Repair of DNA broken ends is similar in embryonic fibroblasts with and without telomerase

Telomeres cap the ends of chromosomes, preventing end-to-end fusions and subsequent chromosome instability. Here we used a telomerase knockout model to investigate whether telomerase participates in the processes of DNA break repair by de novo synthesis of telomere repeats at broken chromosome ends (chromosome healing). Chromosome healing giving rise to new detectable telomeric signals has not been observed in embryonic fibroblasts of telomerase-proficient mice exposed to ionizing radiation. Since the synthesis of telomeric sequences to broken DNA ends would make them refractory to rejoining events, the efficiency of rejoining of broken chromosomes in cell environments with and without telomerase has also been investigated. We conclude that the efficiency of rejoining broken chromosomes is not significantly different in the two cell environments. All together, our results indicate that there is no significant involvement of telomerase in the healing of broken DNA ends by synthesizing new telomeres in mouse embryo fibroblasts after exposure to ionizing radiation.     

The non-homologous end-joining pathway of S. cerevisiae works effectively in G1-phase cells,and religates cognate ends correctly and non-randomly

DNA double-strand breaks (DSBs) are potentially lethal lesions repaired by two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). Homologous recombination preferentially reunites cognate broken ends. In contrast, non-homologous end-joining could ligate together any two ends, possibly generating dicentric or acentric fragments, leading to inviability. Here, we characterize the yeast NHEJ pathway in populations of pure G1 phase cells, where there is no possibility of repair using a homolog. We show that in G1 yeast cells, NHEJ is a highly effective repair pathway for gamma-ray induced breaks, even when many breaks are present. Pulsed-field gel analysis showed chromosome karyotypes following NHEJ repair of cells from populations with multiple breaks. The number of reciprocal translocations was surprisingly low, perhaps zero, suggesting that NHEJ preferentially re-ligates the “correct” broken ends instead of randomly-chosen ends. Although we do not know the mechanism, the preferential correct ligation is consistent with the idea that broken ends are continuously held together by protein–protein interactions or by larger scale chromatin structure.     

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (L?brich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.     

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.     

The endless tale of non-homologous end-joining

DNA double-strand breaks （DSBs） are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V（D）J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs： homologous recombination and non-homologous end-joining （NHEJ）. The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.     

DNA double-strand breaks: linking gene expression to chromosome morphology and mobility

Ionizing radiation can lead to DNA double-strand breaks (DSBs) which belong to the most dangerous forms of damage to the DNA. Cells possess elaborate repair mechanisms and react in a complex manner to the emergence of DSBs. Experiments have shown that gene expression levels in irradiated cells are changed, and thousands of radiation-responsive genes have been identified. On the other hand, recent studies have shown that gene expression is tightly connected to the three-dimensional organization of the genome. In this work, we analyzed the chromatin organization in the cell nuclei before and after exposure to ionizing radiation with an expression-dependent folding model. Our results indicate that the alteration of the chromosome organization on the scale of a complete chromosome is rather limited despite the expression level change of a large number of genes. We further modelled breaks within sub-compartments of the model chromosomes and showed that entropic changes caused by a break lead to increased mobility of the break sites and help to locate break ends further to the periphery of the sub-compartments. We conclude that the changes in the chromatin structure after irradiation are limited to local scales and demonstrate the importance of entropy for the behaviour of break ends.     

Intrachanges as part of complex chromosome-type exchange aberrations

The chromosome-type exchange aberrations induced by ionizing radiation during the G(0)/G(1) phase of the cell cycle are believed to be the result of illegitimate rejoining of chromosome breaks. From numerous studies using chromosome painting, it has emerged that even after a moderate dose of radiation, a substantial fraction of these exchanges is complex. Most of them are derived from the free interaction between the ends of three or more breaks. Other studies have demonstrated that chromosomes occupy distinct territories in the interphase nucleus. Since breaks that are in close proximity have an enhanced interaction probability, it seems likely that after ionizing radiation many of the interacting breaks will be present within one chromosome or chromosome arm. Unfortunately, the majority of these intrachanges remain undetected, even when sophisticated molecular cytogenetic detection methods (i.e. mFISH) are applied to paint all chromosome pairs in distinct colors. In the present paper, we evaluate the limitations of full-color painting for the detection of complex exchanges and the correct interpretations of break interactions.     

Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks

Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway. Cellular dNTP pools are regulated primarily by changes in ribonucleotide reductase activity. We show that an inhibitor of ribonucleotide reductase (hydroxyurea) hypersensitizes NHEJ-deficient cells, but not wild type or HR-deficient cells, to chromosome breaks introduced by ionizing radiation. Hydroxyurea additionally reduces the frequency of irradiated cells with a marker for an early step in HR, Rad51 foci, consistent with reduced initiation of HR under these conditions. Conversely, promotion of ribonucleotide reductase activity protects NHEJ-deficient cells from ionizing radiation. Importantly, promotion of ribonucleotide reductase activity also increases usage of HR in cells proficient in both NHEJ and HR at a targeted chromosome break. Activity of ribonucleotide reductase is thus an important factor in determining how mammalian cells repair broken chromosomes. This may explain in part why G1/G0 cells, which have reduced ribonucleotide reductase activity, rely more on NHEJ for DSB repair.     

Telomere dysfunction and chromosome instability

The ends of chromosomes are composed of a short repeat sequence and associated proteins that together form a cap, called a telomere, that keeps the ends from appearing as double-strand breaks (DSBs) and prevents chromosome fusion. The loss of telomeric repeat sequences or deficiencies in telomeric proteins can result in chromosome fusion and lead to chromosome instability. The similarity between chromosome rearrangements resulting from telomere loss and those found in cancer cells implicates telomere loss as an important mechanism for the chromosome instability contributing to human cancer. Telomere loss in cancer cells can occur through gradual shortening due to insufficient telomerase, the protein that maintains telomeres. However, cancer cells often have a high rate of spontaneous telomere loss despite the expression of telomerase, which has been proposed to result from a combination of oncogene-mediated replication stress and a deficiency in DSB repair in telomeric regions. Chromosome fusion in mammalian cells primarily involves nonhomologous end joining (NHEJ), which is the major form of DSB repair. Chromosome fusion initiates chromosome instability involving breakage-fusion-bridge (B/F/B) cycles, in which dicentric chromosomes form bridges and break as the cell attempts to divide, repeating the process in subsequent cell cycles. Fusion between sister chromatids results in large inverted repeats on the end of the chromosome, which amplify further following additional B/F/B cycles. B/F/B cycles continue until the chromosome acquires a new telomere, most often by translocation of the end of another chromosome. The instability is not confined to a chromosome that loses its telomere, because the instability is transferred to the chromosome donating a translocation. Moreover, the amplified regions are unstable and form extrachromosomal DNA that can reintegrate at new locations. Knowledge concerning the factors promoting telomere loss and its consequences is therefore important for understanding chromosome instability in human cancer.     

Functions and regulation of human artemis in double strand break repair

Cells, which lacked the activity of the nuclease Artemis, retained approximately 10% of unrepaired double strand breaks (DSBs) at later timepoints after ionizing radiation. Ionizing radiation induced hyperphosphorylation of Artemis mainly by ATM and in ATM deficient cells to a minor extent by DNA PK. After induction of DSBs with modified ends by a high dose of calicheamicin gamma1, Artemis was phosphorylated by DNA PK. The type of calicheamicin gamma1-induced DSBs is likely to represent a subclass of DSBs induced by ionizing radiation. DNA PK-dependent phosphorylation of Artemis after treatment with DSB inducing agents increased the cellular retention of Artemis, maintained its interaction with DNA ends and activated its endonucleolytic activity. The following model is suggested: ATM-dependent phosphorylation of Artemis after ionizing radiation could prevent DNA PK-dependent phosphorylation and activation of undesired endonucleolytic activity at DSBs, which do not require endonucleolytic processing by Artemis. The Artemis:DNA PK complex could be involved in the repair of DSBs, which carry modified ends and are refractory to repair by otherwise lesion specific enzymes because of the presence of an inhibitory lesion in the opposite strand.     

Processing of DNA for nonhomologous end-joining is controlled by kinase activity and XRCC4/ligase IV

Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks created by ionizing radiation and V(D)J recombination. To repair the broken ends, NHEJ processes noncompatible ends into a ligatable form but suppresses processing of compatible ends. It is not known how NHEJ controls polymerase and nuclease activities to act exclusively on noncompatible ends. Here, we analyzed processing independently of ligation by using a two-stage assay with extracts that recapitulated the properties of NHEJ in vivo. Processing of noncompatible ends required wortmannin-sensitive kinase activity. Since DNA-dependent protein kinase catalytic subunit (DNA-PKcs) brings the ends together before undergoing activation of its kinase, this suggests that processing occurred after synapsis of the ends. Surprisingly, all polymerase and most nuclease activity required XRCC4/Ligase IV. This suggests a mechanism for how NHEJ suppresses processing to optimize the preservation of DNA sequence.     

Short dysfunctional telomeres impair the repair of arsenite-induced oxidative damage in mouse cells

Telomeres and telomerase appear to participate in the repair of broken DNA ends produced by oxidative damage. Arsenite is an environmental contaminant and a potent human carcinogen, which induces oxidative stress on cells via the generation of reactive oxygen species affecting cell viability and chromosome stability. It promotes telomere attrition and reduces cell survival by apoptosis. In this study, we used mouse embryonic fibroblasts (MEFs) from mice lacking telomerase RNA component (mTERC(-/-) mice) with long (early passage or EP) and short (late passage or LP) telomeres to investigate the extent of oxidative damage by comparing the differences in DNA damage, chromosome instability, and cell survival at 24 and 48 h of exposure to sodium arsenite (As3+; NaAsO2). There was significantly high level of DNA damage in mTERC(-/-) cells with short telomeres as determined by alkaline comet assay. Consistent with elevated DNA damage, increased micronuclei (MN) induction reflecting gross genomic instability was also observed. Fluorescence in situ hybridization (FISH) analysis revealed that increasing doses of arsenite augmented the chromosome aberrations, which contributes to genomic instability leading to possibly apoptotic cell death and cell cycle arrest. Microarray analysis has revealed that As3+ treatment altered the expression of 456 genes of which 20% of them have known functions in cell cycle and DNA damage signaling and response, cell growth, and/or maintenance. Results from our studies imply that short dysfunctional telomeres impair the repair of oxidative damage caused by arsenite. The results will have implications in risk estimation as well as cancer chemotherapy.     

The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

Telomeres distinguish chromosome ends from double-strand breaks (DSBs) and prevent chromosome fusion. However, telomeres can also interfere with DNA repair, as shown by a deficiency in nonhomologous end joining (NHEJ) and an increase in large deletions at telomeric DSBs. The sensitivity of telomeric regions to DSBs is important in the cellular response to ionizing radiation and oncogene-induced replication stress, either by preventing cell division in normal cells, or by promoting chromosome instability in cancer cells. We have previously proposed that the telomeric protein TRF2 causes the sensitivity of telomeric regions to DSBs, either through its inhibition of ATM, or by promoting the processing of DSBs as though they are telomeres, which is independent of ATM. Our current study addresses the mechanism responsible for the deficiency in repair of DSBs near telomeres by combining assays for large deletions, NHEJ, small deletions, and gross chromosome rearrangements (GCRs) to compare the types of events resulting from DSBs at interstitial and telomeric DSBs. Our results confirm the sensitivity of telomeric regions to DSBs by demonstrating that the frequency of GCRs is greatly increased at DSBs near telomeres and that the role of ATM in DSB repair is very different at interstitial and telomeric DSBs. Unlike at interstitial DSBs, a deficiency in ATM decreases NHEJ and small deletions at telomeric DSBs, while it increases large deletions. These results strongly suggest that ATM is functional near telomeres and is involved in end protection at telomeric DSBs, but is not required for the extensive resection at telomeric DSBs. The results support our model in which the deficiency in DSB repair near telomeres is a result of ATM-independent processing of DSBs as though they are telomeres, leading to extensive resection, telomere loss, and GCRs involving alternative NHEJ.     

Hypersensitivity to radiation-induced non-apoptotic and apoptotic death in cell lines from patients with the ICF chromosome instability syndrome

Immunodeficiency, centromeric region instability, and facial anomalies (ICF), a rare recessive chromosome instability syndrome, involves the loss of DNA methyltransferase 3B activity and the consequent hypomethylation of a small portion of the genome. We demonstrate for the first time that ICF cells are strongly hypersensitive to a genotoxic agent, namely, ionizing radiation. However, unlike cell lines from patients with ataxia telangiectasia or Nijmegen breakage syndrome, chromosome instability syndromes also associated with unusual sensitivity to ionizing radiation, ICF cells did not show any deficiencies in their cell cycle checkpoints. ICF lymphoblastoid cell lines demonstrated increased apoptosis, long-term cell cycle arrest, and loss of viability in clonogenicity assays after irradiation compared to analogous normal cell lines. Also, the ICF cell lines were subject to high frequencies of rapid non-apoptotic cell death upon irradiation but not to abnormally high levels of radiation-induced, cytogenetically detectable chromosome abnormalities. ICF-associated undermethylation of some regulatory gene(s) might lead to an exaggerated response to radiation-induced breaks in DNA yielding increased rates of cell death and irreversible cell cycle arrest. As a defense against their frequent spontaneous breaks in chromosomes 1 and 16, ICF patients may be abnormally prone to chromosome break-induced apoptosis, non-apoptotic cell death, and permanent cell cycle arrest so as to minimize the number of cycling cells with spontaneous rearrangements. A similarly increased cell death and cycle-arrest response to chromosome breaks due to cancer-linked DNA hypomethylation might occur during carcinogenesis.     

DNA breaks promote genomic instability by impeding proper chromosome segregation

BACKGROUND: Unrepaired DNA double-stranded breaks (DSBs) can result in the whole or partial loss of chromosomes. Previously, we showed that the ends of broken chromosomes remain associated. Here, we have examined the machinery that holds broken chromosome ends together, and we have explored the behavior of broken chromosomes as they pass through mitosis. RESULTS: Using GFP-localized arrays flanking an HO endonuclease site, we examined the association of broken chromosome ends in yeast cells that are checkpoint-arrested in metaphase. This association is partially dependent upon Rad50 and Rad52. After 6-8 hr, cells adapted to the checkpoint and resumed mitosis, segregating the broken chromosome. When this occurred, we found that the acentric fragments cosegregated into either the mother or daughter cell 95% of the time. Similarly, pedigree analysis showed that postmitotic repair of a broken chromosome (rejoining the centric and acentric fragments) occurred in either the mother or daughter cell, but rarely both, consistent with a model in which both acentric sister chromatid fragments are passaged into the same nucleus. CONCLUSIONS: These data suggest two related phenomena: an intrachromosomal association that holds the halves of a single broken sister chromatid together in metaphase and an interchromosomal force that tethers broken sister chromatids to each other and promotes their missegregation. Strikingly, the interchromosomal association of DNA breaks also promotes the missegregation of centromeric chromosomal fragments, albeit to a lesser extent than acentric fragments. The DNA break-induced missegregation of acentric and centric chromosome fragments provides a novel mechanism for the loss of heterozygosity that precedes tumorigenesis in mammalian cells.     

Histone H2AX is phosphorylated at sites of retroviral DNA integration but is dispensable for postintegration repair

The histone variant H2AX is rapidly phosphorylated (denoted gammaH2AX) in large chromatin domains (foci) flanking double strand DNA (dsDNA) breaks that are produced by ionizing radiation or genotoxic agents and during V(D)J recombination. H2AX-deficient cells and mice demonstrate increased sensitivity to dsDNA break damage, indicating an active role for gammaH2AX in DNA repair; however, gammaH2AX formation is not required for V(D)J recombination. The latter finding has suggested a greater dependence on gammaH2AX for anchoring free broken ends versus ends that are held together during programmed breakage-joining reactions. Retroviral DNA integration produces a unique intermediate in which a dsDNA break in host DNA is held together by the intervening viral DNA, and such a reaction provides a useful model to distinguish gammaH2AX functions. We found that integration promotes transient formation of gammaH2AX at retroviral integration sites as detected by both immunocytological and chromatin immunoprecipitation methods. These results provide the first direct evidence for the association of newly integrated viral DNA with a protein species that is an established marker for the onset of a DNA damage response. We also show that H2AX is not required for repair of the retroviral integration intermediate as determined by stable transduction. These observations provide independent support for an anchoring model for the function of gammaH2AX in chromatin repair.     

Chromatin remodeling finds its place in the DNA double-strand break response

The accurate repair of chromosomal double-strand breaks (DSBs) arising from exposure to exogenous agents, such as ionizing radiation (IR) and radiomimetic drugs is crucial in maintaining genomic integrity, cellular viability and the prevention of tumorigenesis. Eukaryotic cells have evolved efficient mechanisms that sense and respond to DSBs. The DNA DSB response is facilitated by hierarchical signaling networks that orchestrate chromatin structural changes, cell-cycle checkpoints and multiple enzymatic activities to repair the broken DNA ends. Sensors and transducers signal to numerous downstream cellular effectors which function primarily by substrate posttranslational modifications including phosphorylation, acetylation, methylation and ubiquitylation. In particular, the past several years have provided important insight into the role of chromatin remodeling and histones-specific modifications to control DNA damage detection, signaling and repair. This review summarizes recently identified factors that influence this complex process and the repair of DNA DSBs in eukaryotic cells.     

Partners and pathwaysrepairing a double-strand break

Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in meiosis. Broken chromosomes can be repaired either by one of several homologous recombination mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for the repair of a double-strand break. Which of these repair pathways is used appears to be regulated developmentally, genetically and during the cell cycle.