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

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(ADPribosyl)ation, which likely releases DNAS1L3 from inhibition and allows it to catalyze the degradation of genomic DNA.Apoptosis, or programmed cell death, is an evolutionarily conserved process that is important in normal development, physiological homeostasis, and certain pathological conditions. It is mediated by a variety of intracellular enzymes, among which are endonucleases that catalyze the internucleosomal fragmentation of DNA, which is one of the hallmarks of apoptotic death (1, 2). Candidates for such endonucleases include the caspase-activated enzymes DFF40 (or CAD) (3-8) and NUC70 (9), divalent cation-dependent neutral (1, 2) or acidic (10, 11) endonucleases, leukemia-associated endo-exonucleases (12), and Ca 2ϩ -and Mg 2ϩ -dependent endonucleases (CMEs) 1 (13-18). CMEs introduce double strand breaks and single strand nicks into DNA, generating fragments with 5Ј-phosphate and 3Ј-hydroxyl termini, a mode of DNA fragmentation consistent with the products of chromatin degradation in apoptotic cells (19 -21). CME activity is increased by a variety of stimuli that induce apoptosis (17, 21), and treatments that prevent apoptosis also prevent the induction of CME activity. A role for CMEs in apoptosis has also been supported by studies demonstrating the inhibition of DNA fragmentation by Ca 2ϩ chelators or Zn 2ϩ (13)(14)(15)22). 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). PARP catalyzes the modification of histones, topoisomerases I and II, SV40 large T antigen, DNA polymerase ␣, proliferating cell nuclear antigen, and various other DNA-bindi...

Regulation of DNaseY activity by actinin-α4 during apoptosis

“…31 It has been reported that, at early stages of apoptosis, DNaseY can be inactivated by PARP-1 through the poly-ADPribosylation mechanism. 18,19 Here we found a significant upregulation of PARP-1 in the DNaseY-overexpressing cells (Figure 11a). Therefore, it is possible that PARP-1 acts both as a sensor to detect ssb and at the same time to synthesize negatively charged ADP-ribose polymers to inhibit the endonuclease that creates them.…”

“…This is in agreement with the previous studies, which reported that transfection of the same nuclease into HeLa cells or mouse skin fibroblasts increased cell death only in response to apoptotic stimuli, but not by itself. 9,18 In our cell system, overexpression of DNaseY led to the activation of DNA repair pathways. The components of both repair systems, the Polb-XRCC1-Lig3 pathway, which conducts both short and long-patch repair as well as the Pold/e-PCNALig1 pathway, which provides an alternative or 'backup' mechanism for ssb repair and often conducts long-patch break repair, 24 were clearly upregulated in the DNaseYoverexpressing cells (Figure 11e and f).…”

“…Therefore, it is possible that PARP-1 acts both as a sensor to detect ssb and at the same time to synthesize negatively charged ADP-ribose polymers to inhibit the endonuclease that creates them. 20,32 In the execution phase of apoptosis, PARP-1 is cleaved and inactivated by caspaselike proteases relieving inhibition of DNaseY; 18,19 however, the inactivation of PARP-1 may not be sufficient to fully unleash the activity of DNaseY. The cytoplasmic changes, that is, alterations in signalling pathways and cytoskeletal changes, might also be required to accomplish this.…”

“…This negative regulation of DNaseY may be reversed upon the cleavage and inactivation of PARP-1 by a caspase-3 like protease, followed by reactivation of DNaseY by PAR glycohydrolase. 18,19 PARP-1 has long been considered a molecular 'nick sensor' that is activated at the sites of ssb and synthesizes negatively charged poly-ADP-ribose. [20][21][22] Its physical interaction with the base excision repair protein, X-ray repair cross complementing 1 (XRCC1), confirms that PARP-1 is a member of a multiprotein complex involved in the DNA polymerase b, XRCC1 and DNA ligase 3 (Polb-XRCC1-Lig3) repair pathway.…”

Cooperation between C1q and DNase I in the clearance of necrotic cell–derived chromatin

“…The digestion by exogenous DNase I of fixed nuclei derived from viable cells yields a fairly similar initial appearance of the chromatin degradation (45). In the later phases of DNA digestion in apoptotic cells, several endogenous nucleases and auxiliary proteins cooperate to generate an oligonucleosome ladder, a hallmark of apoptosis (19,(46)(47)(48)(49)(50)(51)(52)(53). The nuclei of apoptotic cells are degraded before lysis of the cytoplasmic membrane.…”