conditions, the endonuclease activity of DNase gamma is restricted, but its activity is enhanced in the presence of linker histone H1, which explains DNA cleavage at linker regions of apoptotic nuclei.     

Detection of endonuclease activity and several features of chromatin autolysis in brain cell nuclei]

The presence of Ca2+, Mg2+-dependent endonuclease activity in isolated brain cell nuclei was demonstrated and a comparison of some peculiarities of chromatin autolysis in rat brain and liver cell nuclei was carried out. Endogenous brain nuclease hydrolyzes chromatin into its structural subunits; its specific activity is 10,5 times as low as compared to the endogenous nuclease activity in rat liver nuclei. The dependency of the chromatin autolysis rate on pH and ionic composition of the incubation medium in isolated rate brain and liver nuclei appeared to be the same. The presence of Mn2+ changed the autolysis nature both in brain and in liver cell nuclei, the relative (as compared to Mg2+-dependent) Mn2+-dependent activity being higher in the brain cell nuclei. Possible differences of brain and liver chromatin structure (e. g. the presence of regions free of nucleosomic organization in brain chromatin) are assumed.     

Purification and characterization of a DNase γ-like endonuclease from Xenopus laevis liver

We recently found that two apoptotic DNase γ-like endonucleases (36 and 38kDa DNases) were present in Xenopus laevis larval and adult liver cell nuclei and that their activities increased in metamorphic climax. Here, we purified the main DNase γ-like endonuclease from Xenopus laevis liver cell nuclei and characterized its physical and enzymatic properties in detail. The molecular mass of Xenopus liver nuclear endonuclease was 38,000 daltons as determined by SDS-polyacrylamide gel electrophoresis. A native molecular mass of 35,000 was estimated by gel filtration. The purified Xenopus liver endonuclease was a neutral one and required both Ca2+ and Mg2+ for DNase activity. Unlike the mammalian DNase γ, the Ca2+/Mg2+ requirement could not be supplied by Mn2+. The inhibition profiles by aurintricarboxylic acid, sodium citrate and divalent metal ions such as Co2+, Ni2+, Cu2+ and Zn2+ were similar to those of mammalian DNase γ. These results suggest that this endonuclease is a Xenopus laevis homolog of the mammalian apoptotic endonuclease DNase γ. This revised version was published online in June 2006 with corrections to the Cover Date.     

Solubilization of chromatin by an endogenous enzymic Ca2+, Mg2+-dependent factor. Activity of residual chromatin]

An endogenous Ca2+, Mg2+-dependent factor of enzymic nature (apparently an endonuclease) digests a part of chromatin in the rat liver nuclei producing DNA fragments of an uniform size. After 60 min of incubation at 15 degrees C and pH 7.50 in the presence of 5 mM MgCl2 and 2 mM CaCl2 87-93% of the total chromatin becomes soluble. The insoluble chromatin however contains 70-85% of the in vivo newly synthesized RNA. In regenerating liver the proportion of the insoluble residual chromatin increases while the radioactivity of the newly synthesized DNA in this fraction is highest. Residual chromatin can be solubilized by ultrasonic treatment only. The Ca2+, Mg2+-dependent dissolving factor is not present either in brain or in PMN leucocyte nuclei.     

Electron-cytochemical study of the localization and properties of ATPases in the isolated nuclei of rabbit skeletal muscle under normal conditions and in experimental muscular dystrophy]

The properties and localization of ATPase system in nuclei of skeletal muscle of normal rabbit and of those with experimental muscle dystrophy were studied by electron cytochemistry. The product of cytochemical reaction of ATP hydrolysis, which is a marker of ATPase activity localization in nuclear ultrastructures, was detected on the nuclear membrane, in chromatin and in the nucleolus, ATPase activity in the nuclei was detected in the presence of both, Mg2+ and Ca2+. Addition to the incubation medium, originally containing Mg2+, Na+ and K+, resulted in an increased formation of the product reaction in all the nuclear ultrastructures in both in the norm and under experimental muscle dystrophy. However, specific inhibitor of Mg2+, Na+, K+-ATPase--ouabain--suggests the absence in the nuclei of skeletal muscles of rabbit of transport ATPase working in the "Na-pump" system. The results of experiments with a specific complex of Ca2+--EGTA allow to suppose that Mg2+, Ca2+-ATPase of skeletal muscle nuclei of normal rabbits is localized in the nucleoplasm, whereas Mg2+-ATPase is found on the nuclear membrane. Using EGTA we failed to detected the localization of Mg2+, Ca2+-ATPase in nuclear ultrastructures upon experimental muscular dystrophy.     

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.     

DNA denaturation in situ. Effect of divalent cations and alcohols

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.     

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.     

Ca2+-dependent activity of human DNase I and its hyperactive variants.

We have recently constructed hyperactive human deoxyribonuclease I (DNase I) variants that digest double-stranded DNA more efficiently under physiological saline conditions by introducing positively charged amino acids at eight positions that can interact favorably with the negatively charged DNA phosphates. In this study, we present data from supercoiled DNA nicking, linear DNA digestion, and hyperchromicity assays that distinguish two classes of DNase I hyperactive variants based upon their activity dependence on Ca2+. Class A variants are highly dependent upon Ca2+, having up to 300-fold lower activity in the presence of Mg2+ alone compared to that in the presence of Mg2+ and Ca2+, and include Q9R, H44K, and T205K, in addition to wild-type DNase I. In contrast, the catalytic activity of Class B variants, which comprise the E13R, T14K, N74K, S75K, and N110R hyperactive variants, is relatively Ca2+ independent. A significant proportion of this difference in Ca2+-dependent activity can be attributed to one of the two structural calcium binding sites in DNase I. Compared to wild-type, the removal of Ca2+ binding site 2 by alanine replacements at Asp99, Asp107, and Glu112 decreased activity up to 26-fold in the presence of Mg2+ and Ca2+, but had no effect in the presence of Mg2+ alone. We propose that the rate-enhancing effect of Ca2+ binding at site 2 can be replaced by favorable electrostatic interactions created by proximal positively charged amino acid substitutions such as those found in the Class B variants, thus reducing the dependence on Ca2+.     

Acidocalcisome: A novel Ca2+ storage compartment in trypanosomatids and apicomplexan parasites.

Acidocalcisomes are novel acidic Ca2+ storage organelles found in trypanosomatids and apicomplexan parasites, abundant in the intracellular stages of these parasites, and characterized by their high electron density, and high content of phosphorus, Ca2+, Mg2+, Na+ and Zn2+. A number of energy-utilizing pumps and exchangers have been found in these organelles, which underlines their importance in the homeostasis of different elements, as discussed here by Roberto Docampo and Silvia Moreno.     

Cation-dependent solubilization of rat thymocyte chromatin is closely related to decondensation of the nuclei

The cation-dependent solubilization of rat thymocyte chromatin has been compared with decondensation of the nuclei as a function of sodium phosphate-mediated changes in the concentration of Mg2+ and Na+. After digestion of the nuclei with DNase I or Micrococcus nuclease for a time just sufficient to permit extraction of a maximal amount of chromatin (minimum digestion), solubilization of most of the chromatin was found to occur with the same cation dependency as decondensation of untreated nuclei, while further digestion changed the ionic requirements for solubilization. The cation-dependency of the chromatin solubility and of the nuclear decondensation also exhibited the same variations with temperature. The chromatin in the nuclei became up to 4-times more sensitive to DNase I by decondensation, which also induced a shift in the DNase I cleavage mode from a 200 bp to a 100 bp repeat pattern. In contrast, the sensitivity to Micrococcus nuclease appeared to be nearly unchanged. These results suggest that solubilization of chromatin prepared by a mild endonuclease treatment occurs as a direct consequence of structural changes in the chromatin which take place during decondensation of the nuclei.     

The essential role of Ca2+ in the activity of bovine pancreatic deoxyribonuclease.

DNase requires Ca2+ for activity against DNA with Mg2+. The Ca2+ selective chelating agent, ethylene glycol bis(beta-aminoethyl ether)-N, N'-tetraacetic acid, (EGTA) inhibits DNase completely at pH 7 or 8, and subsequent addition of excess Ca2+ reverses inhibition in less than one second. DNase action can be stopped at any point by the addition of excess EGTA over Ca2+. Ca2+ is required for DNase to bind substrate. Gel filtration experiments fail to show DNase binding to 0.2 mg per ml of DNA at 5 mm Mg2+ and 10-4 M EGTA. The concentration of Ca2+ needed for half of maximum DNase activity decreases with increases DNA concentration, from 1.2 times 10-5 M Ca2+ at 2.3 times 10-5 M DNA-P to about 4 times 10-7 M Ca2+ at 2.3 DNA-P. Kinetic analysis by the titrametic assay of protons releases shows that V max is independent of Ca2+ concentration while Km increases from 7.7 times 10-5 M DNA-P at 5 times 10-4 M Ca2+ to 3.4 times 10-4 M DNA-P at 5 times 10-6 M Ca2+. Both of these results are predicted by a rate equation which is derived from the assumption that DNase must bind Ca2+ before it can bind DNA. The essential Ca2+ atom probably binds to the one of two high affinity Ca2+ binding sites on DNase which cannont bind Mg2+ or Mn2+. The only other divalent metal ions which can bind to this site, Sr2+ and Ba2+, are also the only metal ions which can substitute for Ca2+ in DNase action against DNA with Mg2+. Some DNase activity is obtained in the absence of added Ca2+ with Mg2+ at pH 6 or below and with Mn2+ or Co2+ at pH 8. These assay solutions are contaminated by 1 to 3 muM Ca2+, which may be sufficient to account for the observed activity.