Regulation of DNA double-strand break repair pathway choice: a new focus on 53BP1

Regulation of DNA double-strand break repair pathway choice: a new focus on 53BP1 DNA双链断裂修复途径选择的调控：53BP1蛋白最新研究关注 first-author Zihua GONG, gongz@ccf.org Zihua GONG, gongz@ccf.org gongz@ccf.org https://orcid.org/0000-0002-8008-0615 Department of Cancer Biology, Cleveland Clinic Lerner Research Institute, Cleveland, OH44195, USA Department of Cancer Biology, Cleveland Clinic Lerner Research Institute, Cleveland, OH44195, USA Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining (NHEJ)-mediated DSB repair pathway that rejoins DSB ends. New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. This review focuses on the up- and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair, which in turn promotes the sensitivity of poly(ADP-ribose) polymerase inhibitor (PARPi) in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies. DNA双链断裂（DSB）信号转导的关键作用是维持细胞的稳态和基因组的完整性。P53结合蛋白1（53BP1）在DSB修复信号选择中起到了关键的调节作用：53BP1能够促进DSB末端重新连接，从而促进非同源末端链接（NHEJ）介导的DSB修复途径。53BP1如何被招募到DNA损伤位点以及53BP1如何招募其DNA损伤修复效应子，从而促进NHEJ介导的DSB修复，并抑制同源重组（HR）信号传导？最新的研究对于这些问题有了进一步的阐述。这篇综述将着重于阐述53BP1蛋白是如何促进NHEJ介导的DSB修复途径以及其上下游通路蛋白调控，从而促进BRCA1缺陷型癌症细胞对多聚ADP-核糖聚合酶抑制剂（PARPi）的敏感性，最终为改善癌症治疗策略提供新的方向。 P53-binding protein 1 (53BP1) DNA double-strand break (DSB) Non-homologous end-joining (NHEJ) Homologous recombination (HR) Poly(ADP-ribose) polymerase inhibitor (PARPi) P53结合蛋白1（53BP1） DNA双链断裂（DSB） 非同源末端链接（NHEJ） 同源重组（HR） 多聚ADP-核糖聚合酶抑制剂（PARPi） 1 Introduction 1 Preserving genomic integrity and maintaining cellular homeostasis are crucial functions of DNA double-strand break (DSB) repair. Any unrepaired DSBs can trigger cell growth arrest and cell death, and unrepaired DSBs are potential inducers of oncogene activation and loss of tumor suppressors. Two mechanistically distinct pathways are involved in the elimination of DSBs from the genome: homologous recombination (HR), which requires an identical DNA template in the sister chromatid for DNA repair ( Heyer et al., 2010 Heyer et al., 2010 ), and non-homologous DNA end-joining (NHEJ), which rejoins the broken ends without the use of extensive homology ( Lieber, 2010 Lieber, 2010 ). Fully understanding how DNA repair pathway choice occurs is critical for designing better therapies for cancer patients. Poly(ADP-ribose) polymerase inhibitor (PARPi) is commonly used in patients with breast cancer type 1 susceptibility protein ( Brca1 ) or Brca2 mutations ( Bryant et al., 2005 Bryant et al., 2005 ; Farmer et al., 2005 Farmer et al., 2005 ). Surprisingly, two research groups independently found that loss of p53-binding protein 1 ( 53bp1 ) in the background of Brca1 deficiency could restore HR repair signaling and render Brca1 -deficient tumors resistant to PARP inhibition treatment, which has made resistance to PARPi therapy a major problem in clinical practice ( Bouwman et al., 2010 Bouwman et al., 2010 ; Bunting et al., 2010 Bunting et al., 2010 ). For more than two decades, 53BP1 has been described as an important regulator of DSB signaling. Studies have revealed that although 53BP1 lacks any enzymatic activity, it can recruit other responsive proteins to DNA damage sites to facilitate the NHEJ repair process. In response to DSBs, 53BP1 rapidly accumulates on the chromatin surrounding the DNA damage site ( Schultz et al., 2000 Schultz et al., 2000 ; Anderson et al., 2001 Anderson et al., 2001 ; Rappold et al., 2001 Rappold et al., 2001 ). This accumulation is driven by a signaling cascade that originates with ataxia-telangiectasia mutated (ATM) kinase-mediated phosphorylation of H2AX (known as γH2AX). γH2AX binds directly to the mediator of DNA damage checkpoint protein 1 (MDC1) at the break site and then activates RING finger protein 8 (RNF8)-RNF168-mediated ubiquitination of chromatin ( Bekker-Jensen and Mailand, 2011 Bekker-Jensen and Mailand, 2011 ; Lukas et al., 2011 Lukas et al., 2011 ), which induces the 53BP1 recruitment to DNA lesions. The E3 ubiquitin ligases RNF168 and RNF8 collaborate with ubiquitin-conjugating enzyme E2N (UBC13), an E2-conjugating enzyme, and with other E2 enzymes to ubiquitinate the chromatin around the DNA lesion ( Huen et al., 2007 Huen et al., 2007 ; Kolas et al., 2007 Kolas et al., 2007 ; Mailand et al., 2007 Mailand et al., 2007 ; Wang and Elledge, 2007 Wang and Elledge, 2007 ; Doil et al., 2009 Doil et al., 2009 ; Stewart et al., 2009 Stewart et al., 2009 ). H2A is a key substrate of RNF168, which ubiquitinates H2A on its Lys13 and/or Lys15 in response to DNA damage ( Gatti et al., 2012 Gatti et al., 2012 ; Mattiroli et al., 2012 Mattiroli et al., 2012 ). RNF168 and RNF8 are at the spearhead of a complex and dynamic ubiquitination-dependent signaling cascade that has contact with almost every DSB responsive phase, including damage checkpoint activation, chromatin remodeling, transcriptional silencing, and DNA repair ( Goodarzi and Jeggo, 2013 Goodarzi and Jeggo, 2013 ). The ionizing radiation-induced foci (IRIF) region of 53BP1, necessary for recruitment of 53BP1 to DSBs, contains the ubiquitin-dependent recruitment (UDR) motif, a tandem Tudor domain, and an oligomerization domain ( Iwabuchi et al., 2003 Iwabuchi et al., 2003 ; Charier et al., 2004 Charier et al., 2004 ; Fradet-Turcotte et al., 2013 Fradet-Turcotte et al., 2013 ). The Tudor domain binds H4K20me2 while the UDR motif binds H2AK15Ub. It has been demonstrated that the roles of 53BP1 in DNA repair depend on its accumulation in DNA damage sites via recognition of H4K20me2 ( Botuyan et al., 2006 Botuyan et al., 2006 ; Fradet-Turcotte et al., 2013 Fradet-Turcotte et al., 2013 ). In this review, we highlight 53BP1 as a key determinant of DSB repair pathway choice. In particular, we focus on discoveries that have shed light on how post-transcriptional modifications (PTMs) and upstream factors regulate 53BP1 in the DNA damage response and how 53BP1 induces the downstream responsive effectors involved in the NHEJ signaling pathways. 2 53BP1 structure and interaction partners 1 53BP1 is a large protein of 1972 amino acids and with more than a 200-kD mass which is trans-lated by the TP53BP1 gene ( Adams and Carpenter, 2006 Adams and Carpenter, 2006 ; Panier and Boulton, 2014 Panier and Boulton, 2014 ). Although 53BP1 does not show any enzymatic activity, it consists of multiple interaction surfaces for DSB-responsive proteins. Important structural regions of 53BP1 consist of tandem Tudor domains, a dynein 8-kD light chain binding motif, a glycine-arginine-rich stretch, two BRCA1 carboxyl-terminal (BRCT) domains, and 28 amino-terminal Ser/Thr-Gln sites, which are phosphorylated by ATM kinase ( Adams and Carpenter, 2006 Adams and Carpenter, 2006 ). Three proteins that rely on 53BP1 for their recruitments to DNA damage sites are EXPAND1 (known as mutated melanoma-associated antigen 1 (MUM1)), which binds to the BRCT domains of 53BP1 in a phosphorylation-independent manner ( Huen et al., 2010 Huen et al., 2010 ), and replication timing regulatory factor 1 (RIF1) and Pax transactivation domain-interacting protein (PTIP), which interact with the 53BP1 N-terminal Ser/Thr-Gln sites in an ATM-dependent manner ( Munoz et al., 2007 Munoz et al., 2007 ; Chapman et al., 2013 Chapman et al., 2013 ) ( Fig. 1 Fig. 1 ). 3 Roles of 53BP1 in DSB repair pathway choice 1 Several milestone studies have shown that 53BP1 plays a determinant role in DSB repair pathway choice and in improving the fidelity of DSB repair ( Heyer et al., 2010 Heyer et al., 2010 ; Lieber, 2010 Lieber, 2010 ; Chapman et al., 2012a Chapman et al., 2012a ). The repair pathway selection decision is crucial for maintaining genome integrity and is precisely regulated during different cell cycle phases ( Chapman et al., 2012b Chapman et al., 2012b ). 3.1 53BP1-mediated NHEJ pathway 2 As an important component involved in cellular response to DNA damage, 53BP1 participates in DNA damage checkpoint control and DNA repair ( Abraham, 2002 Abraham, 2002 ; Fitzgerald et al., 2009 Fitzgerald et al., 2009 ; van Gent, 2009 van Gent, 2009 ). Subsequent studies have indicated that a loss of 53bp1 restores HR repair resulting in rescuing embryonic lethality in Brca1 -knockout mice ( Bouwman et al., 2010 Bouwman et al., 2010 ; Bunting et al., 2010 Bunting et al., 2010 ). Moreover, loss of 53bp1 renders Brca1 -deficient cells resistant to PARPi ( Bouwman et al., 2010 Bouwman et al., 2010 ; Bunting et al., 2010 Bunting et al., 2010 ). These significant observations suggest that 53BP1 may counteract HR repair in BRCA1-deficient cells. The current hypothesis is that BRCA1- and 53BP1-dependent pathways compete with each other during the early steps of HR repair, especially at DNA end resection. While BRCA1 promotes DNA end resection, 53BP1 may suppress end resection and therefore promote NHEJ and prevent HR repair. However, 53BP1 is an adaptor protein that recruits other proteins to sites of DNA breaks to facilitate NHEJ repair, which competes with BRCA1-dependent HR-mediated DNA repair. In the past few years, 53BP1-dependent DNA repair pathway was well established, which consists of two downstream sub-pathways that are respectively mediated by RIF1-REV7 and PTIP-Artemis ( Callen et al., 2013 Callen et al., 2013 ; Chapman et al., 2013 Chapman et al., 2013 ; di Virgilio et al., 2013 di Virgilio et al., 2013 ; Escribano-Díaz et al., 2013 Escribano-Díaz et al., 2013 ; Feng et al., 2013 Feng et al., 2013 ; Zimmermann et al., 2013 Zimmermann et al., 2013 ; Wang et al., 2014 Wang et al., 2014 ; Boersma et al., 2015 Boersma et al., 2015 ; Xu et al., 2015 Xu et al., 2015 ). Very recently, several groups reported that the Shieldin complex acts as the downstream effector of 53BP1/RIF1/REV7 to restrict DSB resection and counteract HR ( Dev et al., 2018 Dev et al., 2018 ; Ghezraoui et al., 2018 Ghezraoui et al., 2018 ; Gupta et al., 2018 Gupta et al., 2018 ; Mirman et al., 2018 Mirman et al., 2018 ; Noordermeer et al., 2018 Noordermeer et al., 2018 ; Tomida et al., 2018 Tomida et al., 2018 ). These two sub-pathways may have overlapping functions, but both are involved in the suppression of HR repair in BRCA1-deficient cells. 3.2 BRCA1-mediated HR pathway 2 When DSBs occur, a process termed DNA end resection is activated. End resection produces extensive 3' single-strand overhang, which is a key step in determining repair pathway choice, since ends that bear long 3' DNA tails are destined for HR repair, while at the same time such ends are not substrates for NHEJ and therefore would prevent NHEJ repair. BRCA1 promotes DNA end resection and mediates the HR repair pathway. A critical question is how BRCA1 overcomes the barrier to resection that is enforced by 53BP1. 53BP1 is phosphorylated by ATM kinase in the S phase, which promotes RIF1 recruitment at DSBs and inhibits DNA end-resection. Isono et al. (2017) Isono et al. (2017) have shown that BRCA1 promotes 53BP1 dephosphorylation by PP4C phosphatase and RIF1 release, thus facilitating end resection to mediate HR repair. 53BP1 acetylation has also been implicated in the choice of DNA repair pathways. CREB-binding protein (CBP)-mediated acetylation of 53BP1 inhibits the recruitment of 53BP1 and its downstream factors PTIP and RIF1 to DSBs, which suppresses NHEJ repair and promotes BRCA1-mediated HR repair ( Guo et al., 2018 Guo et al., 2018 ). The meiotic recombination 11 (MRE11)/DNA repair protein RAD50 homolog (RAD50)/Nijmegen breakage syndrome 1 (NBS1) (MRN)-CTBP-interacting protein (CtIP) pathway may potentially promote HR repair in a BRCA1-dependent manner. CtIP has been shown to inhibit RIF1 end resection blocking activity in the S/G2 phase ( Escribano-Díaz et al., 2013 Escribano-Díaz et al., 2013 ; Zimmermann et al., 2013 Zimmermann et al., 2013 ). Recent studies demonstrate that CtIP promotes end resection and mediates the rescue of genomic stability of mouse Brca1/53bp1 -deficient cells ( Polato et al., 2014 Polato et al., 2014 ). 4 Upstream regulators of 53BP1 in DSB signaling 1 As described above, 53BP1 is recruited to the DNA damage sites, a process depends on the Tudor domain of 53BP1 recognizing the lysine methylation of histone. 4.1 53BP1 recruitment to chromatin regulated by Tudor-interacting repair regulator (TIRR) 2 A new protein was recently identified in the 53BP1 signaling pathway ( Drané et al., 2017 Drané et al., 2017 ; Zhang et al., 2017 Zhang et al., 2017 ; Botuyan et al., 2018 Botuyan et al., 2018 ; Dai et al., 2018 Dai et al., 2018 ; Wang et al., 2018 Wang et al., 2018 ). NUDT16L1, now known as the TIRR, which is a member of the family of nucleoside diphosphate-linked moiety X (Nudix) hydrolases, was shown to interact with and regulate the activity of the IRIF region of 53BP1 ( Drané et al., 2017 Drané et al., 2017 ; Zhang et al., 2017 Zhang et al., 2017 ). At a DSB, the DNA ends are marked with histones H4K20me2 and H2AK15ub, which are specific targets of 53BP1. The unique function of TIRR is that it can directly bind the tandem Tudor domain of 53BP1, which prevents the binding of 53BP1 to H4K20me. Moreover, upon DNA damage, ATM phosphorylates 53BP1 and recruits RIF1 to dissociate the 53BP1-TIRR complex from chromatin, which allows 53BP1 to recognize H4K20me2 and direct the repair path choice toward the NHEJ pathway. Further observations support the finding that the interaction between 53BP1 and TIRR is of great importance for the activation of HR in BRCA1-deficient cells, which provides opportunities for developing novel chemotherapeutics ( Wang et al., 2018 Wang et al., 2018 ). X-ray crystallography of TIRR-53BP1 reveals that a TIRR arginine (Arg107) residue can mask 53BP1's histone methyllysine-binding surface ( Drané et al., 2017 Drané et al., 2017 ; Botuyan et al., 2018 Botuyan et al., 2018 ). The TIRR amino-terminal region combined with the TIRR L8-loop can prevent the methylation reader joining surface in Tudor of 53BP1, which in turn abolishes 53BP1 recruitment to nucleosomes bearing H4K20me2 ( Dai et al., 2018 Dai et al., 2018 ). Further, because TIRR is an RNA-binding protein and non-coding RNAs have been implicated in the recruitment of 53BP1 to DSBs, researchers have wondered whether RNA molecules formed in the DNA damage response can disassemble the 53BPl-TIRR complex. X-ray crystallography and biochemistry assay have demonstrated that RNA molecules do disassemble the 53BP1-TIRR complex, which indicates that RNA molecules could act as a trigger for 53BP1 recruitment to chromatin in response to DNA damage ( Botuyan et al., 2018 Botuyan et al., 2018 ). In summary, on the one hand, TIRR stabilizes the 53BP1 protein level; depletion of TIRR decreases nuclear-soluble 53BP1 and weakens the association of 53BP1 with its binding partners ( Zhang et al., 2017 Zhang et al., 2017 ). On the other hand, overexpression of TIRR eliminates the association of H4K20me2 and 53BP1, which consequently suppresses the recruitment of 53BP1 to DSBs, thus disrupting 53BP1-dependent DNA repair ( Drané et al., 2017 Drané et al., 2017 ; Botuyan et al., 2018 Botuyan et al., 2018 ). 4.2 Poly-ADP ribosylation of 53BP1 regulated by NUDT16 2 Recently, researchers have identified a novel post-translational modification of 53BP1 by ADP-ribosylation that is driven by the Nudix enzyme, NUDT16. NUDT16 has been shown to have in vitro hydrolase activity that removes protein ADP-ribosylation ( Palazzo et al., 2015 Palazzo et al., 2015 ). Nudix proteins are characterized by a highly conserved 23-amino acid Nudix motif, GX 5 EX 7 REUXEEXGU , where U is an aliphatic or hydrophobic residue ( McLennan, 2006 McLennan, 2006 ). Zhang et al.(2020) Zhang et al.(2020) have found that NUDT16 regulates the poly-ADP ribosylation of 53BP1, which is targeted by the poly(ADP-ribose) (PAR)-binding E3 ubiquitin ligase, RNF146, leading to 53BP1 poly-ubiquitination and degradation. Overexpression of a catalytically inactive NUDT16 mutant prevented 53BP1 recruitment to DSBs. Because mutant NUDT16 binding of TIRR increased after DNA damage, TIRR/53BP1 Tudor binding was enhanced, thus impairing interaction of the 53BP1 Tudor domain with H4K20me2 ( Zhang et al., 2020 Zhang et al., 2020 ). TIRR and NUDT16 both belong to the Nudix hydrolase family and share a 46% sequence identity with each other. However, TIRR lacks an intact Nudix domain, which is essential for NUDT16 hydrolysis activity. Structural comparisons reveal that a TIRR histidine (H106) essential for TIRR-53BP1 Tudor binding is absent from NUDT16 ( Dai et al., 2018 Dai et al., 2018 ; Wang et al., 2018 Wang et al., 2018 ). The specificity of 53BP1 Tudor recognition by TIRR is unlikely to be conferred by the Nudix domain because this domain is located far from the binding interface of TIRR/53BP1 Tudor. Although these two upstream regulators of 53BP1 stabilize the 53BP1 protein, their mechanisms for regulating 53BP1 in the DNA damage response differ. Firstly, NUDT16, but not TIRR, shows hydrolase activity that could remove ADP-ribosylation from 53BP1, which protects 53BP1 from ADP-ribosylation-dependent ubiquitination and degradation. Secondly, TIRR overexpression inhibits 53BP1 localization to DSBs by blocking its H4K20me2-binding sites, whereas NUDT16 does not affect 53BP1 foci formation after irradiation. NUDT16 also lacks the binding residue critical for binding with 53BP1, which facilitates TIRR directly binding with the Tudor domain of 53BP1 ( Zhang et al., 2020 Zhang et al., 2020 ) ( Fig. 2 Fig. 2 ). 5 Downstream effectors of 53BP1 in the NHEJ pathway 1 53BP1 also regulates DSB repair by suppressing the nucleolytic resection of DNA termini, which controls two downstream sub-pathways that are regulated by the RIF1-REV7/Shieldin complex and PTIP-Artemis, respectively ( Fig. 3 Fig. 3 ). 5.1 Cooperation between 53BP1 and RIF1-REV7/Shieldin complex in NHEJ repair 2 A major step forward in our understanding of how 53BP1 promotes NHEJ signaling came with the discovery that 53BP1 cooperates with RIF1 to promote NHEJ and inhibit HR repair signaling ( Chapman et al., 2013 Chapman et al., 2013 ; di Virgilio et al., 2013 di Virgilio et al., 2013 ; Escribano-Díaz et al., 2013 Escribano-Díaz et al., 2013 ; Feng et al., 2013 Feng et al., 2013 ; Zimmermann et al., 2013 Zimmermann et al., 2013 ). Several observations have shown that RIF1 binds to 53BP1 directly in a DNA damage-dependent manner ( di Virgilio et al., 2013 di Virgilio et al., 2013 ). 53BP1 coimmunoprecipitates with RIF1 and recruits RIF1 to the DNA damaged sites by its N-terminal domain, which contains multiple ATM-phosphorylated Ser/ThrGln sites ( Chapman et al., 2013 Chapman et al., 2013 ; di Virgilio et al., 2013 di Virgilio et al., 2013 ; Escribano-Díaz et al., 2013 Escribano-Díaz et al., 2013 ; Feng et al., 2013 Feng et al., 2013 ; Zimmermann et al., 2013 Zimmermann et al., 2013 ). Furthermore, similarly to the conditional ablation of 53BP1, conditional ablation of mouse Rif1 specifically in B cells resulted in a profound defect in class switch recombination ( Manis et al., 2004 Manis et al., 2004 ; Ward et al., 2004 Ward et al., 2004 ; Chapman et al., 2013 Chapman et al., 2013 ). Although the NHEJ of dysfunctional telomeres is abolished in cells lacking 53BP1 and in cells expressing 53BP1 28A (an allele harboring alanine substitutions in all 28 N-terminal ATM/ATM and Rad3-related (ATR) sites) ( Lottersberger et al., 2013 Lottersberger et al., 2013 ), loss of RIF1 exerts a considerably milder defect. Moreover, although the generation of toxic radial chromosomes in Brca1 -deficient cells is inhibited in 53bp1 -/- or in 53BP1 28A -mutant cells, the loss of RIF1 only partially rescues HR repair in BRCA1-deficient cells ( Bouwman et al., 2010 Bouwman et al., 2010 ; Bunting et al., 2010 Bunting et al., 2010 , 2012 2012 ; Bothmer et al., 2011 Bothmer et al., 2011 ). The function of 53BP1 requires interactions with PTIP ( Munoz et al., 2007 Munoz et al., 2007 ) and RIF1, the latter of which recruits REV7 (also known as MAD2L2) to DSB sites ( Boersma et al., 2015 Boersma et al., 2015 ; Xu et al., 2015 Xu et al., 2015 ). Recently, in several persuasive studies, a number of groups have identified a novel protein complex, named Shieldin, that is a downstream effector of 53BP1-RIF1, which protects DNA ends from resection and facilitates the 53BP1-dependent NHEJ repair pathway ( Dev et al., 2018 Dev et al., 2018 ; Ghezraoui et al., 2018 Ghezraoui et al., 2018 ; Gupta et al., 2018 Gupta et al., 2018 ; Mirman et al., 2018 Mirman et al., 2018 ; Noordermeer et al., 2018 Noordermeer et al., 2018 ). Shieldin, a four-subunit single-stranded DNA (ssDNA)-binding complex, consists of SHLD1 (known as RINN3 or C20ORF196), SHLD2 (known as RINN2 or FAM35A), SHLD3 (known as RINN1 or CTC-534A2.2), and REV7. Among these subunits, SHLD3 is the most apical component, bridging REV7 to RIF1 and filling the gap in the 53BP1-RIF1-REV7 axis ( Gupta et al., 2018 Gupta et al., 2018 ; Noordermeer et al., 2018 Noordermeer et al., 2018 ; Setiaputra and Durocher, 2019 Setiaputra and Durocher, 2019 ). Because of this, REV7 and SHLD3 form a subcomplex that is required for the DSB recruitment unit whereas SHLD1, along with SHLD2, acts as the ssDNA-binding module within the complex. 53BP1-RIF1-Shieldin regulates the 3' overhangs through the CTC1-STN1-TEN1 (CST) complex, which functions as an accessory factor for polymerase-α-primase, and is a downstream effector in the 53BP1 pathway. CST-Pola-mediated fill-in helps to regulate DSB repair by the 53BP1-RIF1-Shieldin axis ( Mirman et al., 2018 Mirman et al., 2018 ). Further, crystal structure analysis of REV7 in complex with the REV7-binding domain of SHLD3 has revealed that SHLD3 binds with REV7 via the SHLD3 N-terminal loop and C-terminal helix, which provides critical insight into how SHLD3 recognizes REV7 ( Dai et al., 2020 Dai et al., 2020 ). Taken together, 53BP1-RIF1 recruits the Shieldin complex to DNA damage sites and mediates NHEJ-related repair of immunoglobulin class-switch recombination, fusion of unprotected telomeres and intrachromosomal breaks. The Shieldin complex acts as a downstream effector of 53BP1-RIF1 to limit DNA end resection and cause BRCA1-deficient cells to be sensitive to PARPi. 5.2 PTIP-Artemis complex mediates 53BP1-dependent DNA repair 2 As described above, RIF1 is one of the critical factors acting as a downstream effector of 53BP1, and it antagonizes the BRCA1 function in DNA repair. However, the level of resection rescued by RIF1 loss is insufficient to rescue the HR defect in BRCA1-deficient cells. Callen et al. (2013) Callen et al. (2013) have revealed that depletion of PTIP provides additional or sustained end resection that is required for rescuing HR repair in BRCA1-deficient cells, which indicates that RIF1 and PTIP may act together and/or inhibit different resection steps during HR repair. PTIP is a ubiquitously expressed nuclear protein that individualistically regulates transcription as part of the MLL3-MLL4 methyltransferase complex that catalyzes H3K4me3 ( Cho et al., 2007 Cho et al., 2007 ; Munoz et al., 2007 Munoz et al., 2007 ). In addition to its well-known function in transcription initiation, a separate PTIP pool performs an unknown capacity in the DNA damage response ( Gong et al., 2009 Gong et al., 2009 ). Indeed, PTIP has been described in both HR ( Wang et al., 2010 Wang et al., 2010 ) and NHEJ ( Callen et al., 2012 Callen et al., 2012 ). PTIP contains BRCT repeats that directly interact with phosphorylated Ser25 of 53BP1 in an ATM-dependent manner ( Manke et al., 2003 Manke et al., 2003 ; Yu et al., 2003 Yu et al., 2003 ; Munoz et al., 2007 Munoz et al., 2007 ). Unlike RIF1, PTIP is dispensable for NHEJ during class-switch recombination, but the loss of PTIP represses toxic NHEJ-mediated DSB repair in BRCA1-defiecient cells and restores RAD51 foci ( Callen et al., 2013 Callen et al., 2013 ). PTIP participates in NHEJ repair and in preventing HR repair by recruiting a bona fide NHEJ protein, Artemis, to sites of DNA damage ( Wang et al., 2014 Wang et al., 2014 ). Artemis is an evolutionally conserved nuclease that possesses endonuclease activity ( Ma et al., 2002 Ma et al., 2002 ). Artemis is a component of the NHEJ pathway, which directly binds to PTIP and accumulates at DNA damage sites ( Wang et al., 2014 Wang et al., 2014 ). The interaction of Artemis and PTIP is induced by DNA damage and phosphorylation dependence. Its endonuclease activity permits Artemis to trim DNA ends to promote NHEJ and, consequently, at the same time to prevent end resection and RAD51-dependent HR repair ( Wang et al., 2014 Wang et al., 2014 ). 6 Concluding remarks 1 DNA damage induces a serious threat to genome integrity, and therefore cells have evolved elegant DNA damage response mechanisms to protect their genome stability. The choice of repair pathways is a crucial step during DSB processing that involves several components such as epigenetic alterations, cell cycle phases, and the DNA end resection. 53BP1, in conjunction with its upstream regulators and downstream effectors, spearheads the restraints of end resection to antagonize BRCA1-mediated HR repair, and consequently promote the NHEJ pathway. Deeper knowledge of up- and downstream regulations of 53BP1 will improve our understanding of how the 53BP1 repair pathway operates in the cell to facilitate DNA repair and through it, mechanisms of cancer-associated PARPi resistance. In the future, this direction of research will offer new targets for improving cancer therapy and overcome therapeutic resistance. References Abraham RT , 2002 . Checkpoint signalling: focusing on 53BP1 . Nat Cell Biol , 4 ( 12 ): E277 - E279 . https://doi.org/10.1038/ncb1202-e277 https://doi.org/10.1038/ncb1202-e277 Checkpoint signalling: focusing on 53BP1 Adams MM , Carpenter PB , 2006 . Tying the loose ends together in DNA double strand break repair with 53BP1 . Cell Div , 1 : 19 . https://doi.org/10.1186/1747-1028-1-19 https://doi.org/10.1186/1747-1028-1-19 Tying the loose ends together in DNA double strand break repair with 53BP1 Anderson L , Henderson C , Adachi Y , 2001 . Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage . Mol Cell Biol , 21 ( 5 ): 1719 - 1729 . https://doi.org/10.1128/MCB.21.5.1719-1729.2001 https://doi.org/10.1128/MCB.21.5.1719-1729.2001 Phosphorylation and rapid relocalization of 53BP1 to nuclear foci upon DNA damage Bekker-Jensen S , Mailand N , 2011 . The ubiquitin- and SUMO-dependent signaling response to DNA double-strand breaks . FEBS Lett , 585 ( 18 ): 2914 - 2919 . https://doi.org/10.1016/j.febslet.2011.05.056 https://doi.org/10.1016/j.febslet.2011.05.056 The ubiquitin- and SUMO-dependent signaling response to DNA double-strand breaks Boersma V , Moatti N , Segura-Bayona S , et al. , 2015 . MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5' end resection . Nature , 521 ( 7553 ): 537 - 540 . https://doi.org/10.1038/nature14216 https://doi.org/10.1038/nature14216 MAD2L2 controls DNA repair at telomeres and DNA breaks by inhibiting 5' end resection Bothmer A , Robbiani DF , di Virgilio M , et al. , 2011 . Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1 . Mol Cell , 42 ( 3 ): 319 - 329 . https://doi.org/10.1016/j.molcel.2011.03.019 https://doi.org/10.1016/j.molcel.2011.03.019 Regulation of DNA end joining, resection, and immunoglobulin class switch recombination by 53BP1 Botuyan MV , Lee J , Ward IM , et al. , 2006 . Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair . Cell , 127 ( 7 ): 1361 - 1373 . https://doi.org/10.1016/j.cell.2006.10.043 https://doi.org/10.1016/j.cell.2006.10.043 Structural basis for the methylation state-specific recognition of histone H4-K20 by 53BP1 and Crb2 in DNA repair Botuyan MV , Cui GF , Drané P , et al. , 2018 . Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein . Nat Struct Mol Biol , 25 ( 7 ): 591 - 600 . https://doi.org/10.1038/s41594-018-0083-z https://doi.org/10.1038/s41594-018-0083-z Mechanism of 53BP1 activity regulation by RNA-binding TIRR and a designer protein Bouwman P , Aly A , Escandell JM , et al. , 2010 . 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers . Nat Struct Mol Biol , 17 ( 6 ): 688 - 695 . https://doi.org/10.1038/nsmb.1831 https://doi.org/10.1038/nsmb.1831 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers Bryant HE , Schultz N , Thomas HD , et al. , 2005 . Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase . Nature , 434 ( 7035 ): 913 - 917 . https://doi.org/10.1038/nature03443 https://doi.org/10.1038/nature03443 Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase Bunting SF , Callen E , Wong N , et al. , 2010 . 53BP1 inhibits homologous recombination in BRCA1 -deficient cells by blocking resection of DNA breaks . Cell , 141 ( 2 ): 243 - 254 . https://doi.org/10.1016/j.cell.2010.03.012 https://doi.org/10.1016/j.cell.2010.03.012 53BP1 inhibits homologous recombination in BRCA1-deficient cells by blocking resection of DNA breaks Bunting SF , Callén E , Kozak ML , et al. , 2012 . Brca1 functions independently of homologous recombination in DNA interstrand crosslink repair . Mol Cell , 46 ( 2 ): 125 - 135 . https://doi.org/10.1016/j.molcel.2012.02.015 https://doi.org/10.1016/j.molcel.2012.02.015 Brca1 functions independently of homologous recombination in DNA interstrand crosslink repair Callen E , Faryabi RB , Luckey M , et al. , 2012 . The DNA damage- and transcription-associated protein Paxip1 controls thymocyte development and emigration . Immunity , 37 ( 6 ): 971 - 985 . https://doi.org/10.1016/j.immuni.2012.10.007 https://doi.org/10.1016/j.immuni.2012.10.007 The DNA damage- and transcription-associated protein Paxip1 controls thymocyte development and emigration Callen E , di Virgilio M , Kruhlak MJ , et al. , 2013 . 53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions . Cell , 153 ( 6 ): 1266 - 1280 . https://doi.org/10.1016/j.cell.2013.05.023 https://doi.org/10.1016/j.cell.2013.05.023 53BP1 mediates productive and mutagenic DNA repair through distinct phosphoprotein interactions Chapman JR , Sossick AJ , Boulton SJ , et al. , 2012a . BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair . J Cell Sci , 125 ( Pt 15 ): 3529 - 3534 . https://doi.org/10.1242/jcs.105353 https://doi.org/10.1242/jcs.105353 BRCA1-associated exclusion of 53BP1 from DNA damage sites underlies temporal control of DNA repair Chapman JR , Taylor MRG , Boulton SJ , 2012b . Playing the end game: DNA double-strand break repair pathway choice . Mol Cell , 47 ( 4 ): 497 - 510 . https://doi.org/10.1016/j.molcel.2012.07.029 https://doi.org/10.1016/j.molcel.2012.07.029 Playing the end game: DNA double-strand break repair pathway choice Chapman JR , Barral P , Vannier JB , et al. , 2013 . RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection . Mol Cell , 49 ( 5 ): 858 - 871 . https://doi.org/10.1016/j.molcel.2013.01.002 https://doi.org/10.1016/j.molcel.2013.01.002 RIF1 is essential for 53BP1-dependent nonhomologous end joining and suppression of DNA double-strand break resection Charier G , Couprie J , Alpha-Bazin B , et al. , 2004 . The Tudor tandem of 53BP1: a new structural motif involved in DNA and RG-rich peptide binding . Structure , 12 ( 9 ): 1551 - 1562 . https://doi.org/10.1016/j.str.2004.06.014 https://doi.org/10.1016/j.str.2004.06.014 The Tudor tandem of 53BP1: a new structural motif involved in DNA and RG-rich peptide binding Cho YW , Hong T , Hong SH , et al. , 2007 . PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4: methyltransferase complex . J Biol Chem , 282 ( 28 ): 20395 - 20406 . https://doi.org/10.1074/jbc.M701574200 https://doi.org/10.1074/jbc.M701574200 PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4: methyltransferase complex Dai YX , Zhang AL , Shan S , et al. , 2018 . Structural basis for recognition of 53BP1 tandem Tudor domain by TIRR . Nat Commun , 9 : 2123 . https://doi.org/10.1038/s41467-018-04557-2 https://doi.org/10.1038/s41467-018-04557-2 Structural basis for recognition of 53BP1 tandem Tudor domain by TIRR Dai YX , Zhang F , Wang LG , et al. , 2020 . Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair . J Biol Chem , 295 ( 1 ): 250 - 262 . https://doi.org/10.1074/jbc.RA119.011464 https://doi.org/10.1074/jbc.RA119.011464 Structural basis for shieldin complex subunit 3-mediated recruitment of the checkpoint protein REV7 during DNA double-strand break repair Dev H , Chiang TWW , Lescale C , et al. , 2018 . Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells . Nat Cell Biol , 20 ( 8 ): 954 - 965 . https://doi.org/10.1038/s41556-018-0140-1 https://doi.org/10.1038/s41556-018-0140-1 Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells di Virgilio M , Callen E , Yamane A , et al. , 2013 . Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching . Science , 339 ( 6120 ): 711 - 715 . https://doi.org/10.1126/science.1230624 https://doi.org/10.1126/science.1230624 Rif1 prevents resection of DNA breaks and promotes immunoglobulin class switching Doil C , Mailand N , Bekker-Jensen S , et al. , 2009 . RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins . Cell , 136 ( 3 ): 435 - 446 . https://doi.org/10.1016/j.cell.2008.12.041 https://doi.org/10.1016/j.cell.2008.12.041 RNF168 binds and amplifies ubiquitin conjugates on damaged chromosomes to allow accumulation of repair proteins Drané P , Brault ME , Cui GF , et al. , 2017 . TIRR regulates 53BP1 by masking its histone methyl-lysine binding function . Nature , 543 ( 7644 ): 211 - 216 . https://doi.org/10.1038/nature21358 https://doi.org/10.1038/nature21358 TIRR regulates 53BP1 by masking its histone methyl-lysine binding function Escribano-Díaz C , Orthwein A , Fradet-Turcotte A , et al. , 2013 . A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice . Mol Cell , 49 ( 5 ): 872 - 883 . https://doi.org/10.1016/j.molcel.2013.01.001 https://doi.org/10.1016/j.molcel.2013.01.001 A cell cycle-dependent regulatory circuit composed of 53BP1-RIF1 and BRCA1-CtIP controls DNA repair pathway choice Farmer H , McCabe N , Lord CJ , et al. , 2005 . Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy . Nature , 434 ( 7035 ): 917 - 921 . https://doi.org/10.1038/nature03445 https://doi.org/10.1038/nature03445 Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy Feng L , Fong KW , Wang JD , et al. , 2013 . RIF1 counteracts BRCA1-mediated end resection during DNA repair . J Biol Chem , 288 ( 16 ): 11135 - 11143 . https://doi.org/10.1074/jbc.M113.457440 https://doi.org/10.1074/jbc.M113.457440 RIF1 counteracts BRCA1-mediated end resection during DNA repair Fitzgerald JE , Grenon M , Lowndes NF , 2009 . 53BP1: function and mechanisms of focal recruitment . Biochem Soc Trans , 37 ( Pt 4 ): 897 - 904 . https://doi.org/10.1042/BST0370897 https://doi.org/10.1042/BST0370897 53BP1: function and mechanisms of focal recruitment Fradet-Turcotte A , Canny MD , Escribano-Díaz C , et al. , 2013 . 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark . Nature , 499 ( 7456 ): 50 - 54 . https://doi.org/10.1038/nature12318 https://doi.org/10.1038/nature12318 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark Gatti M , Pinato S , Maspero E , et al. , 2012 . A novel ubiquitin mark at the N-terminal tail of histone H2As targeted by RNF168 ubiquitin ligase . Cell Cycle , 11 ( 13 ): 2538 - 2544 . https://doi.org/10.4161/cc.20919 https://doi.org/10.4161/cc.20919 A novel ubiquitin mark at the N-terminal tail of histone H2As targeted by RNF168 ubiquitin ligase Ghezraoui H , Oliveira C , Becker JR , et al. , 2018 . 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ . Nature , 560 ( 7716 ): 122 - 127 . https://doi.org/10.1038/s41586-018-0362-1 https://doi.org/10.1038/s41586-018-0362-1 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ Gong ZH , Cho YW , Kim JE , et al. , 2009 . Accumulation of Pax2 transactivation domain interaction protein (PTIP) at sites of DNA breaks via RNF8-dependent pathway is required for cell survival after DNA damage . J Biol Chem , 284 ( 11 ): 7284 - 7293 . https://doi.org/10.1074/jbc.M809158200 https://doi.org/10.1074/jbc.M809158200 Accumulation of Pax2 transactivation domain interaction protein (PTIP) at sites of DNA breaks via RNF8-dependent pathway is required for cell survival after DNA damage Goodarzi AA , Jeggo PA , 2013 . The repair and signaling responses to DNA double-strand breaks . Adv Genet , 82 : 1 - 45 . https://doi.org/10.1016/B978-0-12-407676-1.00001-9 https://doi.org/10.1016/B978-0-12-407676-1.00001-9 The repair and signaling responses to DNA double-strand breaks Guo X , Bai YT , Zhao MM , et al. , 2018 . Acetylation of 53BP1 dictates the DNA double strand break repair pathway . Nucleic Acids Res , 46 ( 2 ): 689 - 703 . https://doi.org/10.1093/nar/gkx1208 https://doi.org/10.1093/nar/gkx1208 Acetylation of 53BP1 dictates the DNA double strand break repair pathway Gupta R , Somyajit K , Narita T , et al. , 2018 . DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity . Cell , 173 ( 4 ): 972 - 988. . E923 . https://doi.org/10.1016/j.cell.2018.03.050 https://doi.org/10.1016/j.cell.2018.03.050 DNA repair network analysis reveals shieldin as a key regulator of NHEJ and PARP inhibitor sensitivity Heyer WD , Ehmsen KT , Liu J , 2010 . Regulation of homologous recombination in eukaryotes . Annu Rev Genet , 44 : 113 - 139 . https://doi.org/10.1146/annurev-genet-051710-150955 https://doi.org/10.1146/annurev-genet-051710-150955 Regulation of homologous recombination in eukaryotes Huen MS , Grant R , Manke I , et al. , 2007 . RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly . Cell , 131 ( 5 ): 901 - 914 . https://doi.org/10.1016/j.cell.2007.09.041 https://doi.org/10.1016/j.cell.2007.09.041 RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly Huen MSY , Huang J , Leung JWC , et al. , 2010 . Regulation of chromatin architecture by the PWWP domain-containing DNA damage-responsive factor EXPAND1/MUM1 . Mol Cell , 37 ( 6 ): 854 - 864 . https://doi.org/10.1016/j.molcel.2009.12.040 https://doi.org/10.1016/j.molcel.2009.12.040 Regulation of chromatin architecture by the PWWP domain-containing DNA damage-responsive factor EXPAND1/MUM1 Isono M , Niimi A , Oike T , et al. , 2017 . BRCA1 directs the repair pathway to homologous recombination by promoting 53BP1 dephosphorylation . Cell Rep , 18 ( 2 ): 520 - 532 . https://doi.org/10.1016/j.celrep.2016.12.042 https://doi.org/10.1016/j.celrep.2016.12.042 BRCA1 directs the repair pathway to homologous recombination by promoting 53BP1 dephosphorylation Iwabuchi K , Basu BP , Kysela B , et al. , 2003 . Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA . J Biol Chem , 278 ( 38 ): 36487 - 36495 . https://doi.org/10.1074/jbc.M304066200 https://doi.org/10.1074/jbc.M304066200 Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA Kolas NK , Chapman JR , Nakada S , et al. , 2007 . Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase . Science , 318 ( 5856 ): 1637 - 1640 . https://doi.org/10.1126/science.1150034 https://doi.org/10.1126/science.1150034 Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase Lieber MR , 2010 . The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway . Annu Rev Biochem , 79 : 181 - 211 . https://doi.org/10.1146/annurev.biochem.052308.093131 https://doi.org/10.1146/annurev.biochem.052308.093131 The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway Lottersberger F , Bothmer A , Robbiani DF , et al. , 2013 . Role of 53BP1 oligomerization in regulating double-strand break repair . Proc Natl Acad Sci USA , 110 ( 6 ): 2146 - 2151 . https://doi.org/10.1073/pnas.1222617110 https://doi.org/10.1073/pnas.1222617110 Role of 53BP1 oligomerization in regulating double-strand break repair Lukas J , Lukas C , Bartek J , 2011 . More than just a focus: the chromatin response to DNA damage and its role in genome integrity maintenance . Nat Cell Biol , 13 ( 10 ): 1161 - 1169 . https://doi.org/10.1038/ncb2344 https://doi.org/10.1038/ncb2344 More than just a focus: the chromatin response to DNA damage and its role in genome integrity maintenance Ma YM , Pannicke U , Schwarz K , et al. , 2002 . Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination . Cell , 108 ( 6 ): 781 - 794 . https://doi.org/10.1016/s0092-8674(02)00671-2 https://doi.org/10.1016/s0092-8674(02)00671-2 Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination Mailand N , Bekker-Jensen S , Faustrup H , et al. , 2007 . RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins . Cell , 131 ( 5 ): 887 - 900 . https://doi.org/10.1016/j.cell.2007.09.040 https://doi.org/10.1016/j.cell.2007.09.040 RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins Manis JP , Morales JC , Xia ZF , et al. , 2004 . 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination . Nat Immunol , 5 ( 5 ): 481 - 487 . https://doi.org/10.1038/ni1067 https://doi.org/10.1038/ni1067 53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination Manke IA , Lowery DM , Nguyen A , et al. , 2003 . BRCT repeats as phosphopeptide-binding modules involved in protein targeting . Science , 302 ( 5645 ): 636 - 639 . https://doi.org/10.1126/science.1088877 https://doi.org/10.1126/science.1088877 BRCT repeats as phosphopeptide-binding modules involved in protein targeting Mattiroli F , Vissers JHA , van Dijk WJ , et al. , 2012 . RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling . Cell , 150 ( 6 ): 1182 - 1195 . https://doi.org/10.1016/j.cell.2012.08.005 https://doi.org/10.1016/j.cell.2012.08.005 RNF168 ubiquitinates K13-15 on H2A/H2AX to drive DNA damage signaling McLennan AG , 2006 . The Nudix hydrolase superfamily . Cell Mol Life Sci , 63 ( 2 ): 123 - 143 . https://doi.org/10.1007/s00018-005-5386-7 https://doi.org/10.1007/s00018-005-5386-7 The Nudix hydrolase superfamily Mirman Z , Lottersberger F , Takai H , et al. , 2018 . 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in . Nature , 560 ( 7716 ): 112 - 116 . https://doi.org/10.1038/s41586-018-0324-7 https://doi.org/10.1038/s41586-018-0324-7 53BP1-RIF1-shieldin counteracts DSB resection through CST- and Polα-dependent fill-in Munoz IM , Jowsey PA , Toth R , et al. , 2007 . Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage . Nucleic Acids Res , 35 ( 16 ): 5312 - 5322 . https://doi.org/10.1093/nar/gkm493 https://doi.org/10.1093/nar/gkm493 Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage Noordermeer SM , Adam S , Setiaputra D , et al. , 2018 . The shieldin complex mediates 53BP1-dependent DNA repair . Nature , 560 ( 7716 ): 117 - 121 . https://doi.org/10.1038/s41586-018-0340-7 https://doi.org/10.1038/s41586-018-0340-7 The shieldin complex mediates 53BP1-dependent DNA repair Palazzo L , Thomas B , Jemth AS , et al. , 2015 . Processing of protein ADP-ribosylation by Nudix hydrolases . Biochem J , 468 ( 2 ): 293 - 301 . https://doi.org/10.1042/BJ20141554 https://doi.org/10.1042/BJ20141554 Processing of protein ADP-ribosylation by Nudix hydrolases Panier S , Boulton SJ , 2014 . Double-strand break repair: 53BP1 comes into focus . Nat Rev Mol Cell Biol , 15 ( 1 ): 7 - 18 . https://doi.org/10.1038/nrm3719 https://doi.org/10.1038/nrm3719 Double-strand break repair: 53BP1 comes into focus Polato F , Callen E , Wong N , et al. , 2014 . CtIP-mediated resection is essential for viability and can operate independently of BRCA1 . J Exp Med , 211 ( 6 ): 1027 - 1036 . https://doi.org/10.1084/jem.20131939 https://doi.org/10.1084/jem.20131939 CtIP-mediated resection is essential for viability and can operate independently of BRCA1 Rappold I , Iwabuchi K , Date T , et al. , 2001 . Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways . J Cell Biol , 153 ( 3 ): 613 - 620 . https://doi.org/10.1083/jcb.153.3.613 https://doi.org/10.1083/jcb.153.3.613 Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways Schultz LB , Chehab NH , Malikzay A , et al. , 2000 . P53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks . J Cell Biol , 151 ( 7 ): 1381 - 1390 . https://doi.org/10.1083/jcb.151.7.1381 https://doi.org/10.1083/jcb.151.7.1381 P53 binding protein 1 (53BP1) is an early participant in the cellular response to DNA double-strand breaks Setiaputra D , Durocher D , 2019 . Shieldin—the protector of DNA ends . EMBO Rep , 20 ( 5 ): e47560 . https://doi.org/10.15252/embr.201847560 https://doi.org/10.15252/embr.201847560 Shieldin—the protector of DNA ends Stewart GS , Panier S , Townsend K , et al. , 2009 . The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage . Cell , 136 ( 3 ): 420 - 434 . https://doi.org/10.1016/j.cell.2008.12.042 https://doi.org/10.1016/j.cell.2008.12.042 The RIDDLE syndrome protein mediates a ubiquitin-dependent signaling cascade at sites of DNA damage Tomida J , Takata KI , Bhetawal S , et al. , 2018 . FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells . EMBO J , 37 ( 12 ): e99543 . https://doi.org/10.15252/embj.201899543 https://doi.org/10.15252/embj.201899543 FAM35A associates with REV7 and modulates DNA damage responses of normal and BRCA1-defective cells van Gent DC , 2009 . Reaching out for the other end with p53-binding protein 1 . Trends Biochem Sci , 34 ( 5 ): 226 - 229 . https://doi.org/10.1016/j.tibs.2009.01.009 https://doi.org/10.1016/j.tibs.2009.01.009 Reaching out for the other end with p53-binding protein 1 Wang B , Elledge SJ , 2007 . Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage . Proc Natl Acad Sci USA , 104 ( 52 ): 20759 - 20763 . https://doi.org/10.1073/pnas.0710061104 https://doi.org/10.1073/pnas.0710061104 Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage Wang JD , Aroumougame A , Lobrich M , et al. , 2014 . PTIP associates with Artemis to dictate DNA repair pathway choice . Genes Dev , 28 ( 24 ): 2693 - 2698 . https://doi.org/10.1101/gad.252478.114 https://doi.org/10.1101/gad.252478.114 PTIP associates with Artemis to dictate DNA repair pathway choice Wang JX , Yuan ZL , Cui YQ , et al. , 2018 . Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR . Nat Commun , 9 : 2689 . https://doi.org/10.1038/s41467-018-05174-9 https://doi.org/10.1038/s41467-018-05174-9 Molecular basis for the inhibition of the methyl-lysine binding function of 53BP1 by TIRR Wang X , Takenaka K , Takeda S , 2010 . PTIP promotes DNA double-strand break repair through homologous recombination . Genes Cells , 15 ( 3 ): 243 - 254 . https://doi.org/10.1111/j.1365-2443.2009.01379.x https://doi.org/10.1111/j.1365-2443.2009.01379.x PTIP promotes DNA double-strand break repair through homologous recombination Ward IM , Reina-San-Martin B , Olaru A , et al. , 2004 . 53BP1 is required for class switch recombination . J Cell Biol , 165 ( 4 ): 459 - 464 . https://doi.org/10.1083/jcb.200403021 https://doi.org/10.1083/jcb.200403021 53BP1 is required for class switch recombination Xu GT , Chapman JR , Brandsma I , et al. , 2015 . REV7 counteracts DNA double-strand break resection and affects PARP inhibition . Nature , 521 ( 7553 ): 541 - 544 . https://doi.org/10.1038/nature14328 https://doi.org/10.1038/nature14328 REV7 counteracts DNA double-strand break resection and affects PARP inhibition Yu XC , Chini CCS , He M , et al. , 2003 . The BRCT domain is a phospho-protein binding domain . Science , 302 ( 5645 ): 639 - 642 . https://doi.org/10.1126/science.1088753 https://doi.org/10.1126/science.1088753 The BRCT domain is a phospho-protein binding domain Zhang AL , Peng B , Huang P , et al. , 2017 . The p53-binding protein 1-Tudor-interacting repair regulator complex participates in the DNA damage response . J Biol Chem , 292 ( 16 ): 6461 - 6467 . https://doi.org/10.1074/jbc.M117.777474 https://doi.org/10.1074/jbc.M117.777474 The p53-binding protein 1-Tudor-interacting repair regulator complex participates in the DNA damage response Zhang F , Lou LH , Peng B , et al. , 2020 . Nudix hydrolase NUDT16 regulates 53BP1 protein by reversing 53BP1 ADP-ribosylation . Cancer Res , 80 ( 5 ): 999 - 1010 . https://doi.org/10.1158/0008-5472.CAN-19-2205 https://doi.org/10.1158/0008-5472.CAN-19-2205 Nudix hydrolase NUDT16 regulates 53BP1 protein by reversing 53BP1 ADP-ribosylation Zimmermann M , Lottersberger F , Buonomo SB , et al. , 2013 . 53BP1 regulates DSB repair using Rif1 to control 5' end resection . Science , 339 ( 6120 ): 700 - 704 . https://doi.org/10.1126/science.1231573 https://doi.org/10.1126/science.1231573 53BP1 regulates DSB repair using Rif1 to control 5' end resection Fan ZHANG wrote and edited the manuscript and drew the figures. Zihua GONG contributed to the revision of the manuscript and the figures. Both authors have read and approved the final manuscript and, therefore, take responsibility for the integrity and security of the manuscript. 10.1631/jzus.B2000306 1673-1581（2021）01-0038-09 ZHANG Fan,GONG Zihua., 2021. Regulation of DNA double-strand break repair pathway choice: a new focus on 53BP1. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(1):38-46. ZHANG Fan,GONG Zihua., 2021. Regulation of DNA double-strand break repair pathway choice: a new focus on 53BP1. Journal of Zhejiang University-SCIENCE B(Biomedicine & Biotechnology), 22(1):38-46. 2020-06-16 2020-08-17 2020-12-15 2021-01-15 2022-05-09 2021 Zhejiang University Press Fan ZHANG and Zihua GONG declare that they have no conflict of interest. This paper does not contain any studies with human or animal subjects performed by either of the authors. Compliance with ethics guidelines 浙江大学学报（英文版）（B辑：生物医学和生物技术） 1 22