Biochemical Association of Poly(ADP‐ribose) Polymerase‐1 and Its Apoptotic Peptide Fragments with DNA Polymerase β

Abstract

We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.

Pleiotropic Cellular Functions of PARP1 in Longevity and Aging: Genome Maintenance Meets Inflammation

“…Thus, PARP1 and PAR are required for the assembly and stability of XRCC1 nuclear foci after DNA damage [83]. Furthermore, XRCC1 and PARP1 interact with DNA polymerase- β and DNA ligase III, forming a multiprotein complex consisting of the major BER factors [84–86]. …”

Multitasking Roles for Poly(ADP-ribosyl)ation in Aging and Longevity

“…Thus, PARP1 and PAR are required for the assembly and stability of XRCC1 nuclear foci after DNA damage [96]. Furthermore, XRCC1 and PARP1 interact with DNA polymerase-β and DNA ligase III, forming a multiprotein complex consisting of the major BER factors [98][99][100]. As mentioned above, PARP1 and PARP-2 work at least partially in a redundant fashion which is evident from the fact that they homo-and heterodimerize and only double knock-out mice show embryonic lethality [35,101].…”