Protein -protein interactions between poly(ADP -ribose) polymerase-1 and DNA polymerase beta

摘要

The mammalian genome is continually subjected to chemical and environmental modifications that are repaired by base excision, and when excessive, may lead to apoptosis. Interestingly, the chromosomal enzyme poly(ADP-ribose) polymerase-1 (PARP-1) appears to modulate both mechanisms, either facilitating DNA repair and/or modulating cell death. In this dissertation project, experiments were performed to address the regulatory potential of PARP-1 in base excision repair (BER) and specifically on DNA polymerase beta (pol beta) function. Activity gels were used to measure the DNA polymerase activity of pol beta following protein-(ADP-ribosyl)ation. However, the fraction of pol beta molecules poly(ADP-ribosyl)ated was never 100% under the reaction conditions employed. In fact, similar results were observed in activity gels specific for PARP-1, even under conditions where this polymerase is the primary nuclear acceptor for poly(ADP-ribose). Here, I also describe a newly developed electrophoretic-mobility-shift-assay (EMSA) to monitor for the specific binding of pol beta to a custom-made five-nucleotide gapped DNA duplex. However, while specific for pol beta, this assay was inefficient to monitor the effects of covalent poly(ADP-ribosyl)ation on pol beta activity. Moreover, I also observed the specific molecular association of PARP-1 with pol beta by co-immunoprecipitation experiments, followed by reciprocal immuno-blotting for the protein partner. Knowing that during apoptosis endogenous PARP-1 is specifically proteolyzed into two peptide fragments by caspases, conditions were established for the efficient proteolysis of PARP-1 by either caspase-3 and/or caspase-7. Experimental results indicated that caspase-3 was more efficient than caspase-7 at splitting unmodified PARP-1 into two peptide fragments. By contrast, caspase-7 appeared best suited for the proteolysis of covalently auto-poly(ADP-ribosyl)ated-(PARP-1). Interestingly, both of the caspase-generated peptide fragments of PARP-1 specifically associated with pol beta as supported by co-immunoprecipitation/immuno-blotting experiments. Taken together, the experimental results presented here support the hypothesis that a molecular mechanism exists that involves interaction(s) of PARP-1 with pol beta that may help to facilitate the decision making process between cell survival and cell death. Thus, upon proteolytic degradation of PARP-1 into a 24-kDa amino-terminal fragment and an 89-kDa carboxy-terminus, each truncated peptide, separately, retains physical association with pol beta, and inhibits DNA repair associated pol beta activity to irreversibly switch the fate of the cell from BER toward chromatin degradation and, eventually, programmed cell death.