A Role of the Ca2+/Mg2+-dependent Endonuclease in Apoptosis and Its Inhibition by Poly(ADP-ribose) Polymerase

Alexander G Yakovlev,Geping Wang,Bogdan A Stoica,Hamid A Boulares,Alexander Y Spoonde,Koichiro Yoshihara,Mark E Smulson

doi:10.1074/jbc.m001087200

Alexander G Yakovlev, Geping Wang + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m001087200

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Abstract

Apoptosis is characterized by various cell morphological and biochemical features, one of which is the internucleosomal degradation of genomic DNA. The role of the human chromatin-bound Ca(2+)- and Mg(2+)-dependent endonuclease (CME) DNAS1L3 and its inhibition by poly(ADP-ribosyl)ation in the DNA degradation that accompanies apoptosis was investigated. The nuclear localization of this endonuclease is the unique feature that distinguishes it from other suggested apoptotic nucleases. Purified recombinant DNAS1L3 was shown to cleave nuclear DNA into both high molecular weight and oligonucleosomal fragments in vitro. Furthermore, exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this CME, as well as in a decrease in cell viability greater than that apparent in the control cells. Recombinant DNAS1L3 was modified by recombinant human poly(ADP-ribose) polymerase (PARP) in vitro, resulting in a loss of nuclease activity. The DNAS1L3 protein also underwent poly(ADP-ribosyl)ation in transfected mouse skin fibroblasts in response to inducers of apoptosis. The cleavage and inactivation of PARP by a caspase-3-like enzyme late in apoptosis were associated with a decrease in the extent of DNAS1L3 poly(ADP-ribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.

Highlights

Apoptosis, or programmed cell death, is an evolutionarily conserved process that is important in normal development, physiological homeostasis, and certain pathological conditions
Exposure of mouse skin fibroblasts expressing DNAS1L3 to inducers of apoptosis resulted in oligonucleosomal DNA fragmentation, an effect not observed in cells not expressing this Ca2؉- and Mg2؉-dependent endonuclease (CME), as well as in a decrease in cell viability greater than that apparent in the control cells
One of earliest nuclear events in apoptosis is the poly(ADPribosyl)ation of various proteins by poly(ADP-ribose) polymerase (PARP), an enzyme that is activated by the presence of DNA strand breaks [23, 24]

Summary

Introduction

Programmed cell death, is an evolutionarily conserved process that is important in normal development, physiological homeostasis, and certain pathological conditions. Our observations suggested that subsequent cleavage of PARP by a caspase-3-like protease releases certain nuclear proteins from poly(ADP-ribosyl)ation-induced inhibition and thereby allows them to mediate DNA fragmentation and cell death [23].

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Conclusion