DNA Double-strand Breaks Repair Pathway Choice Research Articles

Marker-free quantification of repair pathway utilization at Cas9-induced double-strand breaks.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is widely used for targeted genomic and epigenomic modifications and imaging in cells and organisms, and holds tremendous promise in clinical applications. The efficiency and accuracy of the technology are partly determined by the target binding affinity and residence time of Cas9-single-guide RNA (sgRNA) at a given site. However, little attention has been paid to the effect of target binding affinity and residence duration on the repair of Cas9-induced DNA double-strand breaks (DSBs). We propose that the choice of DSB repair pathway may be altered by variation in the binding affinity and residence duration of Cas9-sgRNA at the cleaved target, contributing to significantly heterogeneous mutations in CRISPR/Cas9 genome editing. Here, we discuss the effect of Cas9-sgRNA target binding and residence on the choice of DSB repair pathway in CRISPR/Cas9 genome editing, and the opportunity this presents to optimize Cas9-based technology.

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

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.

Impact of hypoxia on the double-strand break repair after photon and carbon ion irradiation of radioresistant HNSCC cells

DNA double-strand breaks (DSBs) induced by photon irradiation are the most deleterious damage for cancer cells and their efficient repair may contribute to radioresistance, particularly in hypoxic conditions. Carbon ions (C-ions) act independently of the oxygen concentration and trigger complex- and clustered-DSBs difficult to repair. Understanding the interrelation between hypoxia, radiation-type, and DNA-repair is therefore essential for overcoming radioresistance. The DSBs signaling and the contribution of the canonical non-homologous end-joining (NHEJ-c) and homologous-recombination (HR) repair pathways were assessed by immunostaining in two cancer-stem-cell (CSCs) and non-CSCs HNSCC cell lines. Detection and signaling of DSBs were lower in response to C-ions than photons. Hypoxia increased the decay-rate of the detected DSBs (γH2AX) in CSCs after photons and the initiation of DSB repair signaling (P-ATM) in CSCs and non-CSCs after both radiations, but not the choice of DSB repair pathway (53BP1). Additionally, hypoxia increased the NHEJ-c (DNA-PK) and the HR pathway (RAD51) activation only after photons. Furthermore, the involvement of the HR seemed to be higher in CSCs after photons and in non-CSCs after C-ions. Taken together, our results show that C-ions may overcome the radioresistance of HNSCC associated with DNA repair, particularly in CSCs, and independently of a hypoxic microenvironment.

RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection.

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.

Pathway choice for DNA double strand break repair

DNA double strand break (DSB) is a kind of highly toxic damage, causing genomic instability, chromatin aberration and cancer. A number of conserved DSB damage repair pathways have been found in eukaryotes, most notably classical non-homologous end joining (cNHEJ) and homologous recombination (HR). These two repair pathways resolve most DSBs in cells. For different types of DSBs, cells could wittily choose the optimal approach with the lowest risk for genomic instability. Relying on the developing cell imaging techniques and molecular biological methods, sophisticated mechanism of DSB repair pathway choice has been revealed. The regulation mechanism of pathway choice is complex and precise. In this review, the effect factors such as the structures of the broken terminals, the degree of DNA end resection, cell cycle, chromatin environment, histone modification and RNA metabolism on the DSB repair pathway choice are discussed, showing the diversity and flexibility of DSB repair pathway choice.

P31comet promotes homologous recombination by inactivating REV7 through the TRIP13 ATPase

The repair of DNA double strand breaks (DSBs) that arise from external mutagenic agents and routine cellular processes is essential for life. DSBs are repaired by two major pathways, homologous recombination (HR) and classical nonhomologous end joining (C-NHEJ). DSB repair pathway choice is largely dictated at the step of 5'-3' DNA end resection, which is promoted during S phase, in part by BRCA1. Opposing end resection is the 53BP1 protein, which recruits the ssDNA-binding REV7-Shieldin complex to favor C-NHEJ repair. We recently identified TRIP13 as a proresection factor that remodels REV7, causing its dissociation from the Shieldin subunit SHLD3. Here, we identify p31comet, a negative regulator of MAD2 and the spindle assembly checkpoint, as an important mediator of the TRIP13-REV7 interaction. p31comet binds to the REV7-Shieldin complex in cells, promotes REV7 inactivation, and causes PARP inhibitor resistance. p31comet also participates in the extraction of REV7 from the chromatin. Furthermore, p31comet can counteract REV7 function in translesion synthesis (TLS) by releasing it from REV3 in the Pol ζ complex. Finally, p31comet, like TRIP13, is overexpressed in many cancers and this correlates with poor prognosis. Thus, we reveal a key player in the regulation of HR and TLS with significant clinical implications.

The Determinant of DNA Repair Pathway Choices in Ionising Radiation-Induced DNA Double-Strand Breaks.

Ionising radiation- (IR-) induced DNA double-strand breaks (DSBs) are considered to be the deleterious DNA lesions that pose a serious threat to genomic stability. The major DNA repair pathways, including classical nonhomologous end joining, homologous recombination, single-strand annealing, and alternative end joining, play critical roles in countering and eliciting IR-induced DSBs to ensure genome integrity. If the IR-induced DNA DSBs are not repaired correctly, the residual or incorrectly repaired DSBs can result in genomic instability that is associated with certain human diseases. Although many efforts have been made in investigating the major mechanisms of IR-induced DNA DSB repair, it is still unclear what determines the choices of IR-induced DNA DSB repair pathways. In this review, we discuss how the mechanisms of IR-induced DSB repair pathway choices can operate in irradiated cells. We first briefly describe the main mechanisms of the major DNA DSB repair pathways and the related key repair proteins. Based on our understanding of the characteristics of IR-induced DNA DSBs and the regulatory mechanisms of DSB repair pathways in irradiated cells and recent advances in this field, We then highlight the main factors and associated challenges to determine the IR-induced DSB repair pathway choices. We conclude that the type and distribution of IR-induced DSBs, chromatin state, DNA-end structure, and DNA-end resection are the main determinants of the choice of the IR-induced DNA DSB repair pathway.

FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku

The balance between major DNA double-strand break (DSB) repair pathways is influenced by binding of the Ku complex, a XRCC5/6 heterodimer, to DSB ends, initiating non-homologous end joining (NHEJ) but preventing additional DSB end resection and homologous recombination (HR). However, the key molecular cue for Ku recruitment to DSB sites is unknown. Here, we report that FOXL2, a forkhead family transcriptional factor, directs DSB repair pathway choice by acetylation-dependent binding to Ku. Upon DSB induction, SIRT1 translocates to the nucleus and deacetylates FOXL2 at lysine 124, leading to liberation of XRCC5 and XRCC6 from FOXL2 and formation of the Ku complex. FOXL2 ablation enhances Ku recruitment to DSB sites, imbalances DSB repair kinetics by accelerating NHEJ and inhibiting HR, and thus leads to catastrophic genomic events. Our study unveils the SIRT1-(de)acetylated FOXL2-Ku axis that governs the balance of DSB repair pathways to maintain genome integrity.

Mismatch repair dependence of replication stress-associated DSB recognition and repair

Most cancers develop with one of two types of genomic instability, namely, chromosomal instability (CIN) or microsatellite instability (MSI). Both are induced by replication stress-associated DNA double-strand breaks (DSBs). The type of genomic instability that arises is dependent on the choice of DNA repair pathway. Specifically, MSI is induced via a PolQ-dependent repair pathway called microhomology-mediated end joining (MMEJ) in a mismatch repair (MMR)-deficient background. However, it is unclear how the MMR status determines the choice of DSB repair pathway. Here, we show that replication stress-associated DSBs initially targeted by the homologous recombination (HR) system were subsequently hijacked by PolQ-dependent MMEJ in MMR-deficient cells, but persisted as HR intermediates in MMR-proficient cells. PolQ interacting with MMR factors was effectively loaded onto damaged chromatin in an MMR-deficient background, in which merged MRE11/γH2AX foci also effectively formed. Thus, the choice of DNA repair pathway according to the MMR status determines whether CIN or MSI is induced.

Beyond the Trinity of ATM, ATR, and DNA-PK: Multiple Kinases Shape the DNA Damage Response in Concert With RNA Metabolism.

Our genome is constantly exposed to endogenous and exogenous sources of DNA damage resulting in various alterations of the genetic code. DNA double-strand breaks (DSBs) are considered one of the most cytotoxic lesions. Several types of repair pathways act to repair DNA damage and maintain genome stability. In the canonical DNA damage response (DDR) DSBs are recognized by the sensing kinases Ataxia-telangiectasia mutated (ATM), Ataxia-telangiectasia and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK), which initiate a cascade of kinase-dependent amplification steps known as DSB signaling. Recent evidence suggests that efficient recognition and repair of DSBs relies on the transcription and processing of non-coding (nc)RNA molecules by RNA polymerase II (RNAPII) and the RNA interference (RNAi) factors Drosha and Dicer. Multiple kinases influence the phosphorylation status of both the RNAPII carboxy-terminal domain (CTD) and Dicer in order to regulate RNA-dependent DSBs repair. The importance of kinase signaling and RNA processing in the DDR is highlighted by the regulation of p53-binding protein (53BP1), a key regulator of DSB repair pathway choice between homologous recombination (HR) and non-homologous end joining (NHEJ). Additionally, emerging evidence suggests that RNA metabolic enzymes also play a role in the repair of other types of DNA damage, including the DDR to ultraviolet radiation (UVR). RNAi factors are also substrates for mitogen-activated protein kinase (MAPK) signaling and mediate the turnover of ncRNA during nucleotide excision repair (NER) in response to UVR. Here, we review kinase-dependent phosphorylation events on RNAPII, Drosha and Dicer, and 53BP1 that modulate the key steps of the DDR to DSBs and UVR, suggesting an intimate link between the DDR and RNA metabolism.

DNA double-strand break repair-pathway choice in somatic mammalian cells.

The major pathways of DNA double-strand break (DSB) repair are crucial for maintaining genomic stability. However, if deployed in an inappropriate cellular context, these same repair functions can mediate chromosome rearrangements that underlie various human diseases, ranging from developmental disorders to cancer. The two major mechanisms of DSB repair in mammalian cells are non-homologous end joining (NHEJ) and homologous recombination. In this Review, we consider DSB repair-pathway choice in somatic mammalian cells as a series of 'decision trees', and explore how defective pathway choice can lead to genomic instability. Stalled, collapsed or broken DNA replication forks present a distinctive challenge to the DSB repair system. Emerging evidence suggests that the 'rules' governing repair-pathway choice at stalled replication forks differ from those at replication-independent DSBs.

Rif1 S-acylation mediates DNA double-strand break repair at the inner nuclear membrane

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.

DNA double-strand break repair pathway choice: the fork in the road

DNA double-strand breaks (DSBs) are cytotoxic lesions that will lead to genomic instability or even tumorigenesis if left unrepaired or misrepaired. To maintain homeostasis, cells have evolved two major repair pathways to counteract DSBs: classical non-homologous end joining (c-NHEJ) and homologous recombination (HR). Two other modes for repairing DSBs have been described: alternative non-homologous end joining (alt-NHEJ) and single-strand annealing (SSA). c-NHEJ ligates adjacent DSB ends directly with rapid kinetics throughout interphase, while HR meticulously initiates DNA-end resection in late S or G2 phase when sister chromatids are available as repair templates. Although partially sharing the DNA-end resection procedure with HR, alt-NHEJ and SSA often contribute to chromosomal translocation and genome rearrangement. Selection of the appropriate pathway to repair DSBs helps to maintain genome integrity. Here, we review current knowledge of the mechanisms regulating DSB repair pathway choice.

H4K20me0 recognition by BRCA1-BARD1 directs homologous recombination to sister chromatids.

Genotoxic DNA double-strand breaks (DSBs) can be repaired by error-free homologous recombination (HR) or mutagenic non-homologous end-joining1. HR supresses tumorigenesis1, but is restricted to the S and G2 phases of the cell cycle when a sister chromatid is present2. Breast cancer type 1 susceptibility protein (BRCA1) promotes HR by antagonizing the anti-resection factor TP53-binding protein 1(53BP1) (refs. 2-5), but it remains unknown how BRCA1 function is limited to the S and G2 phases. We show that BRCA1 recruitment requires recognition of histone H4 unmethylated at lysine 20 (H4K20me0), linking DSB repair pathway choice directly to sister chromatid availability. We identify the ankyrin repeat domain of BRCA1-associated RING domain protein 1 (BARD1)-the obligate BRCA1 binding partner3-as a reader of H4K20me0 present on new histones in post-replicative chromatin6. BARD1 ankyrin repeat domain mutations disabling H4K20me0 recognition abrogate accumulation of BRCA1 at DSBs, causing aberrant build-up of 53BP1, and allowing anti-resection activity to prevail in S and G2. Consequently, BARD1 recognition of H4K20me0 is required for HR and resistance to poly (ADP-ribose) polymerase inhibitors. Collectively, this reveals that BRCA1-BARD1 monitors the replicative state of the genome to oppose 53BP1 function, routing only DSBs within sister chromatids to HR.

FKBP25 participates in DNA double-strand break repair.

FK506-binding proteins (FKBPs) alter the conformation of proteins via cis-trans isomerization of prolyl-peptide bonds. While this activity can be demonstrated in vitro, the intractability of detecting prolyl isomerization events in cells has limited our understanding of the biological processes regulated by FKBPs. Here we report that FKBP25 is an active participant in the repair of DNA double-strand breaks (DSBs). FKBP25 influences DSB repair pathway choice by promoting homologous recombination (HR) and suppressing single-strand annealing (SSA). Consistent with this observation, cells depleted of FKBP25 form fewer Rad51 repair foci in response to etoposide and ionizing radiation, and they are reliant on the SSA repair factor Rad52 for viability. We find that FKBP25’s catalytic activity is required for promoting DNA repair, which is the first description of a biological function for this enzyme activity. Consistent with the importance of the FKBP catalytic site in HR, rapamycin treatment also impairs homologous recombination, and this effect is at least in part independent of mTor. Taken together these results identify FKBP25 as a component of the DNA DSB repair pathway.

Chromatin control in double strand break repair.

DNA double strand breaks (DSB) are the most deleterious type of damage inflicted on DNA by various environmental factors and as consequences of normal cellular metabolism. The multistep nature of DSB repair and the need to assemble large protein complexes at repair sites necessitate multiple chromatin changes there. This review focuses on the key findings of how chromatin regulators exert temporal and spatial control on DSB repair. These mechanisms coordinate repair with cell cycle progression, lead to DSB repair pathway choice, provide accessibility of repair machinery to damaged sites and move the lesions to nuclear environments permissive for repair.

LMO2 Facilitates Synthetic Lethality after PARP Inhibition (PARPi) in Diffuse Large B-Cell Lymphoma (DLBCL)

Novel therapies for DLBCL are needed to improve patients' outcomes. LIM domain only 2 (LMO2) protein is ubiquitously expressed and plays important roles in endothelial and hematopoietic cell development. LMO2 protein expression is upregulated in germinal center B (GCB) cells, the cell of origin of DLBCL. 73% of GCB and 45% of ABC DLBCLs express LMO2 protein at levels of reactive GCB cells. Although the function of LMO2 in B cells and DLBCL is unknown, expression of LMO2 serves as one of the best prognostic markers of longer survival following R-CHOP therapy. Additionally, LMO2 expression in DLBCL cells results in genomic instability. These observations suggest that LMO2 may decrease DNA repair efficiency. Indeed, here we demonstrate that primary DLBCL tumors and cell lines expressing high levels of LMO2 protein (LMO2HIGH) are defective in DNA double-strand break (DSB) repair via the homologous recombination (HR) pathway. We found that LMO2HIGH DLBCL cells and LMO2LOW DLBCL cells expressing a GFP-LMO2 fusion protein via a doxycycline-inducible system have a defective accumulation of the HR proteins BRCA1 and RAD51 to damaged chromosomes as visualized by immunofluorescence (IF) and Western blot assays. Furthermore, LMO2HIGH DLBCL exposed to ionizing radiation showed decreased levels of phosphorylated (S4 and S8) replication protein A (RPA32 subunit), a marker of DSB end-resection activity and an essential step for HR-dependent DSB repair. Consequently, LMO2HIGH DLBCL showed decreased HR activity as assessed via a DR-GFP reporter system and scoring the frequency of HR-dependent sister chromatid exchanges. Also, in LMO2HIGH vs LMO2LOW DLBCLs, we observed higher levels of ionizing radiation-induced foci (IRIF) for 53BP1 and RIF1 - non-homologous end-joining (NHEJ) core factors playing a critical role in defining DSB repair pathway choice. Similarly, we found a higher accumulation of 53BP1 and RIF1 chromatin-enriched fraction after DNA damage in LMO2HIGH than LMO2LOW DLBCLs. Furthermore, we show that LMO2 forms a complex with 53BP1 but not BRCA1 after DNA damage, as demonstrated by Co-IP, GST pull-down assays and spatial co-localization in IRIFs. This suggests that LMO2 functionally interacts with 53BP1 during DSB repair. Indeed, knockdown of 53BP1 in LMO2HIGH cells increased the levels of BRCA1 and RAD51 IRIF to values similar to LMO2LOW cells without affecting LMO2 levels, revealing that LMO2 depends on 53BP1 to inhibit HR activity.Since LMO2HIGH DLBCL cells exhibit a defective HR-pathway, we next explored the therapeutic potential of PARPi in DLBCL. We found that in LMO2HIGH but not LMO2LOW DLBCL cell lines the PARPi olaparib induced a significant decrease in cell proliferation and colony formation and an increase in cell death via apoptosis. The sensitivity to olaparib directly correlated with LMO2 protein levels. The proliferation defect in LMO2HIGH DLBCL cells was due to the increased DNA damage caused by exposure to PARPi, as observed by an increase in γH2AX foci. Induction of LMO2 expression in LMO2LOW DLBCL cell lines led to sensitivity to olaparib, demonstrating that the proliferation defect induced by olaparib was dependent on LMO2 expression. Silencing of LMO2 via shRNA or CRISP/Cas9 in LMO2HIGH cell lines rescued the proliferation defect induced by olaparib. The proliferation deficiency induced by olaparib was synergistic with doxorubicin in LMO2HIGH but not in LMO2LOW DLBCL cell lines and untreated patient-derived primary DLBCL tumors.We also examined the in vivo efficacy of olaparib in DLBCL mice models. Olaparib treatment prolonged survival of mice harboring LMO2HIGH but not LMO2LOW DLBCL xenograft tumors. Olaparib in combination with RCHOP significantly prolonged survival of mice harboring LMO2HIGH DLBCL tumors compared to cohorts treated with either olaparib or RCHOP alone. Further, analysis of tumors excised from OCI-LY1 bearing animals treated with olaparib for 3 days revealed increased cell death and a higher sensitivity to PARPi in OCI-LY1 LMO2 cells compared to the OCI-LY1 LMO2 negative-GFP control cells, indicating in vivo survival advantage for LMO2LOW cells upon olaparib treatment.In summary, high expression of LMO2 results in HR-dysfunction phenocopying the BRCA1/2 mutations observed in breast and ovarian tumors. In LMO2HIGH DLBCL, PARPi-induced killing is synergistic with doxorubicin, thus providing a clear path for therapeutic development of PARPi in DLBCL. DisclosuresLossos:Affimed: Research Funding.

Understanding the Histone DNA Repair Code: H4K20me2 Makes Its Mark.

Chromatin is a highly compact structure that must be rapidly rearranged in order for DNA repair proteins to access sites of damage and facilitate timely and efficient repair. Chromatin plasticity is achieved through multiple processes, including the posttranslational modification of histone tails. In recent years, the impact of histone posttranslational modification on the DNA damage response has become increasingly well recognized, and chromatin plasticity has been firmly linked to efficient DNA repair. One particularly important histone posttranslational modification process is methylation. Here, we focus on the regulation and function of H4K20 methylation (H4K20me) in the DNA damage response and describe the writers, erasers, and readers of this important chromatin mark as well as the combinatorial histone posttranslational modifications that modulate H4K20me recognition. Finally, we discuss the central role of H4K20me in determining if DNA double-strand breaks (DSB) are repaired by the error-prone, nonhomologous DNA end joining pathway or the error-free, homologous recombination pathway. This review article discusses the regulation and function of H4K20me2 in DNA DSB repair and outlines the components and modifications that modulate this important chromatin mark and its fundamental impact on DSB repair pathway choice. Mol Cancer Res; 16(9); 1335-45. ©2018 AACR.

RNF169 limits 53BP1 deposition at DSBs to stimulate single-strand annealing repair

Unrestrained 53BP1 activity at DNA double-strand breaks (DSBs) hampers DNA end resection and upsets DSB repair pathway choice. RNF169 acts as a molecular rheostat to limit 53BP1 deposition at DSBs, but how this fine balance translates to DSB repair control remains undefined. In striking contrast to 53BP1, ChIP analyses of AsiSI-induced DSBs unveiled that RNF169 exhibits robust accumulation at DNA end-proximal regions and preferentially targets resected, RPA-bound DSBs. Accordingly, we found that RNF169 promotes CtIP-dependent DSB resection and favors homology-mediated DSB repair, and further showed that RNF169 dose-dependently stimulates single-strand annealing repair, in part, by alleviating the 53BP1-imposed barrier to DSB end resection. Our results highlight the interplay of RNF169 with 53BP1 in fine-tuning choice of DSB repair pathways.