Interaction of DNA fragmentation factor (DFF) with DNA reveals an unprecedented mechanism for nuclease inhibition and suggests that DFF can be activated in a DNA-bound state.
DNA fragmentation factor (DFF) is a complex of the DNase DFF40 (CAD) and its chaperone/inhibitor DFF45 (ICAD-L) that can be activated during apoptosis to induce DNA fragmentation. Here, we demonstrate that DFF directly binds to DNA in vitro without promoting DNA cleavage. DNA binding by DFF is mediated by the nuclease subunit, which can also form stable DNA complexes after release from DFF. Recombinant and reconstituted DFF is catalytically inactive yet proficient in DNA binding, demonstrating that the nuclease subunit in DFF is inhibited in DNA cleavage but not in DNA binding, revealing an unprecedented mode of nuclease inhibition. Activation of DFF in the presence of naked DNA or isolated nuclei stimulates DNA degradation by released DFF40 (CAD). In transfected HeLa cells transiently expressed DFF associates with chromatin, suggesting that DFF could be activated during apoptosis in a DNA-bound state.
Korn
Christian
C
Institute of Biochemistry, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Scholz
Sebastian R
SR
Gimadutdinow
Oleg
O
Lurz
Rudi
R
Pingoud
Alfred
A
Meiss
Gregor
G
D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001483:Base Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D008958:Models, Molecular; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D011487:Protein Conformation; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D051017:Apoptosis Regulatory Proteins; D002843:Chromatin; D000075223:Poly-ADP-Ribose Binding Proteins; D021122:Protein Subunits; D011506:Proteins; D011994:Recombinant Proteins; C109664:caspase-activated DNase inhibitor; D004247:DNA; D003851:Deoxyribonucleases; C499445:Dffb protein, mouse; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases