Education:

B.S. Chemistry & Biology
1958
Washington & Lee University
Ph.D. Biochemistry
1964
Cornell University
Postdoctoral Fellow National Cancer Institute

Appointments

Joint Appointment, Department of Radiation Medicine
Past Co-Director, Program of DNA Repair, Lombardi Cancer Center

Research Interest

One of the earliest nuclear events that follows DNA strand breakage caused by agents such as ionizing irradiation, carcinogens, or alkylating agents during DNA repair is the poly(ADP-ribosyl)ation of various proteins that are localized near DNA strand breaks. Poly (ADP-ribose) polymerase (PARP) catalyzes the poly (ADP-ribosyl)ation of various nuclear proteins; however, only when PARP is bound to single -or double stranded DNA ends. We have shown that PARP cycles on and off the DNA ends during DNA repair in vitro. Poly(ADP-ribosyl)ation occurs on nuclear DNA binding proteins, such as histones, topoisomerases I and II SV40 large T antigen, DNA polymerase a, PCNA, and ~15 protein components of the DNA synthesome, as well as the automodification of PARP itself. We have hypothesized that this modification of nucleosomal proteins changes the nucleosomal structure of the DNA surrounding strand breaks and accordingly promotes access of various replicative and repair enzymes to these sites. PARP has been shown to undergo proteolytic cleavage into 89-and 24-kDa fragments that contain the active site and the DNA-binding domain of the enzyme, respectively, during drug-induced and spontaneous apoptosis. Our lab was involved in the purification of caspase-3, a member of the family of aspartate -specific cysteine proteases; and helped verify that caspase-3 also plays a central role in the execution of the apoptotic program, and is responsible for the cleavage of PARP during cell death. We subsequently identified a transient positive requirement for PARP and poly (ADP-ribosyl)ation very early in apoptosis. This was accomplished using well-characterized cell lines stably transfected with inducible PARP-antisense constructs as well as with immortalized fibroblasts derived from PARP knock out mice. Poly(ADP-ribosyl)ation has also been shown by our laboratory to play a unique role in cellular differentiation, since cellular depletion of PARP by PARP antisense induction markedly inhibited differentiation of 3T3-L1 preadipocytes into adipocytes by preventing a transient increase in PARP activity, that appears essential for entering the differentiation process, and precisely correlates with an essential round of DNA replication, required for onset of terminal differentiation. These studies were extended to clarify the role of PARP in DNA replication, and PARP, per se, as a component of a multi-protein DNA replication complex (known as MRC or the DNA synthesome) in cells. Additionally, recently we have established that the tumor suppressor p53, which is required for apoptosis in many cell systems, was shown to be poly(ADP-ribosyl)ated in vitro and in vivo. This work demonstrates for the first time that the modification of p53 by poly(ADP-ribosyl)ation occurs in vivo, and that it represents one of the early acceptors of poly(ADP-ribosyl)ation during apoptosis in human osteosarcoma cells. A major theme of our studies has been based on the hypothesis that when various proteins are in a highly negative poly(ADP-ribosyl)ated state they become "DNA-phobic" and cannot bind to sites in DNA whether on breaks or promoters; however, when poly(ADP-ribose) glycohydrolase cleaves the polymer from these DNA binding proteins they are then able to cycle back to sites in DNA, such as the p53 consensus promoter sequence, which we have shown induces pro-apoptosis genes such as BAX, p21, and FAS. We originally mapped the human PARP gene chromosome to 1q31-q42 and PARP-like sequences to 14q13-q32 and 13q-34. PARP (-/-) mice, with a disrupted PARP gene, neither express immunodetectable PARP nor exhibit significant poly(ADP-ribosyl)ation. Interestingly, it has recently been found that the PARP (-/-) mice are resistant to acquiring murine models of a number of human diseases, including focal cerebral ischemia, stroke, inflammation and toxin-induced diabetes. Our laboratory investigated resistance to MPTP-induced Parkinson’s disease, suggesting that PARP activation, triggered by oxidative stress, plays a role in the pathophysiology of these diseases. Finally, in recent studies, we have used flow cytometry to reveal that immortalized fibroblasts derived from the PARP knockout mice exhibit mixed ploidy, including a tetraploid cell population, which is also indicative of genomic instability. Using comparative genomic hybridization (CGH) analysis, we demonstrated that the genomes of PARP K/O animals, and in cells derived from them, possess specific chromosomal gains and losses, and that these instability markers are involved with both tumor suppressors as well as oncogenes. Using DNA array chip hybridization, we have shown the gene expression patterns of cells derived from the PARP knock out animals versus wild type animals, which will help explain the role of PARP in genomic stability.