Abstract
We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. Here, we identify AN34 as an N-terminally truncated form of human AP endonuclease (Ape1) lacking residues 1-35 (delta35-Ape1). Although Ape1 has hitherto been considered specific for damaged DNA (specific to AP site), recombinant AN34 (delta35-Ape1) possesses significant endonuclease activity on undamaged (normal) DNA and in chromatin. AN34 also displays enhanced 3'-5' exonuclease activity. Caspase-3 activates AN34 in a cell-free system, although caspase-3 cannot cleave Ape1 directly in vitro. We also found that Ape1 itself preferentially cleaves damaged chromatin DNA isolated from cells treated with apoptotic stimuli and that silencing of Ape1 expression decreases apoptotic DNA fragmentation in DFF40/CAD-deficient cells. Thus, we propose that AN34 and Ape1 participate in the process of chromatin fragmentation during apoptosis.
Highlights
Apoptosis is an essential mechanism for maintaining cell number homeostasis in multicellular organisms
DFF40/CAD plays an important role as a major apoptotic nuclease in various experimental systems, mice lacking DFF45/ICAD are viable and still show residual DNA fragmentation [13, 14], suggesting that other endonucleases play an important role during apoptosis [15]
We identified and characterized a novel apoptotic nuclease, apoptotic nuclease 34 kDa (AN34), as an N-terminally truncated form of Ape1 (⌬35-Ape1 comprising residues 36 –318)
Summary
37768 –37776, 2003 Printed in U.S.A. Human Apurinic/Apyrimidinic Endonuclease (Ape1) and Its N-terminal Truncated Form (AN34) Are Involved in DNA Fragmentation during Apoptosis*. We previously isolated a 34-kDa nuclease (AN34) from apoptotic human leukemia cells. We previously purified an apoptotic nuclease 34 kDa (AN34) from human leukemia cells undergoing apoptosis [18]. Ape ( called HAP1 [20], Ref-1 [21], and APEX [22]) is a well characterized, 37-kDa DNA base excision repair protein, highly conserved among different species [23] It cleaves the DNA phosphodiester backbone immediately 5Ј to an AP site. We report that Ape selectively cleaves chromatin DNA from cells treated with apoptotic stimulus and that silencing of Ape expression decreased apoptotic DNA fragmentation in DFF40/CAD-deficient cells. This report is the first demonstration for a role for Ape in degrading DNA during apoptosis
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