DNA binding activity of Ku during chemotherapeutic agent-induced early apoptosis October 17, 2016 Significance Statement This study analyzed the expression of the Ku protein and its DNA-binding activity during early apoptosis. In the study, Ku was neither cleaved nor degraded during early apoptosis induced by etoposide treatment. In addition, Ku was found to bind cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. The DNA-binding properties of Ku were assessed by two methods, each of which is based on electrophoresis mobility shift assay (EMSA). One commonly applied method, EMSA with 15-bp 32P-labeled DNA probe (RI-EMSA), employs radiolabeled DNA probes. The other method, EMSA combined with western blot (WB-EMSA), employs unlabeled DNA probes followed by western blot and detection using anti-Ku antiserum. This study showed that both WB-EMSA and RI-EMSA are useful for evaluating Ku-DNA binding activity. The significance of this work lies in its contribution to the understanding of the mechanism of interaction of Ku with DNA during apoptosis. Figure legends: RI-EMSA: Protein extract obtained from cells treated with etoposide for the duration indicated was incubated with 32P-labeled, 15-bp dsDNA probes in the presence of closed circular DNA. Western blot: Protein extract obtained from either control or etoposide-treated cells was run on a 10% SDS polyacrylamide gel under reducing conditions. Ku70 and Ku80 were detected using human polyclonal antiserum. WB-EMSA: Extracts from etoposide-treated HL-60 cells were separated by 6% native PAGE, and then analyzed by western blot with polyclonal antiserum raised against Ku (lane 1). Extracts from untreated HL-60 cells were incubated with 160-bp DNA, followed by western blot (lane 2). Extracts from untreated or etoposide treated HL-60 cells were incubated with 15-bp DNA, followed by western blot (lanes 3–6). Comparison of WB-EMSA to conventional RI-EMSA. The diagram of RI-EMSA is a part of Figure A, and the figure of WB-EMSA is a part of Figure C. About The Author Katsuya Iuchi is currently an assistant professor at the Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School. His research focuses on the mechanism of cell death and lipid peroxidation of biomembranes. Prior to joining Nippon Medical School, he was a postdoctoral fellow in the Sodeoka Live Cell Chemistry Project, ERATO, Japan Science and Technology Agency. He received his Ph.D. in chemistry from Kwansei Gakuin University, Japan in 2009. Journal Reference Exp Cell Res. 2016;342(2):135-44. Iuchi K1, Yagura T2. Show Affiliations Department of Bioscience, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuin, Sanda-shi, Hyogo-ken 669-1337, Japan. Electronic address: [email protected] Department of Bioscience, Faculty of Science and Technology, Kwansei Gakuin University, 2-1 Gakuin, Sanda-shi, Hyogo-ken 669-1337, Japan. Abstract Ku-protein is a heterodimer composed of two subunits, and is capable of both sequence-independent and sequence-specific DNA binding. The former mode of DNA binding plays a crucial role in DNA repair. The biological role of Ku-protein during apoptosis remains unclear. Here, we show characterization of Ku-protein during apoptosis. In order to study the DNA binding properties of Ku, we used two methods for the electrophoresis mobility shift assay (EMSA). One method, RI-EMSA, which is commonly used, employed radiolabeled DNA probes. The other method, WB-EMSA, employed unlabeled DNA followed by western blot and detection with anti-Ku antiserum. In this study, Ku-DNA probe binding activity was found to dramatically decrease upon etoposide treatment, when examined by the RI-EMSA method. In addition, pre-treatment with apoptotic cell extracts inhibited Ku-DNA probe binding activity in the non-treated cell extract. The inhibitory effect of the apoptotic cell extract was reduced by DNase I treatment. WB-EMSA showed that the Ku in the apoptotic cell extract bound to fragmented endogenous DNA. Interestingly, Ku in the apoptotic cell extract purified by the Resource Q column bound 15-bp DNA in both RI-EMSA and WB-EMSA, whereas Ku in unpurified apoptotic cell extracts did not bind additional DNA. These results suggest that Ku binds cleaved chromosomal DNA and/or nucleosomes in apoptotic cells. In conclusion,Ku is intact and retains DNA binding activity in early apoptotic cells. Copyright © 2016 Elsevier Inc. All rights reserved. Go To Exp Cell Res. 2016-10-17 GMD staff
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