The dynamics of Ku70/80 and DNA-PKcs at DSBs induced by ionizing radiation is dependent on the complexity of damage

DNA double-strand breaks (DSBs) are biologically one of the most important cellular lesions and possess varying degrees of chemical complexity. The notion that the repairability of more chemically complex DSBs is inefficient led to the concept that the extent of DSB complexity underlies the severity of the biological consequences. The repair of DSBs by non-homologous end joining (NHEJ) has been extensively studied but it remains unknown whether more complex DSBs require a different sub-set of NHEJ protein for their repair compared with simple DSBs. To address this, we have induced DSBs in fluorescently tagged mammalian cells (Ku80-EGFP, DNA-PKcs-YFP or XRCC4-GFP, key proteins in NHEJ) using ultra-soft X-rays (USX) or multi-photon near infrared (NIR) laser irradiation. We have shown in real-time that simple DSBs, induced by USX or NIR microbeam irradiation, are repaired rapidly involving Ku70/80 and XRCC4/Ligase IV/XLF. In contrast, DSBs with greater chemical complexity are repaired slowly involving not only Ku70/80 and XRCC4/Ligase IV/XLF but also DNA-PKcs. Ataxia telangiectasia-mutated inhibition only retards repair of the more chemically complex DSBs which require DNA-PKcs. In summary, the repair of DSBs by NHEJ is highly regulated with pathway choice and kinetics of repair dependent on the chemical complexity of the DSB.     

Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays.     

Cellular responses to DNA double-strand breaks after low-dose γ-irradiation

DNA double-strand breaks (DSBs) are a serious threat to genome stability and cell viability. Although biological effects of low levels of radiation are not clear, the risks of low-dose radiation are of societal importance. Here, we directly monitored induction and repair of single DSBs and quantitatively analyzed the dynamics of interaction of DNA repair proteins at individual DSB sites in living cells using 53BP1 fused to yellow fluorescent protein (YFP-53BP1) as a surrogate marker. The number of DSBs formed was linear with dose from 5 mGy to 1 Gy. The DSBs induced by very low radiation doses (5 mGy) were repaired with efficiency similar to repair of DSBs induced at higher doses. The YFP-53BP1 foci are dynamic structures: 53BP1 rapidly and reversibly interacted at these DSB sites. The time frame of recruitment and affinity of 53BP1 for DSB sites were indistinguishable between low and high doses, providing mechanistic evidence for the similar DSB repair after low- and high-dose radiation. These findings have important implications for estimating the risk associated with low-dose radiation exposure on human health.     

DNA double strand break repair in mammalian cells

Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs.     

Up-regulation of HIV-1 transduction in nondividing cells by double-strand DNA break-inducing agents

Efficient HIV-1 transduction depends on a number of cellular co-factors. Cellular double-strand DNA break (DSB) repair proteins have been proposed, by ourselves and others, to be required for efficient HIV-1 transduction. Expression and/or activity of these DNA repair proteins can be induced by the introduction of DSBs into the host cell genome. HIV-1 transduction was up-regulated by treatment with DSB-inducing agents in both drug-arrested cells and differentiated neuronal cells. The presented data support the hypothesis that DSB repair proteins are involved in the early steps of the retroviral life-cycle.     

DNA damage in non-proliferating cells subjected to ionizing irradiation at high or low dose rates

Ionizing radiation damage to the genome of a non-cycling mammalian cell is analyzed using continuous time Markov chains. Immediate damage induced by the radiation is modeled as a batch Poisson arrival process of DNA double strand breaks (DSBs). Different kinds of radiation, for example gamma rays or alpha particles, have different batch probabilities. Enzymatic modulation of the immediate damage is modeled as a Markov process similar to the processes described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete exchange model, which postulates that radiation induced DSBs can subsequently either undergo enzymatically mediated repair (restitution) or can participate pairwise in chromosome exchanges, some of which make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. A complete solution of the Markov chain is known for the case that the exchange rate constant is negligible so that no irremediable chromosome lesions are produced and DSBs are the only damage to the genome. Using PDEs for generating functions, a perturbation calculation is made assuming the exchange rate constant is small compared to the repair rate constant. Some non-perturbative results applicable to very prolonged irradiation are also obtained using matrix methods: Perron-Frobenius theory, variational methods and numerical approximations of eigenvalues. Applications to experimental results on expected values, variances and statistical distributions of DNA lesions are briefly outlined.Continuous time Markov chain models are the most systematic of those current radiation damage models which treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g. ignore diffusion limitations). They contain most other homogeneous models as special cases, limiting cases or approximations. However, applying the continuous time Markov chain models to studying spatial dependence of DSB interactions, which is generally believed to be very important in some situations, presents difficulties.     

Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophila melanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.     

RSC mobilizes nucleosomes to improve accessibility of repair machinery to the damaged chromatin

Repair of DNA double-strand breaks (DSBs) protects cells and organisms, as well as their genome integrity. Since DSB repair occurs in the context of chromatin, chromatin must be modified to prevent it from inhibiting DSB repair. Evidence supports the role of histone modifications and ATP-dependent chromatin remodeling in repair and signaling of chromosome DSBs. The key questions are, then, what the nature of chromatin altered by DSBs is and how remodeling of chromatin facilitates DSB repair. Here we report a chromatin alteration caused by a single HO endonuclease-generated DSB at the Saccharomyces cerevisiae MAT locus. The break induces rapid nucleosome migration to form histone-free DNA of a few hundred base pairs immediately adjacent to the break. The DSB-induced nucleosome repositioning appears independent of end processing, since it still occurs when the 5'-to-3' degradation of the DNA end is markedly reduced. The tetracycline-controlled depletion of Sth1, the ATPase of RSC, or deletion of RSC2 severely reduces chromatin remodeling and loading of Mre11 and Yku proteins at the DSB. Depletion of Sth1 also reduces phosphorylation of H2A, processing, and joining of DSBs. We propose that RSC-mediated chromatin remodeling at the DSB prepares chromatin to allow repair machinery to access the break and is vital for efficient DSB repair.     

Stable maintenance of the Mre11-Rad50-Nbs1 complex is sufficient to restore the DNA double-strand break response in cells lacking RecQL4 helicase activity

The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund–Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.     

Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells

We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced approximately 30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 microm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions.     

New Insights into NHEJ Repair Processes in Prokaryotes

In eukaryotic cells, the repair of DNA double strand breaks (DSBs) by the non-homologous end-joining (NHEJ) pathway is critical for genome stability. Until recently it was assumed that this DSB repair pathway was restricted to the eukarya. However, a functionally homologous prokaryotic NHEJ repair apparatus has now been identified and characterised. In contrast to the complex eukaryotic system, bacterial NHEJ appears to require only two proteins, Ku and a multifunctional DNA ligase, which form a two-component repair complex at the termini of DSBs. Together, these DNA repair factors possess all of the break-recognition, end-processing and ligation activities required to facilitate the complex task of DSB repair, both in vitro and in vivo. Our recent findings lay the foundation for understanding the molecular mechanisms that co-ordinate the processing and joining of DSBs by NHEJ in bacteria and also provides a conceptual framework for delineating the end-processing reactions in eukaryotes.     

Global detection of DNA repair outcomes induced by CRISPR–Cas9

CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.     

Analysis of DNA double-strand break repair pathways in mice

During the last years significant new insights have been gained into the mechanism and biological relevance of DNA double-strand break (DSB) repair in relation to genome stability. DSBs are a highly toxic DNA lesion, because they can lead to chromosome fragmentation, loss and translocations, eventually resulting in cancer. DSBs can be induced by cellular processes such as V(D)J recombination or DNA replication. They can also be introduced by exogenous agents DNA damaging agents such as ionizing radiation or mitomycin C. During evolution several pathways have evolved for the repair of these DSBs. The most important DSB repair mechanisms in mammalian cells are nonhomologous end-joining and homologous recombination. By using an undamaged repair template, homologous recombination ensures accurate DSB repair, whereas the untemplated nonhomologous end-joining pathway does not. Although both pathways are active in mammals, the relative contribution of the two repair pathways to genome stability differs in the different cell types. Given the potential differences in repair fidelity, it is of interest to determine the relative contribution of homologous recombination and nonhomologous end-joining to DSB repair. In this review, we focus on the biological relevance of DSB repair in mammalian cells and the potential overlap between nonhomologous end-joining and homologous recombination in different tissues.     

Assessment of protein dynamics and DNA repair following generation of DNA double-strand breaks at defined genomic sites

The formation of protein aggregates (foci) at sites of DNA double-strand breaks (DSBs) is mainly studied by immunostaining and is hence limited by the low resolution of light microscopy and the availability of appropriate and selective antibodies. Here, we describe a system using enzymatic creation of site-specific DNA DSBs within the human genome combined with chromatin immunoprecipitation (ChIP) that enables molecular probing of a DSB. Following induction of the I-PpoI enzyme and generation of DSBs, cellular DNA and proteins are crosslinked and analyzed by ChIP for specific proteins at the site of the break. The system allows the direct detection of protein and chromatin dynamics at the site of the break with high resolution, as well as direct measurement of DNA repair defects in human cells. Starting with fragmented chromatin, results can be achieved in 2-3 d.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination

DNA double-strand breaks (DSBs) are highly cytotoxic lesions and pose a major threat to genome stability if not properly repaired. We and others have previously shown that a class of DSB-induced small RNAs (diRNAs) is produced from sequences around DSB sites. DiRNAs are associated with Argonaute (Ago) proteins and play an important role in DSB repair, though the mechanism through which they act remains unclear. Here, we report that the role of diRNAs in DSB repair is restricted to repair by homologous recombination (HR) and that it specifically relies on the effector protein Ago2 in mammalian cells. Interestingly, we show that Ago2 forms a complex with Rad51 and that the interaction is enhanced in cells treated with ionizing radiation. We demonstrate that Rad51 accumulation at DSB sites and HR repair depend on catalytic activity and small RNA-binding capability of Ago2. In contrast, DSB resection as well as RPA and Mre11 loading is unaffected by Ago2 or Dicer depletion, suggesting that Ago2 very likely functions directly in mediating Rad51 accumulation at DSBs. Taken together, our findings suggest that guided by diRNAs, Ago2 can promote Rad51 recruitment and/or retention at DSBs to facilitate repair by HR.     

Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair

We developed a novel system to create DNA double-strand breaks (DSBs) at defined endogenous sites in the human genome, and used this system to detect protein recruitment and loss at and around these breaks by chromatin immunoprecipitation (ChIP). The detection of human ATM protein at site-specific DSBs required functional NBS1 protein, ATM kinase activity and ATM autophosphorylation on Ser 1981. DSB formation led to the localized disruption of nucleosomes, a process that depended on both functional NBS1 and ATM. These two proteins were also required for efficient recruitment of the repair cofactor XRCC4 to DSBs, and for efficient DSB repair. These results demonstrate the functional importance of ATM kinase activity and phosphorylation in the response to DSBs, and support a model in which ordered chromatin structure changes that occur after DNA breakage depend on functional NBS1 and ATM, and facilitate DNA DSB repair.     

ERCC1-XPF endonuclease facilitates DNA double-strand break repair

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.