Dependent Nuclease Activity Research Articles

A structural study of TatD from Staphylococcus aureus elucidates a putative DNA-binding mode of a Mg2+-dependent nuclease.

TatD has been thoroughly investigated as a DNA-repair enzyme and an apoptotic nuclease, and still-unknown TatD-related DNases are considered to play crucial cellular roles. However, studies of TatD from Gram-positive bacteria have been hindered by an absence of atomic detail and the resulting inability to determine function from structure. In this study, an X-ray crystal structure of SAV0491, which is the TatD enzyme from the Gram-positive bacterium Staphylococcus aureus (SaTatD), is reported at a high resolution of 1.85 Å with a detailed atomic description. Although SaTatD has the common TIM-barrel fold shared by most TatD-related homologs, and PDB entry 2gzx shares 100% sequence identity with SAV0491, the crystal structure of SaTatD revealed a unique binding mode of two phosphates interacting with two Ni2+ ions. Through a functional study, it was verified that SaTatD has Mg2+-dependent nuclease activity as a DNase and an RNase. In addition, structural comparison with TatD homologs and the identification of key residues contributing to the binding mode of Ni2+ ions and phosphates allowed mutational studies to be performed that revealed the catalytic mechanism of SaTatD. Among the key residues composing the active site, the acidic residues Glu92 and Glu202 had a critical impact on catalysis by SaTatD. Furthermore, based on the binding mode of the two phosphates and structural insights, a putative DNA-binding mode of SaTatD was proposed using in silico docking. Overall, these findings may serve as a good basis for understanding the relationship between the structure and function of TatD proteins from Gram-positive bacteria and may provide critical insights into the DNA-binding mode of SaTatD.

Therapeutic effects of duck Tembusu virus capsid protein fused with staphylococcal nuclease protein to target Tembusu infection in vitro

Tembusu virus (TMUV), a member of the genus flavivirus, primarily causes egg-drop syndrome in ducks and is associated with low disease mortality but high morbidity. The commercially available live vaccines for treating TMUV currently include the main WF100, HB, and FX2010-180P strains, and efficient treatment and/or preventative measures are still urgently needed. Capsid-targeted viral inactivation (CTVI) is a conceptually powerful new antiviral strategy that is based on two proteins from the capsid protein of a virus and a crucial effector molecule. The effector molecule can destroy the viral DNA/RNA or interfere with the proper folding of key viral proteins, while the capsid protein mainly plays a role in viral integration and assembly; the fusion proteins are incorporated into virions during packaging. This study aimed to explore the potential use of this strategy in duck TMUV. Our results revealed that these fusion proteins can be expressed in susceptible BHK21 cells without cytotoxicity and possess excellent Ca2+-dependent nuclease activity, and their expression is also detectable in DF-1 cells. Compared to those in the negative controls (BHK21 and BHK21/pcDNA3.1(+) cells), the numbers of viral RNA copies in TMUV-infected BHK21/Cap-SNase and BHK21/Cap-Linker-SNase cells were reduced by 48 h, and the effect of Cap-Linker-SNase was superior to that of Cap-SNase. As anticipated, these results suggest that these fusion proteins contribute to viral resistance to treatment. Thus, CTVI might be applicable for TMUV inhibition as a novel antiviral therapeutic candidate during viral infection.

Under Pressure: Pressure and Temperature Dependent Nuclease Activity

An intein is an intervening protein segment that can be found in proteins from extremophiles and is typically self‐excised to allow for complete flanking protein activity. We are interested in studying specific inteins that contain a homing endonuclease (HE), an enzyme that cleaves double stranded DNA in order to initiate insertion of an intein gene by homologous recombination. We are focused on the homing endonuclease activity of inteins from two hyperthermophilic archaea: Thermococcus barophilus (Tba), isolated from a deep sea hydrothermal vent, and Thermococcus kodakarensis (Tko), isolated from a volcanic area on the shore of a Japanese island. Because these intein‐containing extremophiles have been isolated from very high temperatures but from different depths, we are interested in the differential homing endonuclease activity of these extremophiles at varied temperatures and pressures. We have shown that Tba HE is active at pressures up to 1000 atm. Given that Tko is a surfaced‐welling organism, we suspect that the Tko HE will be pressure sensitive while Tba, which lives naturally at a pressure near 400 atm, is not. Additionally, we have found that both Tba HE and Tko HE show temperature dependence, which we attribute to the extreme temperatures that both organisms reside in.Support or Funding InformationThis work was supported by the National Science Foundation (grant MCB‐1517138) and by the Camille & Henry Dreyfus Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Leishmania donovani IMPACT-like protein possesses non-specific nuclease activity

IMPACT (Imprinted and Ancient)-like proteins are known to be regulators of GCN2 (General control non-derepressible 2) kinases involved in translation regulation. Here, we report on cloning and characterization of an IMPACT-like protein, LdIMPACT from Leishmania donovani which harbours two domains. ‘RWD domain’ at the N-terminal end that mediates GCN2 regulation, while a conserved ‘ancient domain’ lies at the C-terminal end whose function remains elusive. Interestingly, our observations indicated that LdIMPACT has a novel non-specific nuclease activity. In silico analysis further revealed the resemblance of ancient domain of LdIMPACT to RNase PH domain (known to bind to nucleic acids). The recombinant LdIMPACT exhibited a Mg2+-dependent nuclease activity. Moreover, thermostability and pH stability assays of the protein suggest it to be a stress-responsive protein. Circular dichroism studies elucidated the conformational transitions of the enzyme in response to various temperature and pH conditions which correlated well with the activity profiles. Thus, the current study highlights the structural and functional characteristics of LdIMPACT which interestingly also possesses a novel nuclease activity. With its physiological relevance unresolved, the multifaceted LdIMPACT might therefore lie in a hitherto unknown network, whose perturbation could be an attractive therapeutic approach for treating leishmaniasis.

Probing the potency of triazole tethered Schiff base complexes and the effect of substituents on their biological attributes

Two triazole-derived Schiff base ligands and their eight octahedral metal(II) complexes were designed and synthesized. They were characterized by elemental analysis and various spectroscopic techniques. The compounds were evaluated for their DNA binding tendencies with Calf thymus DNA. The concentration dependent chemical nuclease activity was also explored with supercoiled pBR322 DNA. The in vitro antimicrobial screening was undertaken which showed similar activity to the DNA binding studies. Complex 5 revealed exceptional DNA binding affinity, cleaving propensity and antimicrobial efficacy though all the complexes showed activity. All copper(II) complexes were evaluated for their antiproliferative activity against a panel of human cancer cell lines. Complexes 1 and 5 showed activity against all the cell lines with low toxicity towards normal cell line. The effect of substituents on the ligand system of the complexes is also discussed along with the importance of tuning the ligand system.

Identification and characterization of the novel nuclease activity of human phospholipid scramblase 1.

BackgroundHuman phospholipid scramblase 1 (hPLSCR1) was initially identified as a Ca2+ dependent phospholipid translocator involved in disrupting membrane asymmetry. Recent reports revealed that hPLSCR1 acts as a multifunctional signaling molecule rather than functioning as scramblase. hPLSCR1 is overexpressed in a variety of tumor cells and is known to interact with a number of protein molecules implying diverse functions.ResultsIn this study, the nuclease activity of recombinant hPLSCR1 and its biochemical properties have been determined. Point mutations were generated to identify the critical region responsible for the nuclease activity. Recombinant hPLSCR1 exhibits Mg2+ dependent nuclease activity with an optimum pH and temperature of 8.5 and 37 °C respectively. Experiments with amino acid modifying reagents revealed that histidine, cysteine and arginine residues were crucial for its function. hPLSCR1 has five histidine residues and point mutations of histidine residues to alanine in hPLSCR1 resulted in 60 % loss in nuclease activity. Thus histidine residues could play a critical role in the nuclease activity of hPLSCR1.ConclusionsThis is the first report on the novel nuclease activity of the multi-functional hPLSCR1. hPLSCR1 shows a metal dependent nuclease activity which could play a role in key cellular processes that needs to be further investigated.

Synthesis, characterization, crystal structures and biological activity of set of Cu(II) benzothiazole complexes: Artificial nucleases with cytotoxic activities

A series of Cu(II) complexes with ligand frames based on quinoline derivatives appended with a benzothiazole substituent has been isolated. The complexes, Cu(Q(oBt))(NO3)2(H2O)∙CH3OH (1∙CH3OH), Cu(8OHQ(oBt))Cl2∙CH3OH (2∙CH3OH), Cu(8OQ(oBt))Cl(CH3OH)∙CH3OH (3∙CH3OH) and [Cu(8OH1/2Q(oBt))(CH3OH)(NO3)]2(NO3) (4) have been characterized by single crystal X-ray diffraction, IR and UV-visible spectroscopies, and elemental analysis. The ligand frame within the set of complexes differs in the substituent on the quinoline ring: complex 1 remains unsubstituted at this position while complexes 2–4 have a substituted OH group. In complex 2, the bound phenol remains protonated while in 3 it is a phenolato group. Complex 4 contains two complexes within the unit cell and one NO3− giving rise to an overall ‘half-protonation’. The interaction between complexes 1–3 with CT-DNA was investigated using fluorescence emission spectroscopy and revealed 2 and 3 strongly intercalate DNA with Kapp values of 1.47×107M−1 and 3.09×107M−1, respectively. The ability of complexes 1–3 to cleave SC-DNA was monitored using gel electrophoresis. Each complex exhibits potent, concentration dependent nuclease activity forming single and double-nicked DNA as low as 10μM. The nuclease activity of complexes 1–3 is primarily dependent on 1O2 species while ·OH radicals play a secondary role in the cleavage by complexes 2 and 3. The cytotoxic effects of 1–3 were examined using HeLa cells and show cell death in the micromolar range. The distribution of cell cycle stages remains unchanged when complexes are present indicating DNA damage may be occurring throughout the cell cycle.

The Structure of the Herpes Simplex Virus DNA-Packaging Terminase pUL15 Nuclease Domain Suggests an Evolutionary Lineage among Eukaryotic and Prokaryotic Viruses

Herpes simplex virus 1 (HSV-1), the prototypic member of herpesviruses, employs a virally encoded molecular machine called terminase to package the viral double-stranded DNA (dsDNA) genome into a preformed protein shell. The terminase contains a large subunit that is thought to cleave concatemeric viral DNA during the packaging initiation and completion of each packaging cycle and supply energy to the packaging process via ATP hydrolysis. We have determined the X-ray structure of the C-terminal domain of the terminase large-subunit pUL15 (pUL15C) from HSV-1. The structure shows a fold resembling those of bacteriophage terminases, RNase H, integrases, DNA polymerases, and topoisomerases, with an active site clustered with acidic residues. Docking analysis reveals a DNA-binding surface surrounded by flexible loops, indicating considerable conformational changes upon DNA binding. In vitro assay shows that pUL15C possesses non-sequence-specific, Mg(2+)-dependent nuclease activity. These results suggest that pUL15 uses an RNase H-like, metal ion-mediated catalysis mechanism for cleavage of viral concatemeric DNA. The structure reveals extra structural elements in addition to the RNase H-like fold core and variations in local architecture of the nuclease active site, which are conserved in herpesvirus terminases and bear great similarity to the phage T4 gp17 but are distinct from podovirus and siphovirus orthologs and cellular RNase H, delineating a new evolutionary lineage among a large family of eukaryotic viruses and simple and complex prokaryotic viruses.

PEG-mediated one-pot multicomponent reactions for the efficient synthesis of functionalized dihydropyridines and their functional group dependent DNA cleavage activity

Polyethylene glycol (PEG) has been found to be an inexpensive, non-toxic and useful medium for the one pot synthesis of highly functionalized dihydropyridines using multicomponent reactions (MCRs) at room temperature under catalyst free conditions. The notable features of this protocol are: mild reaction condition, applicability to wide range of substrates, reusability of the PEG and good yields. The interaction of the synthesized compounds with pUC19 plasmid DNA was also analyzed. Some of the synthesized compounds showed interesting functional group dependent nuclease activity for plasmid DNA cleavage under physiological conditions.

Identification of a type I Ca2+/Mg2+-dependent endonuclease induced in maize cells exposed to camptothecin

BackgroundCamptothecin is a plant alkaloid that specifically binds topoisomerase I, inhibiting its activity and inducing double stranded breaks in DNA and activating the cell responses to DNA damage.ResultsMaize cultured cells were incubated in the presence of different concentrations of camptothecin. Camptothecin inhibits cultured cell growth, induces genomic DNA degradation, and induces a 32 kDa Ca2+/Mg2+-dependent nuclease activity. This nuclease, we called CaMNUC32, is inhibited by Zn2+ and by acid pH, it is mainly localized in the nucleus and it cleaves single- and double-stranded DNA, with a higher activity against single-stranded DNA. Two-dimensional electrophoresis combined with mass spectrometry suggests that CaMNUC32 is a member of the type I S1/P1 nuclease family. This type of nucleases are usually Zn2+-dependent but our results support previous indications that S1-type nucleases have a wide variety of enzyme activities, including Ca2+/Mg2+-dependent.ConclusionsWe have identified and characterized CaMNUC32, a 32 kDa Ca2+/Mg2+-dependent nuclease of the S1/P1 family induced by the topoisomerase I inhibitor camptothecin in maize cultured cells.

The hybrid recombinational repair pathway operates in a χ activity deficient recC1004 mutant of Escherichia coli

Homologous recombination is a crucial process for the maintenance of genome integrity. The two main recombination pathways in Escherichia coli (RecBCD and RecF) differ in the initiation of recombination. The RecBCD enzyme is the only component of the RecBCD pathway which acts in the initiation of recombination, and possesses all biochemical activities (helicase, 5'-3' exonuclease, χ cutting and loading of the RecA protein onto single-stranded (ss) DNA) needed for the processing of double stranded (ds) DNA breaks (DSB). When the nuclease and RecA loading activities of the RecBCD enzyme are inactivated, the proteins of the RecF recombination machinery, i.e., RecJ and RecFOR substitute for the missing 5'-3' exonuclease and RecA loading activity respectively. The above mentioned activities of the RecBCD enzyme are regulated by an octameric sequence known as the χ site (5'-GCTGGTGG-3'). One class of recC mutations, designated recC*, leads to reduced χ cutting invitro. The recC1004 strain (a member of the recC* mutant class) is recombination proficient and resistant to UV radiation. In this paper, we studied the effects of mutations in RecF pathway genes on DNA repair (after UV and γ radiation) and on conjugational recombination in recC1004 and recC1004 recD backgrounds. We found that DNA repair after UV and γ radiation in the recC1004 and recC1004 recD backgrounds depends on recFOR and recJ gene products. We also showed that the recC1004 mutant has reduced survival after γ radiation. This phenotype is suppressed by the recD mutation which abolishes the RecBCD dependent nuclease activity. Finally, the genetic requirements for conjugational recombination differ from those for DNA repair. Conjugational recombination in recC1004 recD mutants is dependent on the recJ gene product. Our results emphasize the importance of the canonical χ recognition activity in DSB repair and the significance of interchange between the components of two recombination machineries in achieving efficient DNA repair.

Biochemical Characterization of Bacteriophage T4 Mre11-Rad50 Complex

The Mre11-Rad50 complex (MR) from bacteriophage T4 (gp46/47) is involved in the processing of DNA double-strand breaks. Here, we describe the activities of the T4 MR complex and its modulation by proteins involved in homologous recombination. T4 Mre11 is a Rad50- and Mn(2+)-dependent dsDNA exonuclease and ssDNA endonuclease. ATP hydrolysis is required for the removal of multiple nucleotides via dsDNA exonuclease activity but not for the removal of the first nucleotide or for ssDNA endonuclease activity, indicating ATP hydrolysis is only required for repetitive nucleotide removal. By itself, Rad50 is a relatively inefficient ATPase, but the presence of Mre11 and dsDNA increases ATP hydrolysis by 20-fold. The ATP hydrolysis reaction exhibits positive cooperativity with Hill coefficients ranging from 1.4 for Rad50 alone to 2.4 for the Rad50-Mre11-DNA complex. Kinetic assays suggest that approximately four nucleotides are removed per ATP hydrolyzed. Directionality assays indicate that the prevailing activity is a 3' to 5' dsDNA exonuclease, which is incompatible with the proposed role of MR in the production of 3' ssDNA ends. Interestingly, we found that in the presence of a recombination mediator protein (UvsY) and ssDNA-binding protein (gp32), Mre11 is capable of using Mg(2+) as a cofactor for its nuclease activity. Additionally, the Mg(2+)-dependent nuclease activity, activated by UvsY and gp32, results in the formation of endonuclease reaction products. These results suggest that gp32 and UvsY may alter divalent cation preference and facilitate the formation of a 3' ssDNA overhang, which is a necessary intermediate for recombination-mediated double-strand break repair.

Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation.

Comparative nuclease and anti-cancer properties of the naturally occurring malabaricones

The nuclease activities of the malabaricones have been studied so as to establish a structure–activity correlation and deduce the mechanistic pathway of the process. The inactivity of malabaricone A and malabaricone D revealed that the resorcinol moiety, present in the malabaricones did not contribute to the nuclease activity. Amongst the test compounds, malabaricone C (mal C) containing a B-ring catechol moiety showed significantly better Cu(II)-dependent nuclease activity than the partially methylated catechol derivative, mal B and curcumin. Mal C was found to bind efficiently with Cu(II) and DNA to facilitate the DNA nicking via a site-specifically generated Cu(I)-peroxo complex. Consistent with its Cu(II)-dependent nuclease property, mal C showed better cytotoxicity (IC 50 = 5.26 ± 1.20 μM) than curcumin (IC 50 = 24.46 ± 3.32 μM) against the MCF-7 human breast cancer cell line. The mal C-induced killing of the MCF-7 cells followed an apoptotic pathway involving oxidative damage to the cellular DNA.

In vitro inhibition of the replication of classical swine fever virus by capsid-targeted virus inactivation

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious fatal disease of swine. Few effective antiviral drugs are currently available against CSFV infections. To explore the feasibility of using capsid-targeted viral inactivation (CTVI) as an antiviral strategy against CSFV infections, we expressed the CSFV capsid protein (Cap) fused with the nuclease of Staphylococcus aureus (SN) in Escherichia coli and investigated its effects on the replication of CSFV in PK-15 cells. The results indicated that the fusion protein Cap-SN showed a strong Ca 2+-dependent nuclease activity and inhibited the replication of CSFV in a dose-dependent manner, with complete inhibition at a concentration of 15 μg/ml, whereas the Cap fused with an enzymatically inactive SN (Cap-SN*) showed no nuclease activity or antiviral effects. Thus, the CTVI approach might be applicable to CSFV inhibition as a novel antiviral strategy.

The intermembrane space of plant mitochondria contains a DNase activity that may be involved in programmed cell death.

The key role for mitochondria in mammalian apoptosis, a form of programmed cell death (PCD), is well established, but a similar role for plant mitochondria is just emerging. In order to unravel the molecular mechanisms linking plant mitochondria to the downstream events of PCD, we have developed an Arabidopsis cell-free system that can be used to monitor biochemical and morphological changes in isolated nuclei that are associated with PCD. Using this system, two activities that resulted in nuclear DNA degradation could be distinguished, both of which were facilitated by the addition of mitochondria. One activity mediated the generation of 30 kb DNA fragments within 3 h and chromatin condensation within 6 h, when nuclei were incubated with mitochondria alone. The second activity required cytosolic extract in addition to mitochondria and resulted in oligonucleosome-sized DNA cleavage after >12 h. Submitochondrial fractionation and pharmacological studies suggested the presence of an Mg2+-dependent nuclease activity in the intermembrane space, which is responsible for the former in vitro activity. The evolutionary conservation of the role of mitochondria in PCD in animals and plants is discussed.

An mt(+) gamete-specific nuclease that targets mt(-) chloroplasts during sexual reproduction in C. reinhardtii.

Although the active digestion of mating-type minus (mt-) chloroplast DNA (cpDNA) in young zygotes is considered to be the basis for the uniparental inheritance of cpDNA in Chlamydomonas reinhardtii, little is known about the underlying molecular mechanism. One model of active digestion proposes that nucleases are either synthesized or activated to digest mt- cpDNA. We used a native-PAGE/in gelo assay to investigate nuclease activities in chloroplasts from young zygotes, and identified a novel Ca(2+)-dependent nuclease activity. The timing of activation (approximately 60-90 min after mating) and the localization of the nuclease activity (in mt- chloroplasts) coincided with the active digestion of mt- cpDNA. Furthermore, the activity of the nuclease was coregulated with the maturation of mating-type plus (mt+) gametes, which would enable the efficient digestion of mt- cpDNA. Based on these observations, we propose that the nuclease (designated as Mt(+)-specific DNase, MDN) is a developmentally controlled nuclease that is activated in mt+ gametes and participates in the destruction of mt- cpDNA in young zygotes, thereby ensuring uniparental inheritance of chloroplast traits.

The Escherichia coli ribosomal protein S16 is an endonuclease.

The histone-like protein HU isolated from Escherichia coli exhibited, after several purification steps, a Mg(2+)-dependent nuclease activity. We show here that this activity can be dissociated from HU by a denaturation-renaturation step, and is due to a small fraction of ribosomal protein S16 co-purifying with HU. S16 is an essential component of the 30S ribosomal particles. We have cloned, overproduced, and purified a histidine-tagged S16 and shown that this protein is a DNA-binding protein carrying a Mg(2+)-Mn(2+)-dependent endonuclease activity. This is an unexpected property for a ribosomal protein.

Metal Ion Dependent Binding and Nuclease Activity of Hexaazacyclophane

AbstractThe HAPP‐Fe complex was more active than HAPP‐Cu complex in cleavage of DNA in the presence of dithiothreitol and hydrogen peroxide. In contrast, the HAPP‐Cu complex was found to bind more strongly with λ‐DNA, whereas the HAPP‐Fe binds weakly. It was shown that the metal ion complexed with HAPP ligand can influence the binding and cleaving activity due to the ability of the ligand to undergo geometrical tautomerization.

Types of nuclear endonuclease activity capable of inducing internucleosomal DNA fragmentation are completely different between human CD34+ cells and their granulocytic descendants

A hallmark of apoptosis is internucleosomal DNA fragmentation resulting from the activation of endonucleases. We characterized the endonuclease activity of human myeloid cell nuclei that cleaved their own nuclear chromatin to oligonucleosomal length fragments. Polymorphonuclear leukocytes (PMNs) of normal peripheral blood contained both Ca2+/Mg(2+)- dependent and DNase II-like acidic endonuclease activities in their nuclei. Immature myeloid cells of normal bone marrow at various stages of granulocytic maturation had similar nuclease activities. In contrast, a clear difference was shown in the circulating CD34+ cells, in that only Mg(2+)-dependent, Ca(2+)-independent endonuclease activity was detected. Consistent with these findings is the emergence of the Ca2+/Mg(2+)-dependent and acidic endonuclease concomitantly with the disappearance of the Mg(2+)-dependent endonuclease when CD34+ cells were induced to differentiate in vitro toward granulocytes. Leukemic cell lines of all lineages also had Mg(2+)-dependent nuclease activity. Our results suggest an association of the Mg(2+)-dependent endonuclease with hematopoietic progenitor cells and that the relative activities of the nuclear nuclease in human myeloid cells change substantially during granulocytic differentiation.