This is a repository copy of MRNIP is a replication fork protection factor. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/163246/ Version: Published Version Article: Bennett, L.G., Wilkie, A.M., Antonopoulou, E. et al. (8 more authors) (2020) MRNIP is a replication fork protection factor. Science Advances, 6 (28). eaba5974. ISSN 2375-2548 https://doi.org/10.1126/sciadv.aba5974 Reuse This article is distributed under the terms of the Creative Commons Attribution-NonCommercial (CC BY-NC) licence. This licence allows you to remix, tweak, and build upon this work non-commercially, and any new works must also acknowledge the authors and be non-commercial. You don’t have to license any derivative works on the same terms. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ SCIENCE ADVANCES | RESEARCH ARTICLE CELL BIOLOGY Copyright © 2020 The Authors, some MRNIP is a replication fork protection factor rights reserved; exclusive licensee L. G. Bennett1, A. M. Wilkie1, E. Antonopoulou1, I. Ceppi2,3, A. Sanchez2, E. G. Vernon1, American Association 1 4 4 2,3 1 for the Advancement A. Gamble , K. N. Myers , S. J. Collis , P. Cejka , C. J. Staples * of Science. No claim to original U.S. Government The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the Works. Distributed replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and under a Creative the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological Commons Attribution MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 NonCommercial functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel License 4.0 (CC BY-NC). fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease–dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity. Downloaded from INTRODUCTION derstanding the complex mechanisms at play during the replication Accurate inheritance of genetic information following cell division stress response is an important goal. The MRE11 nuclease can act as depends on the fidelity of genomic DNA replication. This process is either a DNA endonuclease or a 3′-5′ exonuclease, and multiple impaired by damage to or modification of the DNA template or by studies using the MRE11 inhibitor mirin implicate the exonuclease replisome progression through regions that are inherently difficult function of MRE11 in reversed fork degradation (4, 8). MRE11 is http://advances.sciencemag.org/ to replicate. Hence, organisms have evolved elaborate mechanisms recruited to stalled forks by the enzyme PARP1 [poly(adenosine to mitigate the risk of replisome collapse during replication stress (1). diphosphate)–ribose] polymerase 1] (22) in concert with the histone In the context of cancer treatment, many chemotherapies induce cancer methyltransferase MLL3/4 and its binding partner, PTIP (14). How- cell death by modifying genomic DNA to induce replication fork stalling. ever, the mechanisms governing the regulation of MRE11 activity at Mutations in the BRCA1 and BRCA2 tumor suppressor genes reversed forks are poorly understood, and before this study, there predispose individuals to breast, ovarian, and prostate cancers (2). was no evidence that direct dysregulation of nuclease functions at Both BRCA1 and BRCA2 are crucial DNA repair factors that facilitate reversed forks might cause pathological resection of nascent DNA homologous recombination (HR)–mediated repair of DNA double- and genome instability. strand breaks (DSBs) (3, 4). A growing body of evidence demonstrates MRN-interacting protein (C5ORF45/MRNIP) was recently iden- HR-independent genome maintenance functions for BRCA1/2 and tified as a novel factor that promotes repair of radiation-induced several other core DNA repair proteins such as WRN in the protec- lesions via functional interactions with the DSB-binding MRE11- on July 14, 2020 tion of stalled replication forks (5–11). In response to genotoxic RAD50-NBS1 (MRN) complex (23). Here, we have used a combina- stresses, stalled replication forks remodel via rehybridization of nas- tion of iPOND (isolation of proteins on nascent DNA), nuclease cent DNA to form four-way DNA intermediates (12), facilitating assays, and CRISPR-Cas9 and small interfering RNA (siRNA)–based tolerance or bypass of replication impediments. It is now evident loss-of-function studies to identify MRNIP as a novel replication fork that BRCA1/2 and RAD51 act in concert to stabilize stalled forks protection factor. MRNIP associates with nascent DNA and promotes (12) and promote replication stress resistance by preventing the in- replication fork progression, resistance to replication stress agents, appropriate nucleolytic degradation of reversed forks by the nucle- and chromosome stability. MRNIP binds directly to MRE11 and pre- ases MRE11 and EXO1 (8, 13, 14). Several recent publications have vents the MRE11 and DNA2-dependent degradation of reversed forks. yielded further insight, revealing essential roles for the SMARCAL1 MRNIP specifically represses MRE11 exonuclease but not endonu- chromatin remodeler, the DNA translocase ZRANB3, and even the clease activity in vitro. The fork protection functions of MRNIP are RAD51 itself in replication fork reversal (12, 14–16). Loss of func- associated with its ability to bind MRE11, because MRNIP trunca- tion of an additional subset of fork protection factors such as CtIP tions displaying reduced MRE11 interaction are unable to rescue fork (17), BOD1L (18), ABRO1 (19), and RIF1 (20, 21) results in fork deg- degradation induced by MRNIP loss. In summary, our work identi- radation licensed by the nuclease DNA2. fies MRNIP as a novel replication fork protection factor that likely Replication stress–induced genome instability is a major factor functions by repressing the MRE11 exonuclease, and furthers our that drives cancer development and progression, and therefore, un- understanding of the molecular functions underlying the complex processes involved in replication fork protection. 1North West Cancer Research Institute, School of Medical Sciences, Bangor University, Bangor LL57 2UW, UK. 2Institute for Research in Biomedicine, Faculty of Biomedical RESULTS Sciences, Università della Svizzera italiana, 6500 Bellinzona, Switzerland. 3Institute of Deletion of MRNIP using CRISPR-Cas9 results in 4 Biochemistry, Department of Biology, ETH Zürich, 8093 Zürich, Switzerland. Sheffield genome instability Institute for Nucleic Acids, Department of Oncology and Metabolism, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK. The novel DNA repair factor MRNIP was identified in an siRNA-based *Corresponding author. Email: [email protected] screen for novel regulators of genome stability and characterized as Bennett et al., Sci. Adv. 2020; 6 : eaba5974 10 July 2020 1 of 10 SCIENCE ADVANCES | RESEARCH ARTICLE a functional MRN complex interactor that promotes Ataxia A F Telangiectasia-Mutated (ATM) signaling in response to radiation- 60 ParentalMRNIP-3MRNIP-7 ParentalMRNIP A H2AX +ve ** induced DNA damage (23). All published work on MRNIP to date MRNIP MRNIP 50 53BP1 +ve ** ** Tubulin Tubulin has been based on studies using RNA interference (RNAi). We examined 40 ** the consequences of total deletion of MRNIP using homology-directed HCT116 HeLa 30 repair (HDR)–directed CRISPR-Cas9 protocols. Cells were trans- B N.S. N.S. ParentalClone Clone3 7 ParentalClone A fected with Cas9 and HDR constructs expressing MRNIP-directed 500 bp 20 400 bp Cells with >5 foci (%) guide RNAs (gRNAs), and then insertion-positive cells were selected HCT116 HeLa 10 with puromycin. Surviving clones were isolated, subcultured, and C 0 *** Parental MRNIP-3MRNIP-7 MRNIP-3 + screened for homozygous MRNIP deletion and complete loss of the 20 *** H2AX +ve HeLa G FLAG-MRNIP MRNIP protein by a combination of genomic polymerase chain 53BP1+ve 15 reaction (PCR), quantitative reverse transcription PCR (qRT-PCR), Parental MRNIP-A and Western blot. We successfully generated three cancer cell lines 10 lacking MRNIP in two different backgrounds, hereafter referred to as 5 MRNIP-A (HeLa) and MRNIP-3 or MRNIP-7 (HCT116) (Fig. 1, A and B). Cells with >5 foci (%) All MRNIP knockout (KO) cell lines exhibited evidence of DNA dam- * 0 age accumulation evidenced by increased accumulation of 53BP1 and Parental MRNIP-A D gH2AX-positive foci (Fig. 1, C to F) and increased neutral COMET COMETassay 53BP1 H2AX Parental Downloaded from score (Fig. 1G). MRNIP KO cells also displayed an increased fre- ** 1.2 MRNIPCRISPR quency of radial chromosomes and chromosome fusions (Fig. 1H). ** Levels of DNA damage were fully