The role of the Mre11–Rad50–Nbs1 complex in double-strand break repair—facts and myths

Journal of Radiation Research, Vol. 57, No. S1, 2016, pp. i25–i32 doi: 10.1093/jrr/rrw034 Advance Access Publication: 15 June 2016 Supplement – ICRR highlights The role of the Mre11–Rad50–Nbs1 complex in double-strand break repair—facts and myths Shunichi Takeda*, Nguyen Ngoc Hoa and Hiroyuki Sasanuma Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606–8501, Japan *Corresponding author. Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Yoshida Konoe, Sakyo-ku, Kyoto 606–8501, Japan. Tel: +81+75-753-4411; Fax: +81+75-753-4419; Email: Received January 19, 2016; Revised February 16, 2016; Accepted February 23, 2016 A B S T R AC T Homologous recombination (HR) initiates double-strand break (DSB) repair by digesting 5′-termini at DSBs, the biochemical reaction called DSB resection, during which DSBs are processed by nucleases to generate 3′ singlestrand DNA. Rad51 recombinase polymerizes along resected DNA, and the resulting Rad51–DNA complex undergoes homology search. Although DSB resection by the Mre11 nuclease plays a critical role in HR in Saccharomyces cerevisiae, it remains elusive whether DSB resection by Mre11 significantly contributes to HR-dependent DSB repair in mammalian cells. Depletion of Mre11 decreases the efficiency of DSB resection only by 2- to 3-fold in mammalian cells. We show that although Mre11 is required for efficient HR-dependent repair of ionizing-radiation–induced DSBs, Mre11 is largely dispensable for DSB resection in both chicken DT40 and human TK6 B cell lines. Moreover, a 2- to 3-fold decrease in DSB resection has virtually no impact on the efficiency of HR. Thus, although a large number of researchers have reported the vital role of Mre11-mediated DSB resection in HR, the role may not explain the very severe defect in HR in Mre11-deficient cells, including their lethality. We here show experimental evidence for the additional roles of Mre11 in (i) elimination of chemical adducts from DSB ends for subsequent DSB repair, and (ii) maintaining HR intermediates for their proper resolution. KE YWOR DS: double-strand break repair, double-strand break resection, etoposide, homologous recombination, ionizing radiation, Mre11, non-homologous end joining, topoisomerase T H E C H O I C E OF T H E T WO M A J O R D S B RE PA I R PAT H WAY S : H O M O LO G O US RE CO M B I N AT I O N A N D N O N H O M O LO G O U S E N D J O I N I N G DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage, as a single unrepaired DSB can trigger apoptosis. DSBs are generated during physiological replication, and are induced by radiotherapy and chemotherapeutic reagents such as the topoisomerase poison. There are two major DSB repair pathways in eukaryotic cells: homologous recombination (HR) and non-homologous end joining (NHEJ). The choice of the two pathways in Saccharomyces cerevisiae is alternative so that DSB resection can inhibit NHEJ, because DSBs containing 3′ single-strand DNA cannot be repaired by canonical NHEJ (Fig. 1A). It has been widely believed that the alternative choice model is also relevant to mammalian cells [1], though the relevance has not yet been demonstrated. The method of examining the precise structure of resected DSB sites is currently available only for meiotic HR in S. cerevisiae, but not for mammalian cells [2, 3]. Although DSB resection would strongly inhibit canonical NHEJ, quick initiation of DSB resection does not reduce the overall efficiency of DSB repair in S. cerevisiae due to the very small contribution of NHEJ to DSB repair [3]. By contrast, DSB resection from DSB ends (Fig. 1A) would result in a considerable decrease in the overall efficiency of DSB repair in mammalian cells due to the major role of NHEJ in DSB repair. A structural study of resected DSBs during meiotic HR in S. cerevisiae revealed that DSB resection is initiated by a single-strand break (SSB) on the strand to be resected up to 300 bases from the 5′-terminus of the DSB [2] (Fig. 1B). This SSB is subjected to subsequent bidirectional resection, both in the 5′–3′ direction away from the DSB and in the 3′–5′ direction towards the DSB end. The Mre11 nuclease forms a complex with Rad50 and Xrs2 (the yeast ortholog of mammalian Nbs1) [4]. The resulting MRX complex is responsible for the formation of the SSB, followed by the 5′–3′ direction resection in the meiotic HR of S. cerevisiae. This model agrees with the in vitro nuclease activity of purified Mre11. It © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/bync/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact • i25 i26 • S. Takeda et al. Fig. 1. (A) Double strand break (DSB) repair in Saccharomyces cerevisiae. The choice of homologous recombination (HR) or nonhomologous end-joining (NHEJ) is determined by the presence or absence of DSB resection, respectively. Rad54 facilitates homology search and strand exchange of resected DSB sites. (B) Bidirectional processing of DSBs during DSB resection for meiotic HR in S. cerevisiae. Spo11 protein, a topoisomerase, generates DSBs, where Spo11 is covalently associated with the 5′ end of DSBs leading to formation of blocked ends. Single-strand break formation on the Spo11-associating strand by Mre11 initiates DSB resection, enabling resection in a bidirectional manner, where Exo1 digests in the 5′–3′ direction away from the DSB, and Mre11 digests in the 3′–5′ direction towards the DSB end. (C) Measurement of unrepaired DSBs [6]. We exposed the indicated genotypes of chicken DT40 cells to doses equivalent to LD50% (the dose that reduces cellular survival to 50%) of γ-rays in the G2 phase, harvested mitotic cells at 3 h, and counted the number of chromosomal breaks in mitotic chromosome spreads. The x-axis shows the number of chromosome aberrations per 100 mitotic cells and per Gray. (D) In contrast with meiotic HR in S. cerevisiae (B), vertebrate Mre11 has a very minor role in DSB resection for subsequent HR in somatic vertebrate cells. Dna2 plays the dominant role in DSB resection in chicken DT40 cells among the CtIP, Dna2, Exo1 and Mre11 nucleases [12]. remains elusive whether this bidirectional resection also plays a role in mitotic HR in mammalian cells as well as in yeast [5]. Based on findings in chicken DT40 cells (Fig. 1C) [6], we here propose another model for DSB resection (Fig. 1D). If resection from the SSB is carried out only in the 5′–3′ direction away from the DSB, but not in the 3′–5′ direction, DSB ends would be maintained as duplex DNA (Fig. 1D). The absence of homologous si (...truncated)