Deoxyribonuclease IIα is required during the phagocytic phase of apoptosis and its loss causes perinatal lethality

Krieser, Ronald J.; MacLea, Kyle S.; Longnecker, Daniel S.; Fields, Jennifer; Fiering, Steven; Eastman, Alan

doi:10.1038/sj.cdd.4401056

Cited by 86 publications

(80 citation statements)

References 26 publications

(19 reference statements)

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“…Additionally in development, it is hypothesized that phagocytosis is a critical component in which the DNAse II plays a critical role. 35 Therefore, the existence of multiple redundant pathways may obscure understanding the role of a single endonuclease through targeted disruption.…”

Section: Discussionmentioning

confidence: 99%

EndoG is dispensable in embryogenesis and apoptosis

Sasaki

et al. 2005

Cell Death Differ

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The mitochondrial protein, endonuclease G (EndoG), is one of the endonucleases implicated in DNA fragmentation during apoptosis. It has been shown to translocate from the mitochondria to the nucleus upon cell death stimuli. These observations suggest that EndoG is a mitochondrial cell death effector, and that it possibly acts as a cell death nuclease, similar to DNA fragmentation factor. To better understand the role of EndoG in development and apoptosis, we generated EndoG null mice by homologous gene targeting without disruption of D2Wsu81e. EndoG null mice are viable and develop to adulthood with no obvious abnormalities. Fibroblasts generated from the EndoG null mice show no difference in susceptibility when induced to die by a variety of intrinsic and extrinsic apoptotic stimuli. Additionally, EndoG null mice are equally sensitive to excitotoxic stress. These data suggest that EndoG is not essential for early embryogenesis and apoptosis.

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Section: Discussionmentioning

confidence: 99%

EndoG is dispensable in embryogenesis and apoptosis

Sasaki

et al. 2005

Cell Death Differ

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“…In our previous work, we described the development of a knockout mouse model for DNase IIα [9]. To produce primary MEFs, we harvested day-12.5 mouse embryos from a breeding of mice heterozygous for the null allele.…”

Section: Isolation Of Primary Mouse Embryonic Fibroblasts (Mefs) Delementioning

confidence: 99%

“…DNase II has been reported in lysosomes, nuclei and various secretions [4][5][6][7][8], and is critical to the process of mammalian development [9,10]. The activity of DNase II is distinguished from other endonucleases by its acidic pH optimum, the absence of a requirement for bivalent cations and its ability to hydrolyse double-stranded DNA to yield short oligonucleotides bearing 3h-phosphate groups, rather than 3h-hydroxyl groups.…”

Section: Introductionmentioning

confidence: 99%

Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging

MacLea

Krieser

Eastman

2003

Biochemical Journal

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DNase IIα (EC 3.1.22.1) is an endonuclease, which is active at low pH, that cleaves double-stranded DNA to short 3′-phosphoryl oligonucleotides. Although its biochemistry is well understood, its structure–activity relationship has been largely unexamined. Recently, we demonstrated that active DNase IIα consists of one contiguous polypeptide, heavily glycosylated, and containing at least one intrachain disulphide linkage [MacLea, Krieser and Eastman (2002) Biochem. Biophys. Res. Commun. 292, 415–421]. The present paper describes further work to examine the elements of DNase IIα protein required for activity. Truncated forms and site-specific mutants were expressed in DNase IIα-null mouse cells. Results indicate that the signal-peptide leader sequence is required for correct glycosylation and that N-glycosylation is important for formation of the active enzyme. Despite this, enzymic deglycosylation of wild-type protein with peptide N-glycosidase F reveals that glycosylation is not intrinsically required for DNase activity. DNase IIα contains six evolutionarily conserved cysteine residues, and mutations in any one of these cysteines completely ablated enzymic activity, consistent with the importance of disulphide bridging in maintaining correct protein structure. We also demonstrate that a mutant form of DNase IIα that lacks the purported active-site His295 can still bind DNA, indicating that this histidine residue is not simply involved in DNA binding, but may have a direct role in catalysis. These results provide a more complete model of the DNase IIα protein structure, which is important for three-dimensional structural analysis and for production of DNase IIα as a potential protein therapeutic for cystic fibrosis or other disorders.

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“…Mutation of the C. elegans dnase II homologue, nuc-1, resulted in persistent dead-cell nuclei within neighboring phagocytic cells as well as accumulation of DNA within the gut and ovaries [7,8]. Interestingly, targeted mutation of the dnase II gene in the germline of mice resulted in perinatal lethality presumably due to loss definitive erythropoiesis [9,10]. Subsequent analyses of these mutant mice revealed that dnase II-deficient macrophages accumulate ingested DNA and overexpress β-interferon (INFβ) resulting in embryonic lethality [11,12].…”

Section: Introductionmentioning

confidence: 99%

DNase II deficiency impairs innate immune function in Drosophila

Seong

Varela-Ramı́rez

Aguilera

2006

Cellular Immunology

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DNase II enzymes are highly conserved proteins that are required for the degradation of DNA within phagolysosomes. Engulfment of apoptotic cells and/or bacteria by phagocytic cells requires the function of DNase II to completely destroy ingested DNA. Mutation of the dnase II gene results in an increase of undegraded apoptotic DNA within phagocytic cells in mice and nematodes. Additionally, reduction of DNase II enzymatic activity in Drosophila melanogaster has been shown to lead to increased accumulation of DNA in the ovaries. Due to the importance of DNA clearance during infection, we hypothesized that a severe reduction of DNase II activity would result in diminished immune function and viability. To test this hypothesis, we knocked down DNase II activity in flies using RNAi. As expected, expression of a dnase II-RNAi construct in flies resulted in a dramatic reduction of DNase II activity and a significant decrease in total hemocyte numbers. Furthermore, infection of dnase II-RNAi flies with Gram negative or positive bacteria resulted in a severe reduction in fly viability. These results confirm that DNase II and the ability to clear macromolecular DNA is essential for maintaining proper immune function in Drosophila.

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Deoxyribonuclease IIα is required during the phagocytic phase of apoptosis and its loss causes perinatal lethality

Cited by 86 publications

References 26 publications

EndoG is dispensable in embryogenesis and apoptosis

EndoG is dispensable in embryogenesis and apoptosis

Structural requirements of human DNase IIalpha for formation of the active enzyme: the role of the signal peptide, N-glycosylation, and disulphide bridging

DNase II deficiency impairs innate immune function in Drosophila

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