Loss of Dna2 fidelity results in decreased Exo1-mediated resection at DNA double-strand breaks.

A DNA double-strand break (DSB) is one of the most dangerous types of DNA damage that is repaired largely by homologous recombination or nonhomologous end-joining (NHEJ). The interplay of repair factors at the break directs which pathway is used, and a subset of these factors also function in more mutagenic alternative (alt) repair pathways. Resection is a key event in repair pathway choice and extensive resection, which is a hallmark of homologous recombination, and it is mediated by two nucleases, Exo1 and Dna2. We observed differences in resection and repair outcomes in cells harboring nuclease-dead dna2-1 compared with dna2Δ pif1-m2 that could be attributed to the level of Exo1 recovered at DSBs. Cells harboring dna2-1 showed reduced Exo1 localization, increased NHEJ, and a greater resection defect compared with cells where DNA2 was deleted. Both the resection defect and the increased rate of NHEJ in dna2-1 mutants were reversed upon deletion of KU70 or ectopic expression of Exo1. By contrast, when DNA2 was deleted, Exo1 and Ku70 recovery levels did not change; however, Nej1 increased as did the frequency of alt-end joining/microhomology-mediated end-joining repair. Our findings demonstrate that decreased Exo1 at DSBs contributed to the resection defect in cells expressing inactive Dna2 and highlight the complexity of understanding how functionally redundant factors are regulated in vivo to promote genome stability.

Limiting the DNA Double-Strand Break Resectosome for Genome Protection.

DNA double-strand break (DSB) resection, once thought to be a simple enzymatic process, is emerging as a highly complex series of coordinated activities required to maintain genome integrity. Progress in cell biology, biochemistry, and genetics has deciphered the precise resecting activities, the regulatory components, and their ability to properly channel the resected DNA to the appropriate DNA repair pathway. Herein, we review the mechanisms of regulation of DNA resection, with an emphasis on negative regulators that prevent single-strand (ss)DNA accumulation to maintain genome stability. Interest in targeting DNA resection inhibitors is emerging because their inactivation leads to poly(ADP-ribose) polymerase inhibitor (PARPi) resistance. We also present detailed regulation of DNA resection machineries, their analysis by functional assays, and their impact on disease and PARPi resistance.

Increased Gene Targeting in Hyper-Recombinogenic LymphoBlastoid Cell Lines Leaves Unchanged DSB Processing by Homologous Recombination.

In the cells of higher eukaryotes, sophisticated mechanisms have evolved to repair DNA double-strand breaks (DSBs). Classical nonhomologous end joining (c-NHEJ), homologous recombination (HR), alternative end joining (alt-EJ) and single-strand annealing (SSA) exploit distinct principles to repair DSBs throughout the cell cycle, resulting in repair outcomes of different fidelity. In addition to their functions in DSB repair, the same repair pathways determine how cells integrate foreign DNA or rearrange their genetic information. As a consequence, random integration of DNA fragments is dominant in somatic cells of higher eukaryotes and suppresses integration events at homologous genomic locations, leading to very low gene-targeting efficiencies. However, this response is not universal, and embryonic stem cells display increased targeting efficiency. Additionally, lymphoblastic chicken and human cell lines DT40 and NALM6 show up to a 1000-fold increased gene-targeting efficiency that is successfully harnessed to generate knockouts for a large number of genes. We inquired whether the increased gene-targeting efficiency of DT40 and NALM6 cells is linked to increased rates of HR-mediated DSB repair after exposure to ionizing radiation (IR). We analyzed IR-induced γ-H2AX foci as a marker for the total number of DSBs induced in a cell and RAD51 foci as a marker for the fraction of those DSBs undergoing repair by HR. We also evaluated RPA accretion on chromatin as evidence for ongoing DNA end resection, an important initial step for all pathways of DSB repair except c-NHEJ. We finally employed the DR-GFP reporter assay to evaluate DSB repair by HR in DT40 cells. Collectively, the results obtained, unexpectedly show that DT40 and NALM6 cells utilized HR for DSB repair at levels very similar to those of other somatic cells. These observations uncouple gene-targeting efficiency from HR contribution to DSB repair and suggest the function of additional mechanisms increasing gene-targeting efficiency. Indeed, our results show that analysis of the contribution of HR to DSB repair may not be used as a proxy for gene-targeting efficiency.

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.

Analysis of chromatid-break-repair detects a homologous recombination to non-homologous end-joining switch with increasing load of DNA double-strand breaks.

We recently reported that when low doses of ionizing radiation induce low numbers of DNA double-strand breaks (DSBs) in G2-phase cells, about 50 % of them are repaired by homologous recombination (HR) and the remaining by classical non-homologous end-joining (c-NHEJ). However, with increasing DSB-load, the contribution of HR drops to undetectable (at ∼10 Gy) as c-NHEJ dominates. It remains unknown whether the approximately equal shunting of DSBs between HR and c-NHEJ at low radiation doses and the predominant shunting to c-NHEJ at high doses, applies to every DSB, or whether the individual characteristics of each DSB generate processing preferences. When G2-phase cells are irradiated, only about 10 % of the induced DSBs break the chromatids. This breakage allows analysis of the processing of this specific subset of DSBs using cytogenetic methods. Notably, at low radiation doses, these DSBs are almost exclusively processed by HR, suggesting that chromatin characteristics awaiting characterization underpin chromatid breakage and determine the preferential engagement of HR. Strikingly, we also discovered that with increasing radiation dose, a pathway switch to c-NHEJ occurs in the processing of this subset of DSBs. Here, we confirm and substantially extend our initial observations using additional methodologies. Wild-type cells, as well as HR and c-NHEJ mutants, are exposed to a broad spectrum of radiation doses and their response analyzed specifically in G2 phase. Our results further consolidate the observation that at doses <2 Gy, HR is the main option in the processing of the subset of DSBs generating chromatid breaks and that a pathway switch at doses between 4-6 Gy allows the progressive engagement of c-NHEJ. PARP1 inhibition, irrespective of radiation dose, leaves chromatid break repair unaffected suggesting that the contribution of alternative end-joining is undetectable under these experimental conditions.

Establishment of a Transformation Coupled in vitro End Joining Assay to Estimate Radiosensitivity in Tumor Cells.

Here, we present a modified in vitro end-joining (EJ) assay to quantify EJ capacity, accuracy as well as pathway switch to alternative end-joining (Alt-EJ) or single strand annealing (SSA). A novel transformation assay was established to specifically measure circular repair products, which correlate with classical EJ efficiency. The EJ assay was validated using EJ-deficient mammalian cell lines (Ku80, DNA-PKcs, LigIV, or XRCC4 mutants). A pathway switch to Alt-EJ and SSA was seen exclusively in Ku-deficient cells. Circular EJ product formation correlated with cell survival and DSB repair capacity after X-irradiation. Investigation of 14 HNSCC cell lines revealed differences in the total EJ capacity but a broader variation in the amount of circular repair products. Sequencing of repair junctions in HNSCC cells demonstrated a predominance of high-fidelity EJ and an avoidance of both Alt-EJ and SSA. A significant correlation was observed between the amount of circular repair products, repair of IR-induced DSB and radiosensitivity. Collectively, these data indicate that the presented in vitro-EJ-assay can not only estimate the repair capacity of cancer cells to enable the stratification into radiosensitive or radioresistant, but can also identify repair pathway deregulation such as a switch to Alt-EJ or SSA, which enables tumor targeting.

A Paradigm Revolution or Just Better Resolution-Will Newly Emerging Superresolution Techniques Identify Chromatin Architecture as a Key Factor in Radiation-Induced DNA Damage and Repair Regulation?

DNA double-strand breaks (DSBs) have been recognized as the most serious lesions in irradiated cells. While several biochemical pathways capable of repairing these lesions have been identified, the mechanisms by which cells select a specific pathway for activation at a given DSB site remain poorly understood. Our knowledge of DSB induction and repair has increased dramatically since the discovery of ionizing radiation-induced foci (IRIFs), initiating the possibility of spatiotemporally monitoring the assembly and disassembly of repair complexes in single cells. IRIF exploration revealed that all post-irradiation processes-DSB formation, repair and misrepair-are strongly dependent on the characteristics of DSB damage and the microarchitecture of the whole affected chromatin domain in addition to the cell status. The microscale features of IRIFs, such as their morphology, mobility, spatiotemporal distribution, and persistence kinetics, have been linked to repair mechanisms. However, the influence of various biochemical and structural factors and their specific combinations on IRIF architecture remains unknown, as does the hierarchy of these factors in the decision-making process for a particular repair mechanism at each individual DSB site. New insights into the relationship between the physical properties of the incident radiation, chromatin architecture, IRIF architecture, and DSB repair mechanisms and repair efficiency are expected from recent developments in optical superresolution microscopy (nanoscopy) techniques that have shifted our ability to analyze chromatin and IRIF architectures towards the nanoscale. In the present review, we discuss this relationship, attempt to correlate still rather isolated nanoscale studies with already better-understood aspects of DSB repair at the microscale, and consider whether newly emerging "correlated multiscale structuromics" can revolutionarily enhance our knowledge in this field.

UBQLN4 promotes non-homologous end joining by repressing DNA end-resection.

Ataxia-telangiectasia-mutated (ATM) promotes homologous recombination (HR)-mediated DNA double-strand break repair. It was recently shown that the proteasomal shuttle factor UBQLN4 facilitates MRE11 degradation to repress HR. Surprisingly, the UBQLN4-MRE11 interaction is ATM-dependent, suggesting that the proximal DNA damage kinase ATM does not only initiate HR, but also limits excessive end resection.

DNA double-strand break repair pathway choice - from basic biology to clinical exploitation.

Mutations in genes encoding components of the DNA damage response (DDR) are among the most frequent aberrations in human tumors. Moreover, a large array of human syndromes is caused by mutations in genes involved in DDR pathways. Among others, homologous recombination repair (HR) of DNA double-strand breaks (DSB) is frequently affected by disabling mutations. While impaired HR is clearly promoting tumorigenesis, it is also associated with an actionable sensitivity against PARP inhibitors. PARP inhibitors have recently received FDA approval for the treatment of breast- and ovarian cancer. However, as with all molecularly targeted agents, acquired resistance limits its use. Both pharmaco-genomic approaches and the study of human genome instability syndromes have led to a profound understanding of PARP inhibitor resistance. These experiments have revealed new insights into the molecular mechanisms that drive mammalian DSB repair. Here, we review recent discoveries in the field and provide a clinical perspective.

Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin.

The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1's target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR.

Cancer risk at low doses of ionizing radiation: artificial neural networks inference from atomic bomb survivors.

Cancer risk at low doses of ionizing radiation remains poorly defined because of ambiguity in the quantitative link to doses below 0.2 Sv in atomic bomb survivors in Hiroshima and Nagasaki arising from limitations in the statistical power and information available on overall radiation dose. To deal with these difficulties, a novel nonparametric statistics based on the 'integrate-and-fire' algorithm of artificial neural networks was developed and tested in cancer databases established by the Radiation Effects Research Foundation. The analysis revealed unique features at low doses that could not be accounted for by nominal exposure dose, including (i) the presence of a threshold that varied with organ, gender and age at exposure, and (ii) a small but significant bumping increase in cancer risk at low doses in Nagasaki that probably reflects internal exposure to (239)Pu. The threshold was distinct from the canonical definition of zero effect in that it was manifested as negative excess relative risk, or suppression of background cancer rates. Such a unique tissue response at low doses of radiation exposure has been implicated in the context of the molecular basis of radiation-environment interplay in favor of recently emerging experimental evidence on DNA double-strand break repair pathway choice and its epigenetic memory by histone marking.