Detection of DNA breaks in apoptotic cells utilizing the DNA binding domain of poly(ADP-ribose) polymerase with fluorescence microscopy.
The DNA binding domain (DBD) of poly(ADP-ribose) polymerase (PARP) has proved to be a novel, highly sensitive probe for detecting DNA breaks in intact cells undergoing apoptosis. A recombinant peptide spanning the DNA binding domain of PARP was expressed, purified and used to detect DNA strand breaks in fixed cells. Fluorescence microscopy with this probe followed by detection with anti-PARP antisera initially revealed an increased binding following treatment of cells with DNA strand-breaking agents (such asN-methyl-N'-nitro-N-nitrosoguanidine) and, subsequently, using biotinylated PARP DBD, during the later stages of apoptosis in several cell systems, when internucleosomal strand breaks became evident. This procedure was found to be at least as sensitive and required fewer steps to detect DNA strand breaks than those utilizing Klenow incorporation of biotinylated nucleotides.
Rosenthal
D S
DS
Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Ding
R
R
Simbulan-Rosenthal
C M
CM
Cherney
B
B
Vanek
P
P
Smulson
M
M
D017209:Apoptosis; D001710:Biotin; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D006801:Humans; D008856:Microscopy, Fluorescence; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D001710:Biotin; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases