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Irreversible Binding of Poly(ADP)Ribose Polymerase Cleavage Product to DNA Ends Revealed by Atomic Force Microscopy: Possible Role in Apoptosis¹

Departments of Biochemistry and Molecular Biology [M. E. S., C. S-R., D. R., A. Y.], and Radiation Medicine [D. P., M. J., A. Di., S. C., A. Dr.], Georgetown University, Washington, DC 20007, and Pacific Northwest National Laboratories, Richland, Washington 99352 [A. S.]

During apoptosis, DNA undergoes fragmentation and caspase-3 cleaves poly(ADP-ribose) polymerase (PARP) into both a 24-kDa fragment containing the DNA binding domain and an 89-kDa fragment containing the catalytic and automodification domains. Atomic force microscopy revealed that recombinant full-length PARP bound to plasmid DNA fragments and linked them into chainlike structures. Automodification of PARP in the presence of NAD⁺ resulted in its dissociation from the DNA fragments, which, nevertheless, remained physically aligned. A recombinant 28-kDa fragment of PARP containing the DNA binding domain but lacking the automodification domain irreversibly bound to and linked DNA fragments in the absence or presence of NAD⁺. Identical results were obtained on incubation of internucleosomal DNA fragments from apoptotic cells with the products of cleavage of recombinant PARP by purifled caspase-3. The 24-kDa product of PARP cleavage by caspase-3 may contribute to the irreversibility of apoptosis by blocking the access of DNA repair enzymes to DNA strand breaks.

¹ Supported in part by USPHS Grants CA45408 (A. Dr.), CA74175 (M. E. S. and A. Dr.), and CA25344 (M. E. S.); United States Army Medical Research and Material Command Grant DAMD17-95-C-5001 (M. E. S.); and Air Force Office of Scientific Research Grant AFOSR-89-0053 (M. E. S.).

² To whom requests for reprints should be addressed, at Department of Radiation Medicine, Division of Radiation Research, Georgetown University School of Medicine, TRB E202A-B, 3970 Reservoir Road NW, Washington, DC 20007. Phone: (202) 687-2144; Fax: (202) 687-0400; E-mail: dritscha@gunet.georgetown.

Received 4/27/98. Accepted 7/ 1/98.

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