ADP-ribosyl transferase Diptheria toxin-like 1 (ARTD1), also known as Poly(ADP-ribose) polymerase 1 (PARP1), is a 116 kDa, multi-domain regulatory protein that catalyzes the iterative transfer of ADP-ribose from NAD+ onto target residues, the phenomenon called poly(ADP-ribosyl)ation (PARylation). PARP1 can either PARylate itself (self-PARylation) or other proteins (trans-PARylation), leading to building linear or branched chains of Poly(ADP-ribose) (PAR) [1]. PARP1 consists of regulatory domains, which include three zinc finger domains, ZnF1, ZnF2, and ZnF3, followed by the BRCT (Breast cancer-associated C-terminal) domain, the WGR (Trp-Gly-Arg rich) domain, and the signature catalytic (CAT) domain which comprises two sub domains, helical domain (HD) and ADP-ribosyl transferase domain (ART) (Fig. 1A).

PARP1, the founding member of the PARP family, was first identified for its involvement in single-strand breaks (SSB) repair through break recognition [2], [3], [4]. The role of PARP1 in maintaining genome integrity through detecting DNA damage and PAR-mediated recruitment of repair factors to initiate the DNA repair process is well-established [5], [6], [7]. The DNA breaks are recognized by zinc finger domains of PARP1, leading to robust stimulation of its catalytic activity [8], [9]. Contrarily, when unrepaired, cells undergo either caspase-dependent (apoptosis) or caspase-independent (parthanatos) programmed cell death [10], [11].

During apoptosis, PARP1 is proteolytically cleaved by caspase 3 and caspase 7 into a 24 kDa fragment, which includes ZnF1 and ZnF2 (ZnF1-2PARP1) DNA-binding domain (DBD) or regulatory fragment, and an 89 kDa fragment (PARP1ΔZnF1-2) containing the ZnF3, BRCT, WGR and CAT domains, called as catalytic fragment (Fig. 1A) [12], [13]. The PARylated-PARP1ΔZnF1-2 translocates to the cytoplasm to induce the release of apoptosis-inducing factor (AIF) from mitochondria [14]. On the other hand, the ZnF1-2PARP1 apoptotic fragment remains bound to DNA breaks, which competes with incoming PARP1 resulting in a trans-dominant inhibition of PAR synthesis leading to total abrogation of DNA repair [15], [16], [17]. Although, when levels of caspase 3 increase in the cell, unPARylated-PARP1 could also get cleaved [17], [18].

In parthanatos, PAR is the key signalling molecule for cell death. Over-activation of PARP1 causes over-production of PAR, leading to depletion of the NAD+ pool in the cell (10). The excessive PAR translocates to cytoplasm and induces the release of AIF (19). The involvement of PAR and the apoptotic variants of PARP1 (PARP1ΔZnF1-2 and ZnF1-2PARP1) in mediating parthanatos and apoptosis, respectively, is well established [11], [14], [15], [19].

Recently, it has been shown that (A) ZnF3-BRCT-WGR domains of PARP1 can bind to PAR and stimulate PARP1 catalytic activity [20] and (B) PARP1ΔZnF1-2 is incapable of DNA-dependent stimulation due to absence of DBD but shows basal enzymatic activity [15], [16]. Since the apoptotic variant PARP1ΔZnF1-2 lacks the DBD but contains all the three high-affinity PAR-binding domains (Fig. 1A), we wanted to understand if PAR can also regulate apoptotic fragment activity. Therefore, to decipher the effect of PAR and DNA on the activity of apoptotic fragments of PARP1, we investigated (A) the effect of PAR on the catalytic activity of apoptotic fragment PARP1ΔZnF1-2? and (B) Can the apoptotic fragments, PARP1ΔZnF1-2 and ZnF1-2PARP1, assemble and respond to DNA dependent stimulation of catalytic activity? Upon cleavage, the N-terminal apoptotic fragment ZnF1-2PARP1 fragment remains bound to the DNA break and trans-dominantly inhibits the activity of PARP1 [14] and the DNA break recognition by PARP2 stimulates its PARylation activity that recruits the repair proteins at the site of DNA damage which may hinder the apoptosis [21], [22], [23]. Therefore, we studied whether ZnF1-2PARP1 can compete with PARP2 for the DNA break binding? If so, then does this affect the DNA-dependent stimulation of PARP2?