Nucleolin Participates in DNA Double-Strand Break- Induced Damage Response through MDC1-Dependent Pathway Junya Kobayashi1*, Hiroko Fujimoto1, Jun Sato1, Ikue Hayashi2, Sandeep Burma3, Shinya Matsuura4, David J. Chen3, Kenshi Komatsu1 1 Department of Genome Repair Dynamics, Radiation Biology Center, Kyoto University, Kyoto, Japan, 2 Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan, 3 Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States of America, 4 Department of Genetics and Cell Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan Abstract H2AX is an important factor for chromatin remodeling to facilitate accumulation of DNA damage-related proteins at DNA double-strand break (DSB) sites. In order to further understand the role of H2AX in the DNA damage response (DDR), we attempted to identify H2AX-interacting proteins by proteomics analysis. As a result, we identified nucleolin as one of candidates. Here, we show a novel role of a major nucleolar protein, nucleolin, in DDR. Nucleolin interacted with c-H2AX and accumulated to laser micro-irradiated DSB damage sites. Chromatin Immunoprecipitation assay also displayed the accumulation of nucleolin around DSB sites. Nucleolin-depleted cells exhibited repression of both ATM-dependent phosphorylation following exposure to c-ray and subsequent cell cycle checkpoint activation. Furthermore, nucleolin- knockdown reduced HR and NHEJ activity and showed decrease in IR-induced chromatin accumulation of HR/NHEJ factors, agreeing with the delayed kinetics of c-H2AX focus. Moreover, nucleolin-knockdown decreased MDC1-related events such as focus formation of 53 BP1, RNF168, phosphorylated ATM, and H2A ubiquitination. Nucleolin also showed FACT-like activity for DSB damage-induced histone eviction from chromatin. Taken together, nucleolin could promote both ATM- dependent cell cycle checkpoint and DSB repair by functioning in an MDC1-related pathway through its FACT-like function. Citation: Kobayashi J, Fujimoto H, Sato J, Hayashi I, Burma S, et al. (2012) Nucleolin Participates in DNA Double-Strand Break-Induced Damage Response through MDC1-Dependent Pathway. PLoS ONE 7(11): e49245. doi:10.1371/journal.pone.0049245 Editor: Thomas G. Hofmann, German Cancer Research Center, Germany Received May 9, 2012; Accepted October 5, 2012; Published November 7, 2012 Copyright: 2012 Kobayashi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan to JK (21310035) and KK (18101002). SB is supported by grants from the National Institutes of Health (RO1 CA149461), National Aeronautics and Space Administration (NNX10AE08G) and the Cancer Prevention and Research Institute of Texas (RP100644). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jkobayashi@house.rbc.kyoto-u.ac.jp Introduction DNA double-strand breaks (DSBs) are often generated in genomic DNA upon exposure to ionizing radiation, DNA damaging agents such as bleomycin and neocarzinostatin, or due to the stalling or collapse of DNA replication forks. As unrepaired DSBs induce genome instability and promote apopto- sis or tumorigenesis, cells recognize DSBs immediately, allow DNA repair factors to access DSBs and then activate DNA repair mechanisms. However, eukaryotic genomic DNA is organized into stable chromatin structures comprised of higher-order folding and condensed nucleosomes. As this stable structure impedes access of DNA-modifying factors for replication, transcription and repair, cells have evolved various mechanisms to mark and modify the chromatin landscape, including histone modifications and local recruitment of chromatin remodeling factors before activation of DNA-modifying machinery. Likewise, in order to promote access of DNA repair factors to DNA damage sites, both modifications of histones and remodeling of chromatin structure are required. Although histones undergo different kinds of modification such as methylation, acetylation and phosphorylation, several reports suggest a tight relationship between histone acetylation and DNA damage response [1,2]. Tamburini and Tyler showed that acetylation of histone H3 and H4 increased at HO endonucle- ase-generated DSB sites in yeasts, and also showed that the histone acetyltransferases Gcn5 and Esa1 were recruited to these damage sites [3]. Downs and his colleagues also reported that Arp4, a subunit of NuA4 HAT (histone acetyltransferase) complex, is recruited to HO endonuclease-induced DSB sites and interacts with phosphorylated H2A directly [4]. In addition, Ikura et al. reported that the human homolog of NuA4 HAT, Tip60, interacts with and acetylates histone H2AX, and also showed that acetylation of histone H2AX increased in response to DSB damage, and dominant-negative Tip60-expressing HeLa cells exhibit attenuated DSB repair following IR [5,6]. Also, we reported previously that acetylation of histone H2A is indispens- able for DNA damage-induced focus formation and homologous recombination (HR) repair [7]. Furthermore, chromatin remodel- ing factors such as INO80 and SWI/SNF appear to be important for the DNA damage response. The yeast Ino80 and Swr1 chromatin-remodeling complexes interact with the phosphorylated form of histone H2AX (cH2AX) and facilitate DSB repair [8–10]. Wu and his colleagues showed that knockdowns of human INO80 PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49245