The major apoptotic endonuclease DFF40/CAD is a deoxyribose-specific and double-strand-specific enzyme

Abstract

DFF40/CAD endonuclease is primarily responsible for internucleosomal DNA cleavage during the terminal stages of apoptosis. The nuclease specifically introduces DNA double strand breaks into chromatin substrates. Here we performed a detailed study on the specificity of the nuclease using synthetic single-stranded and double-stranded ribo- and deoxyribo-oligonucleotides as substrates. We have found that neither single-stranded DNA, single-stranded RNA, double-stranded RNA nor RNA-DNA heteroduplexes are cleaved by the DFF40/CAD nuclease. Noteworthy, all types of oligonucleotides that are not cleaved by the nuclease inhibit cleavage of double-stranded DNA. We have also observed that in cells undergoing apoptosis in vivo neither the activation of DFF40/CAD nor oligonucleosomal chromatin fragmentation was temporally correlated with either total cellular or nuclear RNA degradation. We conclude that DFF40/CAD is exclusively specific for double-stranded DNA.

Roles of the Major Apoptotic Nuclease-DNA Fragmentation Factor-in Biology and Disease

“…The nuclease is inhibited by several poly-anions, like heparin, polyglutamic acid [66], poly(ADP-ribose) [67] and nucleic acids that are not its substrates (single stranded DNA, RNA and DNA-RNA heteroduplexes) [34,66]. Such polyanions, which are thought to bind to the positively charged surface formed by the a4 helices of DFF40 and impair its ability to bind to double-stranded DNA, are competitive inhibitors of the nuclease [66,68].…”

“…Hypothetically, RNA might suppress DFF40 activity to avoid premature apoptotic DNA breakdown that would inhibit the induction of genes encoding pro-apoptotic factors, and certain apoptotic RNases could co-ordinate the proper timing of RNA and DNA breakdown. However, degradation of RNA does not precede DNA cleavage in cells undergoing apoptosis, and thus the integrity of cellular RNA unlikely affects the activity of DFF in vivo [34]. Low-molecular-weight compounds that affect the activity of DFF are much less studied at present.…”

“…DFF40/CAD (hereafter termed DFF40) is a magnesium dependent endonuclease that cuts DNA to yield 5-phosphate residues and 3-hydroxyl groups that are substrates for terminal deoxynucleotide transferase, which is the foundation of the apoptotic TUNEL assay [33]. It is both a deoxyribose-specific and double-strand specific enzyme [34] that generates exclusively double strand breaks (primarily blunt ends) [33]. In addition, the nuclease exhibits an extraordinary preference for cleaving the internucleosomal linker regions in chromatin [28,33].…”

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells

“…It is now well-documented that DFF40 is the only nuclease that cuts double-stranded DNA [8] in the DNA internucleosomal linker region [9], thus producing oligonucleosomes. This type of degradation can be detected by agarose gel electrophoresis as a characteristic ''DNA ladder'' [6,9,10] as opposed to the ''smear-type'' appearance after nonspecific DNA fragmentation, when other apoptotic endonucleases are activated which cut single-stranded and double-stranded DNA in both intra-and inter-nucleosomal regions.…”

Contemporary Views on the Pathogenesis of Trauma to the Spinal Cord and Peripheral Nerve Trunks

“…DNA fragmentation in cells in which programmed death has been triggered starts with occasional single-stranded breaks and later involves frequent double-stranded breaks [62]. This is apparent as a characteristic ladder on electrophoresis, due to the formation of DNA fragments [57]. It is estimated that about 40 double-stranded breaks per cell, which is one per chromosome, is suffi cient for cell death [11].…”