DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of transcription-coupled repair enzymes, and inhibition of RNA synthesis1,2. During the S phase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication3,4. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (Pol I) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in Pol I transcription. Surprisingly, we find Pol I inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with Pol I initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.
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We thank Y. Shiloh for reagents and comments. We are grateful to Z. Lou, J. Chen and C. Deng for cell lines, J. Ionita, K. Zaal and S. Twitty for technical assistance, and I. Grummt for antibodies and protocols. We also thank S. Wincovitch for access to the multi-photon microscope. This research was supported (in part) by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the Center for Cancer Research of the National Cancer Institute, National Institutes of Health.
Author Contributions M.K. and R.C. planned the project and did experimental work, data analysis, and wrote the manuscript. E.E.C., M.O., C.M. and R.D.P. did experimental work, data analysis, and provided assistance in writing the manuscript. S.A.G. shared unpublished data and provided advice during experimental design. A.N. and T.M. provided advice and expertise and proof-read the manuscript.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
This file contains Supplementary Figures S1-S10 with Legends, Supplementary Methods 1-2 and Supplementary Table 1. The Supplementary Figures provide data that corroborate the main findings of the text. The Supplementary Methods include a detailed description of Pol I kinetic studies and statistical analysis applied during FRAP analysis. The Supplementary Table 1 provides the kinetic constant values described in the assembly/displacement model. (PDF 6341 kb)
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Kruhlak, M., Crouch, E., Orlov, M. et al. The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks. Nature 447, 730–734 (2007). https://doi.org/10.1038/nature05842
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