Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates.
Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.
Widlak
P
P
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Li
P
P
Wang
X
X
Garrard
W T
WT
D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D053938:DNA Fragmentation / Q000502:physiology; D004273:DNA, Neoplasm / Q000378:metabolism*; D003851:Deoxyribonucleases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D000075223:Poly-ADP-Ribose Binding Proteins; D013379:Substrate Specificity
D002843:Chromatin; D004273:DNA, Neoplasm; D000075223:Poly-ADP-Ribose Binding Proteins; C113696:DFFB protein, human; D003851:Deoxyribonucleases; C109663:caspase-activated deoxyribonuclease; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D008274:Magnesium