Double-strand Breaks In Mammalian Cells Research Articles

Repair Kinetics of DNA Double-Strand Breaks in Mammalian Cells under Photon Irradiation

Repair Kinetics of DNA Double-Strand Breaks in Mammalian Cells under Photon Irradiation

Identification and characterization of novel SCR7-based small-molecule inhibitor of DNA end-joining, SCR130 and its relevance in cancer therapeutics.

Targeting DNA repair with small-molecule inhibitors is an attractive strategy for cancer therapy. Majority of DNA double-strand breaks in mammalian cells are repaired through nonhomologous end-joining (NHEJ). It has been shown that small-molecule inhibitors of NHEJ can block efficient repair inside cancer cells, leading to cell death. Previously, we have reported that SCR7, an inhibitor of NHEJ can induce tumor regression in mice. Later studies have shown that different forms of SCR7 can inhibit DNA end-joining in Ligase IV-dependent manner. Recently, we have derivatized SCR7 by introducing spiro ring into core structure. Here, we report the identification of a novel inhibitor of NHEJ, named SCR130 with 20-fold higher efficacy in inducing cytotoxicity in cancer cell lines. SCR130 inhibited DNA end-joining catalyzed by rat tissue extract. Specificity analysis revealed that while SCR130 was specific to Ligase IV, it showed minimal or no effect on Ligase III and Ligase I mediated joining. Importantly, SCR130 exhibited the least cytotoxicity in Ligase IV-null cell line as compared with wild type, confirming Ligase IV-specificity. Furthermore, we demonstrate that SCR130 can potentiate the effect of radiation in cancer cells when used in combination with γ-radiation. Various cellular assays in conjunction with Western blot analysis revealed that treatment with SCR130 led to loss of mitochondrial membrane potential leading to cell death by activating both intrinsic and extrinsic pathways of apoptosis. Thus, we describe a novel inhibitor of NHEJ with higher efficacy and may have the potential to be developed as cancer therapeutic.

Detection of DNA Damage-Induced DSBs by the Contour-Clamped Homogeneous Electric Field (CHEF) System in Mammalian Cells.

Double-strand breaks (DSBs) and their repair mechanisms are essential for normal cell life. However, quantitative analysis of DSBs on mammalian whole chromosomes remains difficult. The method described here enables the quantitative detection of mammalian chromosomal DSBs by pulsed-field gel electrophoresis (PFGE) using a contour-clamped homogeneous electric field (CHEF). We illustrate this method by measuring DNA damage-induced DSBs in mammalian cells. The electrophoresis conditions presented here enabled the visualization of fragmented DNA (several mega-base pairs down to 500kbp) as a single band. Using this protocol, about 10-45 samples can be analyzed on a single gel, depending on the direction of electrophoresis.

Optogenetics and CRISPR: A New Relationship Built to Last.

Since the breakthrough discoveries that CRISPR-Cas9 nucleases can be easily programmed and employed to induce targeted double-strand breaks in mammalian cells, the gene editing field has grown exponentially. Today, CRISPR technologies based on engineered class II CRISPR effectors facilitate targeted modification of genes and RNA transcripts. Moreover, catalytically impaired CRISPR-Cas variants can be employed as programmable DNA binding domains and used to recruit effector proteins, such as transcriptional regulators, epigenetic modifiers or base-modifying enzymes, to selected genomic loci. The juxtaposition of CRISPR and optogenetics enables spatiotemporally confined and highly dynamic genome perturbations in living cells and animals and holds unprecedented potential for biology and biomedicine.Here, we provide an overview of the state-of-the-art methods for light-control of CRISPR effectors. We will detail the plethora of exciting applications enabled by these systems, including spatially confined genome editing, timed activation of endogenous genes, as well as remote control of chromatin-chromatin interactions. Finally, we will discuss limitations of current optogenetic CRISPR tools and point out routes for future innovation in this emerging field.

Homologous recombination preferentially repairs heat-induced DNA double-strand breaks in mammalian cells

Purpose: Heat shock induces DNA double-strand breaks (DSBs), but the precise mechanism of repairing heat-induced damage is unclear. Here, we investigated the DNA repair pathways involved in cell death induced by heat shock.Materials and methods: B02, a specific inhibitor of human RAD51 (homologous recombination; HR), and NU7026, a specific inhibitor of DNA-PK (non-homologous end-joining; NHEJ), were used for survival assays of human cancer cell lines with different p53-gene status. Mouse embryonic fibroblasts (MEFs) lacking Lig4 (NHEJ) and/or Rad54 (HR) were used for survival assays and a phosphorylated histone H2AX at Ser139 (γH2AX) assay. MEFs lacking Rad51d (HR) were used for survival assays. SPD8 cells were used to measure HR frequency after heat shock.Results: Human cancer cells were more sensitive to heat shock in the presence of B02 despite their p53-gene status, and the effect of B02 on heat sensitivity was specific to the G2 phase. Rad54-deficient MEFs were sensitive to heat shock and showed prolonged γH2AX signals following heat shock. Rad51d-deficient MEFs were also sensitive to heat shock. Moreover, heat shock-stimulated cells had increased HR.Conclusions: The HR pathway plays an important role in the survival of mammalian cells against death induced by heat shock via the repair of heat-induced DNA DSBs.

Convergent and Modular Synthesis of Candidate Precolibactins. Structural Revision of Precolibactin A.

The colibactins are hybrid polyketide-nonribosomal peptide natural products produced by certain strains of commensal and extraintestinal pathogenic Escherichia coli. The metabolites are encoded by the clb gene cluster as prodrugs termed precolibactins. clb(+) E. coli induce DNA double-strand breaks in mammalian cells in vitro and in vivo and are found in 55-67% of colorectal cancer patients, suggesting that mature colibactins could initiate tumorigenesis. However, elucidation of their structures has been an arduous task as the metabolites are obtained in vanishingly small quantities (μg/L) from bacterial cultures and are believed to be unstable. Herein we describe a flexible and convergent synthetic route to prepare advanced precolibactins and derivatives. The synthesis proceeds by late-stage union of two complex precursors (e.g., 28 + 17 → 29a, 90%) followed by a base-induced double dehydrative cascade reaction to form two rings of the targets (e.g., 29a → 30a, 79%). The sequence has provided quantities of advanced candidate precolibactins that exceed those obtained by fermentation, and is envisioned to be readily scaled. These studies have guided a structural revision of the predicted metabolite precolibactin A (from 5a or 5b to 7) and have confirmed the structures of the isolated metabolites precolibactins B (3) and C (6). Synthetic precolibactin C (6) was converted to N-myristoyl-d-asparagine and its corresponding colibactin by colibactin peptidase ClbP. The synthetic strategy outlined herein will facilitate mechanism of action and structure-function studies of these fascinating metabolites, and is envisioned to accommodate the synthesis of additional (pre)colibactins as they are isolated.

Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

Homologous recombination and human health: the roles of BRCA1, BRCA2, and associated proteins.

Homologous recombination (HR) is a major pathway for the repair of DNA double-strand breaks in mammalian cells, the defining step of which is homologous strand exchange directed by the RAD51 protein. The physiological importance of HR is underscored by the observation of genomic instability in HR-deficient cells and, importantly, the association of cancer predisposition and developmental defects with mutations in HR genes. The tumor suppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutated in familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51 paralogs, have also been identified as tumor suppressors. This review summarizes recent findings on BRCA1, BRCA2, and associated proteins involved in human disease with an emphasis on their molecular roles and interactions.

Effect of Ku70 expression on radiosensitivity in renal carcinoma 786-O cells.

BackgroundRadiotherapy plays an important role in cancer therapy. However, the radioresistance of some human cancers, particularly renal carcinoma, often results in radiotherapy failure. The Ku protein is essential for the repair of a majority of DNA double-strand breaks in mammalian cells, but effect of Ku70 expression on radiosensitivity in renal carcinoma is unclear. Here, we investigate the impact of Ku70 on radiosensitivity in renal carcinoma cells through regulating the expression of Ku70.MethodsThe stable overexpression of Ku70 or suppression of Ku70 in renal carcinoma cell line (786-O) was generated by retrovirus-mediated Ku70 cDNA or shRNA targeting Ku70. Ku70 expression was determined by RT-PCR and Western blot analysis, the apoptosis of the stable cells was assayed with flow cytometry and TUNEL assay and the effect of radiation on the livability of stable cells was assessed by MTT assay.ResultsUp-regulation of Ku70 expression of 786-O cells could inhibit cell apoptosis and reduce susceptibility to radiation. On the contrary, 786-O cells with suppression of Ku70 expression could induce cell apoptosis and significantly enhance the sensitivity to radiation.ConclusionsThese findings indicated that Ku70 might play an important role in radioresistance of renal carcinoma, and inhibition of Ku70 can increase the radiosensitivity of 786-O cells by enhancing apoptosis, suggesting down-regulation of Ku70 expression combined with radiotherapy will be a potential strategy for renal cell carcinoma therapy.

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells.

Microhomology-mediated end joining (MMEJ) is a major pathway for Ku-independent alternative nonhomologous end joining, which contributes to chromosomal translocations and telomere fusions, but the underlying mechanism of MMEJ in mammalian cells is not well understood. In this study, we demonstrated that, distinct from Ku-dependent classical nonhomologous end joining, MMEJ--even with very limited end resection--requires cyclin-dependent kinase activities and increases significantly when cells enter S phase. We also showed that MMEJ shares the initial end resection step with homologous recombination (HR) by requiring meiotic recombination 11 homolog A (Mre11) nuclease activity, which is needed for subsequent recruitment of Bloom syndrome protein (BLM) and exonuclease 1 (Exo1) to DNA double-strand breaks (DSBs) to promote extended end resection and HR. MMEJ does not require S139-phosphorylated histone H2AX (γ-H2AX), suggesting that initial end resection likely occurs at DSB ends. Using a MMEJ and HR competition repair substrate, we demonstrated that MMEJ with short end resection is used in mammalian cells at the level of 10-20% of HR when both HR and nonhomologous end joining are available. Furthermore, MMEJ is used to repair DSBs generated at collapsed replication forks. These studies suggest that MMEJ not only is a backup repair pathway in mammalian cells, but also has important physiological roles in repairing DSBs to maintain cell viability, especially under genomic stress.

SNF2 family ATPase LSH promotes phosphorylation of H2AX and efficient repair of DNA double-strand breaks in mammalian cells

LSH, a protein related to the SNF2 family of chromatin-remodelling ATPases, is essential for the correct establishment of DNA methylation levels and patterns in plants and mammalian cells. However, some of the phenotypes resulting from LSH deficiency cannot be explained easily by defects in DNA methylation. Here we show that LSH-deficient mouse and human fibroblasts show reduced viability after exposure to ionizing radiation and repair DNA double-strand breaks less efficiently than wild-type cells. A more detailed characterisation of this phenotype revealed that, in the absence of LSH, the histone variant H2AX is not efficiently phosphorylated in response to DNA damage. This results in impaired recruitment of MDC1 and 53BP1 proteins to DNA double-strand breaks and compromises phosphorylation of checkpoint kinase CHK2. Furthermore, we demonstrate that the ability of LSH to hydrolyse ATP is necessary for efficient phosphorylation of H2AX at DNA double-strand breaks and successful repair of DNA damage. Taken together, our data reveal a previously unsuspected role of LSH ATPase in the maintenance of genome stability in mammalian somatic cells, which is independent of its function in de novo DNA methylation during development.

Chronic exposure to low-dose of X-rays induces DNA double-strand breaks in mammalian cells

The purpose of present study is to evaluate the induction of clastogenic effect after chronic exposure to low dose of ionising radiation in both CHO-K1 cells and in their DNA Double-Strand Breaks (DSBs)-repair deficient mutant xrs-5. Cells were cultured for 14 dilution cycles and grown to confluency. Radiation treatment was performed once per cycle with 10 mGy X-rays to carry out the structural chromosome aberration test. The results showed a significant increase in the frequency of achromatic lesions ( p < 0.001) in CHO-K1 cells. In addition, xrs-5 cells showed an increase of chromatid and chromosome-type aberrations ( p < 0.001). Sequential exposure produced a level of DNA damage that is amplified through successive cycles triggering the efficient repair of DSBs. The increased damage in xrs-5 cells provides evidence that the non-homologous end-joining repair of DNA DSBs is involved after low dose X-ray exposure.

Radiation induced DNA DSBs: Contribution from stalled replication forks?

When cells are exposed to radiation serious lesions are introduced into the DNA including double strand breaks (DSBs), single strand breaks (SSBs), base modifications and clustered damage sites (a specific feature of ionizing radiation induced DNA damage). Radiation induced DNA damage has the potential to initiate events that can lead ultimately to mutations and the onset of cancer and therefore understanding the cellular responses to DNA lesions is of particular importance. Using γH2AX as a marker for DSB formation and RAD51 as a marker of homologous recombination (HR) which is recruited in the processing of frank DSBs or DSBs arising from stalled replication forks, we have investigated the contribution of SSBs and non-DSB DNA damage to the induction of DSBs in mammalian cells by ionizing radiation during the cell cycle. V79-4 cells and human HF19 fibroblast cells have been either irradiated with 0–20 Gy of γ radiation or, for comparison, treated with a low concentration of hydrogen peroxide, which is known to induce SSBs but not DSBs. Inhibition of the repair of oxidative DNA lesions by poly(ADP ribose) polymerase (PARP) inhibitor leads to an increase in radiation induced γH2AX and RAD51 foci which we propose is due to these lesions colliding with replication forks forming replication induced DSBs. It was confirmed that DSBs are not induced in G 1 phase cells by treatment with hydrogen peroxide but treatment does lead to DSB induction, specifically in S phase cells. We therefore suggest that radiation induced SSBs and non-DSB DNA damage contribute to the formation of replication induced DSBs, detected as RAD51 foci.

Cyclin A overexpression induces chromosomal double-strand breaks in mammalian cells

Cyclin A is a major regulator in vertebrate cell cycle, associated with cyclin-dependent kinase (Cdk), and involved in S-phase progression and entry into mitosis. It has been known that cyclin A overexpression not only causes premature S-phase entry but also induces prolongation of S phase. Here we show that ectopic expression of cyclin A leads to extensive γ−H2AX focus formation, which is indicative of DNA double-strand breaks. Likewise, cyclin E, but not cyclin B1 and cyclin D1, also induced the γ−H2AX focus formation, suggesting that these DNA lesions may be induced via aberrant DNA replication process. Moreover, the γ−H2AX focus formation was suppressed by co-expressing p21Cip1/Waf1 or dominant-negative Cdk2 mutant, suggesting that aberrant cyclin A-Cdk2 activation induces the chromosomal double-strand breaks.

The Complexity of Double-Strand Break Ends is a Factor in the Repair Pathway Choice

The repair of double-strand breaks in mammalian cells is carried out by two pathways: homologous recombination and nonhomologous end joining. The factors that regulate the mechanism through which a specific repair pathway is activated are still not clearly defined. To study whether the complexity of the double-strand break ends is a factor that determines the choice of the repair pathway, we examined the involvement of homologous recombination by the formation of Rad51 foci in human HeLa cells treated with bleomycin and ionizing radiation. The quantity of double-strand breaks was determined by gel electrophoresis and the formation of gamma-H2AX foci. Two hours after treatment with low doses of the agents that induced similar quantities of double-strand breaks that could be repaired effectively by the cells, Rad51 foci were observed only in the irradiated cells. Rad51 foci appeared in bleomycin-treated cells after prolonged exposure to the drug when the cells were arrested in the G2 phase of the cell cycle. Since bleomycin produces double-strand breaks that are less complex than the breaks induced by ionizing radiation, these results indicate that the complexity of the break ends is a factor in the choice of repair pathway and that homologous recombination is recruited in the repair of breaks with more complex multiply damaged ends during the late S and G2 phases of the cell cycle.

Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining.

DNA DSBs (double-strand breaks) are considered the most cytotoxic type of DNA lesion. They can be introduced by external sources such as IR (ionizing radiation), by chemotherapeutic drugs such as topoisomerase poisons and by normal biological processes such as V(D)J recombination. If left unrepaired, DSBs can cause cell death. If misrepaired, DSBs may lead to chromosomal translocations and genomic instability. One of the major pathways for the repair of IR-induced DSBs in mammalian cells is NHEJ (non-homologous end-joining). The main proteins required for NHEJ in mammalian cells are the Ku heterodimer (Ku70/80 heterodimer), DNA-PKcs [the catalytic subunit of DNA-PK (DNA-dependent protein kinase)], Artemis, XRCC4 (X-ray-complementing Chinese hamster gene 4), DNA ligase IV and XLF (XRCC4-like factor; also called Cernunnos). Additional proteins, including DNA polymerases mu and lambda, PNK (polynucleotide kinase) and WRN (Werner's Syndrome helicase), may also play a role. In the present review, we will discuss our current understanding of the mechanism of NHEJ in mammalian cells and discuss the roles of DNA-PKcs and DNA-PK-mediated phosphorylation in NHEJ.

Influence of Chromatin Structure on Induction of Double-Strand Breaks in Mammalian Cells Irradiated with DNA-Incorporated125I

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter (125)I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with (125)IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at -70 degrees C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with gamma rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated (125)I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by (125)IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.

Positional stability of single double-strand breaks in mammalian cells

Formation of cancerous translocations requires the illegitimate joining of chromosomes containing double-strand breaks (DSBs). It is unknown how broken chromosome ends find their translocation partners within the cell nucleus. Here, we have visualized and quantitatively analysed the dynamics of single DSBs in living mammalian cells. We demonstrate that broken ends are positionally stable and unable to roam the cell nucleus. Immobilization of broken chromosome ends requires the DNA-end binding protein Ku80, but is independent of DNA repair factors, H2AX, the MRN complex and the cohesion complex. DSBs preferentially undergo translocations with neighbouring chromosomes and loss of local positional constraint correlates with elevated genomic instability. These results support a contact-first model in which chromosome translocations predominantly form among spatially proximal DSBs.

Involvement of DNA polymerase mu in the repair of a specific subset of DNA double-strand breaks in mammalian cells.

The repair of DNA double-strand breaks (DSB) requires processing of the broken ends to complete the ligation process. Recently, it has been shown that DNA polymerase μ (polμ) and DNA polymerase λ (polλ) are both involved in such processing during non-homologous end joining in vitro. However, no phenotype was observed in animal models defective for either polμ and/or polλ. Such observations could result from a functional redundancy shared by the X family of DNA polymerases. To avoid such redundancy and to clarify the role of polμ in the end joining process, we generated cells over-expressing the wild type as well as an inactive form of polμ (polμD). We observed that cell sensitivity to ionizing radiation (IR) was increased when either polμ or polμD was over-expressed. However, the genetic instability in response to IR increased only in cells expressing polμD. Moreover, analysis of intrachromosomal repair of the I-SceI-induced DNA DSB, did not reveal any effect of either polμ or polμD expression on the efficiency of ligation of both cohesive and partially complementary ends. Finally, the sequences of the repaired ends were specifically affected when polμ or polμD was over-expressed, supporting the hypothesis that polμ could be involved in the repair of a DSB subset when resolution of junctions requires some gap filling.

Extensive ssDNA end formation at DNA double-strand breaks in non-homologous end-joining deficient cells during the S phase

BackgroundEfficient and correct repair of DNA damage, especially DNA double-strand breaks, is critical for cellular survival. Defects in the DNA repair may lead to cell death or genomic instability and development of cancer. Non-homologous end-joining (NHEJ) is the major repair pathway for DNA double-strand breaks in mammalian cells. The ability of other repair pathways, such as homologous recombination, to compensate for loss of NHEJ and the ways in which contributions of different pathways are regulated are far from fully understood.ResultsIn this report we demonstrate that long single-stranded DNA (ssDNA) ends are formed at radiation-induced DNA double-strand breaks in NHEJ deficient cells. At repair times ≥ 1 h, processing of unrejoined DNA double-strand breaks generated extensive ssDNA at the DNA ends in cells lacking the NHEJ protein complexes DNA-dependent protein kinase (DNA-PK) or DNA Ligase IV/XRCC4. The ssDNA formation was cell cycle dependent, since no ssDNA ends were observed in G1-synchronized NHEJ deficient cells. Furthermore, in wild type cells irradiated in the presence of DNA-PKcs (catalytic subunit of DNA-PK) inhibitors, or in DNA-PKcs deficient cells complemented with DNA-PKcs mutated in six autophosphorylation sites (ABCDE), no ssDNA was formed. The ssDNA generation also greatly influences DNA double-strand break quantification by pulsed-field gel electrophoresis, resulting in overestimation of the DNA double-strand break repair capability in NHEJ deficient cells when standard protocols for preparing naked DNA (i. e., lysis at 50°C) are used.ConclusionWe provide evidence that DNA Ligase IV/XRCC4 recruitment by DNA-PK to DNA double-strand breaks prevents the formation of long ssDNA ends at double-strand breaks during the S phase, indicating that NHEJ components may downregulate an alternative repair process where ssDNA ends are required.