Time-Dependent Predominance of Nonhomologous DNA End-Joining Pathways during Embryonic Development in Mice Kishore K. Chiruvella 1 †, Robin Sebastian 1 †, Sheetal Sharma 1 , Anjali A. Karande 1 , Bibha Choudhary 1,2 ⁎ and Sathees C. Raghavan 1 ⁎ 1 Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India 2 Institute of Bioinformatics and Applied Biotechnology, Biotech Park, Electronics City, Bangalore 560 100, India Received 21 December 2011; received in revised form 18 January 2012; accepted 20 January 2012 Available online 27 January 2012 Edited by M. Yaniv Keywords: NHEJ; double-strand break repair; DNA damage; genomic instability; cell-free repair system Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplanta- tion embryos has been largely unknown. In the present study, using a cell- free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development. © 2012 Elsevier Ltd. All rights reserved. Introduction DNA double-strand breaks (DSBs) are the most deleterious forms of DNA damage that result in a loss or rearrangement of genomic material, thereby leading to mutations, genomic instability, cancer or cell death. 1–4 Hence, repair of DSBs is essential for maintaining genomic stability and cell viability. DSBs are induced by a number of agents and mechanisms, including exposure to ionizing radiation and radiomi- metic drugs, collapse of replication forks, programmed cleavage by specific endonucleases during meiotic recombination, and immunoglobulin–TCR gene rearrangements. 5–8 Eukaryotic cells have evolved two major pathways for repairing DSBs, namely homologous recombination (HR) and nonhomologous *Corresponding authors. B. Choudhary is to be contacted at Institute of Bioinformatics and Applied Biotechnology, Biotech Park, Electronics City, Bangalore 560 100, India; S. Raghavan, Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India. E-mail addresses: vibha@ibab.ac.in; sathees@biochem.iisc.ernet.in. † K.K.C. and R.S. contributed equally to this work. Abbreviations used: DSB, double-strand break; NHEJ, nonhomologous end joining; HR, homologous recombination; MMEJ, microhomology-mediated end joining; EDTA, ethylenediaminetetraacetic acid. doi:10.1016/j.jmb.2012.01.029 J. Mol. Biol. (2012) 417, 197–211 Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb 0022-2836/$ - see front matter © 2012 Elsevier Ltd. All rights reserved.