Cell cycle-regulated centers of DNA double-strand break repair

In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms foci specifically during S phase. We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize. Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein. In addition, we show that the two ends of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair.     

Cell Cycle-Regulated Centers of DNA Double-Strand Break Repair

In eukaryotes, homologous recombination is an important pathway for the repair of DNA double-strand breaks. We have studied this process in living cells in the yeast Saccharomyces cerevisiae using Rad52 as a cell biological marker. In response to DNA damage, Rad52 redistributes itself and forms foci specifically during S phase. We have shown previously that Rad52 foci are centers of DNA repair where multiple DNA double-strand breaks colocalize. Here we report a correlation between the timing of Rad52 focus formation and modification of the Rad52 protein. In addition, we show that the two ends of a double-strand break are held tightly together in the majority of cells. Interestingly, in a small but significant fraction of the S phase cells, the two ends of a break separate suggesting that mechanisms exist to reassociate and align these ends for proper DNA repair.     

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents.     

Split-dose recovery is due to the repair of DNA double-strand breaks

DNA double-strand breaks are the molecular lesions the repair of which leads to the reappearance of the shoulder observed in split-dose experiments. This conclusion is based on results obtained with the help of a diploid yeast mutant rad 54-3 which is temperature-conditional for the repair of DNA double-strand breaks. Two repair steps must be met to yield the reappearance of the shoulder on a split-dose survival curve: the repair of double-strand breaks during the interval between two doses and on the nutrient agar plate after the second dose. In yeast lethality may be attributable to either an unrepaired double-strand break (i.e. a double-strand break is a potentially lethal lesion) or to the interaction of two double-strand breaks (misrepair of double-strand breaks). Evidence is presented that the two cellular phenomena of liquid holding recovery (repair of potentially lethal damage) and of split-dose recovery (repair of sublethal damage) are based on the repair of the same molecular lesion, the DNA double-strand break.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

细胞DNA损伤检控系统在维持端粒稳定中的功能

真核生物的DNA损伤检控系统是维持细胞基因组稳定的一个重要机制,该系统能检测细胞在生命活动过程中出现的DNA损伤并引发细胞周期阻滞,对DNA损伤进行修复,以维持细胞遗传的稳定性。端粒是位于真核细胞染色体末端由重复DNA序列和蛋白质组成的复合物,具有保护染色体、介导染色体复制、引导减数分裂时的同源染色体配对和调节细胞衰老等作用。虽然端粒与DNA双链断裂都具有作为线性染色体末端的共同特点,但正常端粒并不像DNA双链断裂那样激活DNA损伤检控系统。另一方面,端粒又与DNA损伤相似,因为多种DNA损伤检控蛋白在端粒长度稳定中起重要作用。因此DNA损伤检控系统既参与了维持正常端粒的完整性,又可对端粒损伤作出应答。现就DNA损伤检控系统在维持端粒稳定中的作用及其对功能缺陷端粒的应答作一简要综述。     

Double-strand DNA break formation mediated by flap endonuclease-1

Double-strand DNA breaks are the most lethal type of DNA damage induced by ionizing radiations. Previously, we reported that double-strand DNA breaks can be enzymatically produced from two DNA damages located on opposite DNA strands 18 or 30 base pairs apart in a cell-free double-strand DNA break formation assay (Vispé, S., and Satoh, M. S. (2000) J. Biol. Chem. 275, 27386-27392). In the assay that we developed, these two DNA damages are converted into single-strand interruptions by enzymes involved in base excision repair. We showed that these single-strand interruptions are converted into double-strand DNA breaks; however, it was not due to spontaneous denaturation of DNA. Thus, we proposed a model in which DNA polymerase delta/epsilon, by producing repair patches at single-strand interruptions, collide, resulting in double-strand DNA break formation. We tested the model and investigated whether other enzymes/factors are involved in double-strand DNA break formation. Here we report that, instead of DNA polymerase delta/epsilon, flap endonuclease-1 (FEN-1), an enzyme involved in base excision repair, is responsible for the formation of double-strand DNA break in the assay. Furthermore, by transfecting a flap endonuclease-1 expression construct into cells, thus altering their flap endonuclease-1 content, we found an increased number of double-strand DNA breaks after gamma-ray irradiation of these cells. These results suggest that flap endonuclease-1 acts as a double-strand DNA break formation factor. Because FEN-1 is an essential enzyme that plays its roles in DNA repair and DNA replication, DSBs may be produced in cells as by-products of the activity of FEN-1.     

Ornithine decarboxylase antizyme upregulates DNA-dependent protein kinase and enhances the nonhomologous end-joining repair of DNA double-strand breaks in human oral cancer cells

Ornithine decarboxylase (ODC) antizyme targets ODC for ubiquitin-independent proteosome degradation, thereby inhibiting polyamine synthesis. It has been shown to regulate DNA methylation and has tumor suppressor activity. Increasing evidence suggested that antizyme may also have ODC-independent functions. Here, we report that antizyme plays a role in DNA double-strand break repairs. A zinc-inducible human antizyme gene expression vector was transfected into UM1 human oral squamous cancer cells that do not express endogenous antizyme. The resultant upregulated genes were screened by cDNA arrays and confirmed by quantitative real-time polymerase chain reaction. DNA-dependent protein kinase including its catalytic subunit DNA-PKcs and regulatory subunit Ku70, two key proteins of the DNA damage repair machinery, was significantly upregulated after ectopic expression of antizyme. Consistently, we found that UM1 cells are sensitive to gamma irradiation and deficient in DNA damage repairs, as shown by radio-sensitivity and Comet assays. Ectopic expression of antizyme increased radio-resistance of UM1 cells and restored their capacity of DNA damage repairs to the level of UM2 cells that have an identical genetic background but express endogenous antizyme. Plasmid end-joining assays confirmed that antizyme enhances the ability of UM1 cells to repair DNA double-strand breaks by the nonhomologous end-joining pathway.     

Collaboration and competition between DNA double-strand break repair pathways

DNA double-strand breaks resulting from normal cellular processes including replication and exogenous sources such as ionizing radiation pose a serious risk to genome stability, and cells have evolved different mechanisms for their efficient repair. The two major pathways involved in the repair of double-strand breaks in eukaryotic cells are non-homologous end joining and homologous recombination. Numerous factors affect the decision to repair a double-strand break via these pathways, and accumulating evidence suggests these major repair pathways both cooperate and compete with each other at double-strand break sites to facilitate efficient repair and promote genomic integrity.     

Unraveling DNA Repair in Human: Molecular Mechanisms and Consequences of Repair Defect

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders.     

AT细胞DNA双链断裂重接修复效率及其忠实性

用脉冲电场凝胶电泳和双标记基因质粒ＤＮＡ转染技术研究辐射敏感的毛细血管扩张性共济失调症患者皮肤成纤维细胞（ＡＴ５ＢＩＶＡ）和正常辐射抗性的人宫颈癌细胞（ＨｅＬａＳ３）ＤＮＡ双链断裂重接修复率及其忠实性。结果表明γ射线照射诱发ＤＮＡ双链断裂的产额和重接修复率,在两株细胞间无差别．而ＡＴ细胞对导入的限制性内切酶ＥｃｏＲＶ产生双链断裂质粒ＤＮＡ的重接修复忠实性显著低于ＨｅｌａＳ３ｔｅ胞,表明ＡＴ细胞易发生ＤＮＡ错误修复,这很可能就是ＡＴ细胞高度辐射敏感性的主要原因。     

The Caenorhabditis elegans Homolog of Gen1/Yen1 Resolvases Links DNA Damage Signaling to DNA Double-Strand Break Repair

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR), which can involve Holliday junction (HJ) intermediates that are ultimately resolved by nucleolytic enzymes. An N-terminal fragment of human GEN1 has recently been shown to act as a Holliday junction resolvase, but little is known about the role of GEN-1 in vivo. Holliday junction resolution signifies the completion of DNA repair, a step that may be coupled to signaling proteins that regulate cell cycle progression in response to DNA damage. Using forward genetic approaches, we identified a Caenorhabditis elegans dual function DNA double-strand break repair and DNA damage signaling protein orthologous to the human GEN1 Holliday junction resolving enzyme. GEN-1 has biochemical activities related to the human enzyme and facilitates repair of DNA double-strand breaks, but is not essential for DNA double-strand break repair during meiotic recombination. Mutational analysis reveals that the DNA damage-signaling function of GEN-1 is separable from its role in DNA repair. GEN-1 promotes germ cell cycle arrest and apoptosis via a pathway that acts in parallel to the canonical DNA damage response pathway mediated by RPA loading, CHK1 activation, and CEP-1/p53–mediated apoptosis induction. Furthermore, GEN-1 acts redundantly with the 9-1-1 complex to ensure genome stability. Our study suggests that GEN-1 might act as a dual function Holliday junction resolvase that may coordinate DNA damage signaling with a late step in DNA double-strand break repair.     

DNA resection at chromosome breaks promotes genome stability by constraining non-allelic homologous recombination

DNA double-strand breaks impact genome stability by triggering many of the large-scale genome rearrangements associated with evolution and cancer. One of the first steps in repairing this damage is 5'→3' resection beginning at the break site. Recently, tools have become available to study the consequences of not extensively resecting double-strand breaks. Here we examine the role of Sgs1- and Exo1-dependent resection on genome stability using a non-selective assay that we previously developed using diploid yeast. We find that Saccharomyces cerevisiae lacking Sgs1 and Exo1 retains a very efficient repair process that is highly mutagenic to genome structure. Specifically, 51% of cells lacking Sgs1 and Exo1 repair a double-strand break using repetitive sequences 12-48 kb distal from the initial break site, thereby generating a genome rearrangement. These Sgs1- and Exo1-independent rearrangements depend partially upon a Rad51-mediated homologous recombination pathway. Furthermore, without resection a robust cell cycle arrest is not activated, allowing a cell with a single double-strand break to divide before repair, potentially yielding multiple progeny each with a different rearrangement. This profusion of rearranged genomes suggests that cells tolerate any dangers associated with extensive resection to inhibit mutagenic pathways such as break-distal recombination. The activation of break-distal recipient repeats and amplification of broken chromosomes when resection is limited raise the possibility that genome regions that are difficult to resect may be hotspots for rearrangements. These results may also explain why mutations in resection machinery are associated with cancer.     

Inter-individual variation in DNA repair capacity: A need for multi-pathway functional assays to promote translational DNA repair research

Why does a constant barrage of DNA damage lead to disease in some individuals, while others remain healthy? This article surveys current work addressing the implications of inter-individual variation in DNA repair capacity for human health, and discusses the status of DNA repair assays as potential clinical tools for personalized prevention or treatment of disease. In particular, we highlight research showing that there are significant inter-individual variations in DNA repair capacity (DRC), and that measuring these differences provides important biological insight regarding disease susceptibility and cancer treatment efficacy. We emphasize work showing that it is important to measure repair capacity in multiple pathways, and that functional assays are required to fill a gap left by genome wide association studies, global gene expression and proteomics. Finally, we discuss research that will be needed to overcome barriers that currently limit the use of DNA repair assays in the clinic.     

The comet assay for DNA damage and repair

The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.     

Securing genome stability by orchestrating DNA repair: removal of radiation-induced clustered lesions in DNA

In addition to double- and single-strand DNA breaks and isolated base modifications, ionizing radiation induces clustered DNA damage, which contains two or more lesions closely spaced within about two helical turns on opposite DNA strands. Post-irradiation repair of single-base lesions is routinely performed by base excision repair and a DNA strand break is involved as an intermediate. Simultaneous processing of lesions on opposite DNA strands may generate double-strand DNA breaks and enhance nonhomologous end joining, which frequently results in the formation of deletions. Recent studies support the possibility that the mechanism of base excision repair contributes to genome stability by diminishing the formation of double-strand DNA breaks during processing of clustered lesions.     

Ku, Artemis, and ataxia-telangiectasia-mutated: signalling networks in DNA damage

Cell death linked to DNA damage has been implicated in various diseases caused by environmental stress and infection. Severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. Accumulation of DNA damage has been proposed to be a principal mechanism of infection, inflammation, cancer, and aging. The most deleterious form of DNA damage is double-strand breaks (DSBs), where ataxia-telangiectasia-mutated (ATM) is the main transducer of the double-strand DNA break signal. Once the DNA is damaged, the DNA repair protein Ku70/80 translocates into the nucleus, a process which may be mediated by ataxia-telangiectasia-mutated, a member of the phosphoinositide-3-kinase-like family. The function and stability of Artemis may also be regulated by ataxia-telangiectasia-mutated through its phosphorylation upon the occurrence of DNA damage. Interestingly, both Artemis and Ku70/80 are substrates of DNA-dependent protein kinase (DNA-PK), another member of the phosphoinositide-3-kinase-like family. In this review, we show how Ku and Artemis function in the DNA damage response and the ataxia-telangiectasia-mutated signaling pathway and discuss potential applications of agents targeting these DNA damage response molecules in the treatment of inflammation and cancer.     

Genetic analysis of BRCA1 function in a defined tumor cell line

Retrovirally expressed, wild-type BRCA1 decreased the gamma radiation (IR) sensitivity and increased the efficiency of double-strand DNA break repair (DSBR) of the BRCA1-/- human breast cancer line, HCC1937. It also reduced its susceptibility to DSB generation by IR. By contrast, multiple, clinically validated, missense mutant BRCA1 products were nonfunctional in these assays. These data constitute the basis for a BRCA1 functional assay and suggest that efficient repair of double-strand DNA breaks is linked to BRCA1 tumor suppression function.     

In Vitro Repair of Gaps in Bacteriophage T7 DNA

An in vitro system based upon extracts of Escherichia coli infected with bacteriophage T7 was used to study the mechanism of double-strand break repair. Double-strand breaks were placed in T7 genomes by cutting with a restriction endonuclease which recognizes a unique site in the T7 genome. These molecules were allowed to repair under conditions where the double-strand break could be healed by (i) direct joining of the two partial genomes resulting from the break, (ii) annealing of complementary versions of 17-bp sequences repeated on either side of the break, or (iii) recombination with intact T7 DNA molecules. The data show that while direct joining and single-strand annealing contributed to repair of double-strand breaks, these mechanisms made only minor contributions. The efficiency of repair was greatly enhanced when DNA molecules that bridge the region of the double-strand break (referred to as donor DNA) were provided in the reaction mixtures. Moreover, in the presence of the donor DNA most of the repaired molecules acquired genetic markers from the donor DNA, implying that recombination between the DNA molecules was instrumental in repairing the break. Double-strand break repair in this system is highly efficient, with more than 50% of the broken molecules being repaired within 30 min under some experimental conditions. Gaps of 1,600 nucleotides were repaired nearly as well as simple double-strand breaks. Perfect homology between the DNA sequence near the break site and the donor DNA resulted in minor (twofold) improvement in the efficiency of repair. However, double-strand break repair was still highly efficient when there were inhomogeneities between the ends created by the double-strand break and the T7 genome or between the ends of the donor DNA molecules and the genome. The distance between the double-strand break and the ends of the donor DNA molecule was critical to the repair efficiency. The data argue that ends of DNA molecules formed by double-strand breaks are typically digested by between 150 and 500 nucleotides to form a gap that is subsequently repaired by recombination with other DNA molecules present in the same reaction mixture or infected cell.     

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.