RAP80 suppresses the vulnerability of R-loops during DNA double-strand break repair.

Single-stranded DNA (ssDNA) arising as an intermediate of cellular processes on DNA is a potential vulnerability of the genome unless it is appropriately protected. Recent evidence suggests that R-loops, consisting of ssDNA and DNA-RNA hybrids, can form in the proximity of DNA double-strand breaks (DSBs) within transcriptionally active regions. However, how the vulnerability of ssDNA in R-loops is overcome during DSB repair remains unclear. Here, we identify RAP80 as a factor suppressing the vulnerability of ssDNA in R-loops, chromosome translocations, and deletions during DSB repair. Mechanistically, RAP80 prevents unscheduled nucleolytic processing of ssDNA in R-loops by CtIP. This mechanism promotes efficient DSB repair via transcription-associated end joining dependent on BRCA1, Polθ, and LIG1/3. Thus, RAP80 suppresses the vulnerability of R-loops during DSB repair, thereby precluding genomic abnormalities in a critical component of the genome caused by deleterious R-loop processing.

RIF1 Links Replication Timing with Fork Reactivation and DNA Double-Strand Break Repair.

Replication timing (RT) is a cellular program to coordinate initiation of DNA replication in all origins within the genome. RIF1 (replication timing regulatory factor 1) is a master regulator of RT in human cells. This role of RIF1 is associated with binding G4-quadruplexes and changes in 3D chromatin that may suppress origin activation over a long distance. Many effects of RIF1 in fork reactivation and DNA double-strand (DSB) repair (DSBR) are underlined by its interaction with TP53BP1 (tumor protein p53 binding protein). In G1, RIF1 acts antagonistically to BRCA1 (BRCA1 DNA repair associated), suppressing end resection and homologous recombination repair (HRR) and promoting non-homologous end joining (NHEJ), contributing to DSBR pathway choice. RIF1 is an important element of intra-S-checkpoints to recover damaged replication fork with the involvement of HRR. High-resolution microscopic studies show that RIF1 cooperates with TP53BP1 to preserve 3D structure and epigenetic markers of genomic loci disrupted by DSBs. Apart from TP53BP1, RIF1 interact with many other proteins, including proteins involved in DNA damage response, cell cycle regulation, and chromatin remodeling. As impaired RT, DSBR and fork reactivation are associated with genomic instability, a hallmark of malignant transformation, RIF1 has a diagnostic, prognostic, and therapeutic potential in cancer. Further studies may reveal other aspects of common regulation of RT, DSBR, and fork reactivation by RIF1.

The 3'-flap endonuclease XPF-ERCC1 promotes alternative end joining and chromosomal translocation during B cell class switching.

Robust alternative end joining (A-EJ) in classical non-homologous end joining (c-NHEJ)-deficient murine cells features double-strand break (DSB) end resection and microhomology (MH) usage and promotes chromosomal translocation. The activities responsible for removing 3' single-strand overhangs following resection and MH annealing in A-EJ remain unclear. We show that, during class switch recombination (CSR) in mature mouse B cells, the structure-specific endonuclease complex XPF-ERCC1SLX4, although not required for normal CSR, represents a nucleotide-excision-repair-independent 3' flap removal activity for A-EJ-mediated CSR. B cells deficient in DNA ligase 4 and XPF-ERCC1 exhibit further impaired class switching, reducing joining to the resected S region DSBs without altering the MH pattern in S-S junctions. In ERCC1-deficient A-EJ cells, 3' single-stranded DNA (ssDNA) flaps that are generated predominantly in S/G2 phase of the cell cycle are susceptible to nuclease resolution. Moreover, ERCC1 promotes c-myc-IgH translocation in Lig4-/- cells. Our study reveals an important role of the flap endonuclease XPF-ERCC1 in A-EJ and oncogenic translocation in mouse B cells.

Autophosphorylation and Self-Activation of DNA-Dependent Protein Kinase.

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related kinase family, phosphorylates serine and threonine residues of substrate proteins in the presence of the Ku complex and double-stranded DNA. Although it has been established that DNA-PKcs is involved in non-homologous end-joining, a DNA double-strand break repair pathway, the mechanisms underlying DNA-PKcs activation are not fully understood. Nevertheless, the findings of numerous in vitro and in vivo studies have indicated that DNA-PKcs contains two autophosphorylation clusters, PQR and ABCDE, as well as several autophosphorylation sites and conformational changes associated with autophosphorylation of DNA-PKcs are important for self-activation. Consistent with these features, an analysis of transgenic mice has shown that the phenotypes of DNA-PKcs autophosphorylation mutations are significantly different from those of DNA-PKcs kinase-dead mutations, thereby indicating the importance of DNA-PKcs autophosphorylation in differentiation and development. Furthermore, there has been notable progress in the high-resolution analysis of the conformation of DNA-PKcs, which has enabled us to gain a visual insight into the steps leading to DNA-PKcs activation. This review summarizes the current progress in the activation of DNA-PKcs, focusing in particular on autophosphorylation of this kinase.

Regulatory and Functional Involvement of Long Non-Coding RNAs in DNA Double-Strand Break Repair Mechanisms.

Protection of genome integrity is vital for all living organisms, particularly when DNA double-strand breaks (DSBs) occur. Eukaryotes have developed two main pathways, namely Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR), to repair DSBs. While most of the current research is focused on the role of key protein players in the functional regulation of DSB repair pathways, accumulating evidence has uncovered a novel class of regulating factors termed non-coding RNAs. Non-coding RNAs have been found to hold a pivotal role in the activation of DSB repair mechanisms, thereby safeguarding genomic stability. In particular, long non-coding RNAs (lncRNAs) have begun to emerge as new players with vast therapeutic potential. This review summarizes important advances in the field of lncRNAs, including characterization of recently identified lncRNAs, and their implication in DSB repair pathways in the context of tumorigenesis.

Nonhomologous end joining: new accessory factors fine tune the machinery.

Nonhomologous DNA end joining (NHEJ) is one of the major DNA double-strand break (DSB) repair pathways in eukaryotes. The well-known critical proteins involved in NHEJ include Ku70/80, DNA-PKcs, Artemis, DNA pol λ/µ, DNA ligase IV-XRCC4, and XLF. Recent studies have added a number of new proteins to the NHEJ repertoire namely paralog of XRCC4 and XLF (PAXX), modulator of retroviral infection (MRI)/ cell cycle regulator of NHEJ (CYREN), transactivation response DNA-binding protein (TARDBP) of 43 kDa (TDP-43), intermediate filament family orphan (IFFO1), ERCC excision repair 6 like 2 (ERCC6L2), and RNase H2. PAXX acts as a stabilizing factor for the main NHEJ components. MRI/CYREN seems to play a dual role stimulating NHEJ in the G1 phase of the cell cycle, while inhibiting the pathway in the S and G2 phases. TDP-43 can recruit the ligase IV-XRCC4 complex to the DSB sites and stimulate ligation in neuronal cells. RNase H2 excises out the ribonucleotides inserted during repair by DNA polymerase µ/TdT. This review provides a brief glimpse into how these new partners were discovered and their contribution to the mechanism and regulation of NHEJ.

Repair of DNA Double-Strand Breaks by the Nonhomologous End Joining Pathway.

DNA double-strand breaks pose a serious threat to genome stability. In vertebrates, these breaks are predominantly repaired by nonhomologous end joining (NHEJ), which pairs DNA ends in a multiprotein synaptic complex to promote their direct ligation. NHEJ is a highly versatile pathway that uses an array of processing enzymes to modify damaged DNA ends and enable their ligation. The mechanisms of end synapsis and end processing have important implications for genome stability. Rapid and stable synapsis is necessary to limit chromosome translocations that result from the mispairing of DNA ends. Furthermore, end processing must be tightly regulated to minimize mutations at the break site. Here, we review our current mechanistic understanding of vertebrate NHEJ, with a particular focus on end synapsis and processing.

SUMOylation mediates CtIP's functions in DNA end resection and replication fork protection.

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP's function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP's carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.

Role of deubiquitinating enzymes in DNA double-strand break repair.

DNA is the hereditary material in humans and almost all other organisms. It is essential for maintaining accurate transmission of genetic information. In the life cycle, DNA replication, cell division, or genome damage, including that caused by endogenous and exogenous agents, may cause DNA aberrations. Of all forms of DNA damage, DNA double-strand breaks (DSBs) are the most serious. If the repair function is defective, DNA damage may cause gene mutation, genome instability, and cell chromosome loss, which in turn can even lead to tumorigenesis. DNA damage can be repaired through multiple mechanisms. Homologous recombination (HR) and non-homologous end joining (NHEJ) are the two main repair mechanisms for DNA DSBs. Increasing amounts of evidence reveal that protein modifications play an essential role in DNA damage repair. Protein deubiquitination is a vital post-translational modification which removes ubiquitin molecules or polyubiquitinated chains from substrates in order to reverse the ubiquitination reaction. This review discusses the role of deubiquitinating enzymes (DUBs) in repairing DNA DSBs. Exploring the molecular mechanisms of DUB regulation in DSB repair will provide new insights to combat human diseases and develop novel therapeutic approaches.

The end protection problem-an unexpected twist in the tail.

In this perspective, we introduce shelterin and the mechanisms of ATM activation and NHEJ at telomeres, before discussing the following questions: How are t-loops proposed to protect chromosome ends and what is the evidence for this model? Can other models explain how TRF2 mediates end protection? Could t-loops be pathological structures? How is end protection achieved in pluripotent cells? What do the insights into telomere end protection in pluripotent cells mean for the t-loop model of end protection? Why might different cell states have evolved different mechanisms of end protection? Finally, we offer support for an updated t-loop model of end protection, suggesting that the data is supportive of a critical role for t-loops in protecting chromosome ends from NHEJ and ATM activation, but that other mechanisms are involved. Finally, we propose that t-loops are likely dynamic, rather than static, structures.

Plant Organellar DNA Polymerases Evolved Multifunctionality through the Acquisition of Novel Amino Acid Insertions.

The majority of DNA polymerases (DNAPs) are specialized enzymes with specific roles in DNA replication, translesion DNA synthesis (TLS), or DNA repair. The enzymatic characteristics to perform accurate DNA replication are in apparent contradiction with TLS or DNA repair abilities. For instance, replicative DNAPs incorporate nucleotides with high fidelity and processivity, whereas TLS DNAPs are low-fidelity polymerases with distributive nucleotide incorporation. Plant organelles (mitochondria and chloroplast) are replicated by family-A DNA polymerases that are both replicative and TLS DNAPs. Furthermore, plant organellar DNA polymerases from the plant model Arabidopsis thaliana (AtPOLIs) execute repair of double-stranded breaks by microhomology-mediated end-joining and perform Base Excision Repair (BER) using lyase and strand-displacement activities. AtPOLIs harbor three unique insertions in their polymerization domain that are associated with TLS, microhomology-mediated end-joining (MMEJ), strand-displacement, and lyase activities. We postulate that AtPOLIs are able to execute those different functions through the acquisition of these novel amino acid insertions, making them multifunctional enzymes able to participate in DNA replication and DNA repair.

Deletion of yeast TPK1 reduces the efficiency of non-homologous end joining DNA repair.

Non-homologous end joining (NHEJ) is a highly conserved mechanism of DNA double-stranded break (DSB) repair. Here we utilize a computational protein-protein interaction method to identify human PRKACB as a potential candidate interacting with NHEJ proteins. We show that the deletion of its yeast homolog, TPK1 that codes for the protein kinase A catalytic subunit reduces the efficiency of NHEJ repair of breaks with overhangs and blunt ends in plasmid-based repair assays. Additionally, tpk1Δ mutants showed defects in the repair of chromosomal breaks induced by HO-site specific endonuclease. Our double deletion mutant analyses suggest that TPK1 and YKU80, a key player in NHEJ could function in parallel pathways. Altogether, here we report a novel involvement for TPK1 in NHEJ.

The molecular basis and disease relevance of non-homologous DNA end joining.

Non-homologous DNA end joining (NHEJ) is the predominant repair mechanism of any type of DNA double-strand break (DSB) during most of the cell cycle and is essential for the development of antigen receptors. Defects in NHEJ result in sensitivity to ionizing radiation and loss of lymphocytes. The most critical step of NHEJ is synapsis, or the juxtaposition of the two DNA ends of a DSB, because all subsequent steps rely on it. Recent findings show that, like the end processing step, synapsis can be achieved through several mechanisms. In this Review, we first discuss repair pathway choice between NHEJ and other DSB repair pathways. We then integrate recent insights into the mechanisms of NHEJ synapsis with updates on other steps of NHEJ, such as DNA end processing and ligation. Finally, we discuss NHEJ-related human diseases, including inherited disorders and neoplasia, which arise from rare failures at different NHEJ steps.

TRIM29 is required for efficient recruitment of 53BP1 in response to DNA double-strand breaks in vertebrate cells.

Tripartite motif-containing protein 29 (TRIM29) is involved in DNA double-strand break (DSB) repair. However, the specific roles of TRIM29 in DNA repair are not clearly understood. To investigate the involvement of TRIM29 in DNA DSB repair, we disrupted TRIM29 in DT40 cells by gene targeting with homologous recombination (HR). The roles of TRIM29 were investigated by clonogenic survival assays and immunofluorescence analyses. TRIM29 triallelic knockout (TRIM29-/-/-/+) cells were sensitive to etoposide, but resistant to camptothecin. Foci formation assays to assess DNA repair activities showed that the dissociation of etoposide-induced phosphorylated H2A histone family member X (É£-H2AX) foci was retained in TRIM29-/-/-/+ cells, and the formation of etoposide-induced tumor suppressor p53-binding protein 1 (53BP1) foci in TRIM29-/-/-/+ cells was slower compared with wild-type (WT) cells. Interestingly, the kinetics of camptothecin-induced RAD51 foci formation of TRIM29-/-/-/+ cells was higher than that of WT cells. These results indicate that TRIM29 is required for efficient recruitment of 53BP1 to facilitate the nonhomologous end-joining (NHEJ) pathway and thereby suppress the HR pathway in response to DNA DSBs. TRIM29 regulates the choice of DNA DSB repair pathway by facilitating 53BP1 accumulation to promote NHEJ and may have potential for development into a therapeutic target to sensitize refractory cancers or as biomarker of personalized therapies.

Impaired NHEJ repair in amyotrophic lateral sclerosis is associated with TDP-43 mutations.

BACKGROUND: Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response. METHODS: We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants (A315T, Q331K), in which DNA damage was induced by etoposide or H2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage. RESULTS: We demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm. CONCLUSIONS: This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS.

ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks.

Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos.

Introducing useful traits into livestock breeding programs through gene knock-ins has proven challenging. Typically, targeted insertions have been performed in cell lines, followed by somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce genome editing reagents and a homologous recombination (HR) donor template into embryos to trigger homology directed repair (HDR). However, the HR pathway is primarily restricted to actively dividing cells (S/G2-phase) and its efficiency for the introduction of large DNA sequences in zygotes is low. The homology-mediated end joining (HMEJ) approach has been shown to improve knock-in efficiency in non-dividing cells and to harness HDR after direct injection of embryos. The knock-in efficiency for a 1.8 kb gene was contrasted when combining microinjection of a gRNA/Cas9 ribonucleoprotein complex with a traditional HR donor template or an HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach will facilitate the one-step introduction of gene constructs at a specific location of the bovine genome and contribute to the next generation of elite cattle.

BCAS2, a protein enriched in advanced prostate cancer, interacts with NBS1 to enhance DNA double-strand break repair.

BACKGROUND: Breast cancer amplified sequence 2 (BCAS2) plays crucial roles in pre-mRNA splicing and androgen receptor transcription. Previous studies suggested that BCAS2 is involved in double-strand breaks (DSB); therefore, we aimed to characterise its mechanism and role in prostate cancer (PCa). METHODS: Western blotting and immunofluorescence microscopy were used to assay the roles of BCAS2 in the DSBs of PCa cells and apoptosis in Drosophila, respectively. The effect of BCAS2 dosage on non-homologous end joining (NHEJ) and homologous recombination (HR) were assayed by precise end-joining assay and flow cytometry, respectively. Glutathione-S-transferase pulldown and co-immunoprecipitation assays were used to determine whether and how BCAS2 interacts with NBS1. The expression of BCAS2 and other proteins in human PCa was determined by immunohistochemistry. RESULTS: BCAS2 helped repair radiation-induced DSBs efficiently in both human PCa cells and Drosophila. BCAS2 enhanced both NHEJ and HR, possibly by interacting with NBS1, which involved the BCAS2 N-terminus as well as both the NBS1 N- and C-termini. The overexpression of BCAS2 was significantly associated with higher Gleason and pathology grades and shorter survival in patients with PCa. CONCLUSION: BCAS2 promotes two DSB repair pathways by interacting with NBS1, and it may affect PCa progression.

Methods Favoring Homology-Directed Repair Choice in Response to CRISPR/Cas9 Induced-Double Strand Breaks.

Precise gene editing is-or will soon be-in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs-nonhomologous end joining (NHEJ) and homology-directed repair (HDR)-and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.

XRN2 interactome reveals its synthetic lethal relationship with PARP1 inhibition.

Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5'-3'-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.