Regulation of the cellular DNA double-strand break response.

DNA double-strand breaks occur frequently in cycling cells, and are also induced by exogenous sources, including ionizing radiation. Cells have developed integrated double-strand break response pathways to cope with these lesions, including pathways that initiate DNA repair (either via homologous recombination or nonhomologous end joining), the cell-cycle checkpoints (G1-S, intra-S phase, and G2-M) that provide time for repair, and apoptosis. However, before any of these pathways can be activated, the damage must first be recognized. In this review, we will discuss how the response of mammalian cells to DNA double-strand breaks is regulated, beginning with the activation of ATM, the pinnacle kinase of the double-strand break signalling cascade.     

Single-cell microarray enables high-throughput evaluation of DNA double-strand breaks and DNA repair inhibitors

A key modality of non-surgical cancer management is DNA damaging therapy that causes DNA double-strand breaks that are preferentially toxic to rapidly dividing cancer cells. Double-strand break repair capacity is recognized as an important mechanism in drug resistance and is therefore a potential target for adjuvant chemotherapy. Additionally, spontaneous and environmentally induced DSBs are known to promote cancer, making DSB evaluation important as a tool in epidemiology, clinical evaluation and in the development of novel pharmaceuticals. Currently available assays to detect double-strand breaks are limited in throughput and specificity and offer minimal information concerning the kinetics of repair. Here, we present the CometChip, a 96-well platform that enables assessment of double-strand break levels and repair capacity of multiple cell types and conditions in parallel and integrates with standard high-throughput screening and analysis technologies. We demonstrate the ability to detect multiple genetic deficiencies in double-strand break repair and evaluate a set of clinically relevant chemical inhibitors of one of the major double-strand break repair pathways, non-homologous end-joining. While other high-throughput repair assays measure residual damage or indirect markers of damage, the CometChip detects physical double-strand breaks, providing direct measurement of damage induction and repair capacity, which may be useful in developing and implementing treatment strategies with reduced side effects.     

Inhibition of DNA damage repair by artificial activation of PARP with siDNA

One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.     

Mammalian DNA single-strand break repair: an X-ra(y)ted affair

The genetic stability of living cells is continuously threatened by the presence of endogenous reactive oxygen species and other genotoxic molecules. Of particular threat are the thousands of DNA single-strand breaks that arise in each cell, each day, both directly from disintegration of damaged sugars and indirectly from the excision repair of damaged bases. If un-repaired, single-strand breaks can be converted into double-strand breaks during DNA replication, potentially resulting in chromosomal rearrangement and genetic deletion. Consequently, cells have adopted multiple pathways to ensure the rapid and efficient removal of single-strand breaks. A general feature of these pathways appears to be the extensive employment of protein-protein interactions to stimulate both the individual component steps and the overall repair reaction. Our current understanding of DNA single-strand break repair is discussed, and testable models for the architectural coordination of this important process are presented.     

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.     

Effect of Ku86 and DNA-PKcs deficiency on non-homologous end-joining and homologous recombination using a transient transfection assay

In mammalian cells, DNA double-strand breaks are repaired by non-homologous end-joining and homologous recombination, both pathways being essential for the maintenance of genome integrity. We determined the effect of mutations in Ku86 and DNA-PK on the efficiency and the accuracy of double-strand break repair by non-homologous end-joining and homologous recombination in mammalian cells. We used an assay, based on the transient transfection of a linearized plasmid DNA, designed to simultaneously detect transfection and recombination markers. In agreement with previous results non-homologous end-joining was largely compromised in Ku86 deficient cells, and returned to normal in the Ku86-complemented isogenic cell line. In addition, analysis of DNA plasmids recovered from Ku86 mutant cells showed an increased use of microhomologies at the nonhomologous end joining junctions, and displayed a significantly higher frequency of DNA insertions compared to control cells. On the other hand, the DNA-PKcs deficient cell lines showed efficient double-strand break repair by both mechanisms.     

DNA double-strand break repair: from mechanistic understanding to cancer treatment

Accurate repair of DNA double-strand breaks is essential to life. Indeed, defective DNA double-strand break repair can lead to toxicity and large scale sequence rearrangements that cause cancer and promote premature aging. Here, we highlight the two major repair systems for handling DNA double-strand breaks: homologous recombination and non-homologous end joining. To clarify recombination mechanisms, we present animations that illustrate DNA strand movements. In addition to describing how these pathways operate, we also describe why appropriate pathway choice is critical to genomic stability, and we summarize key pathway control features related to cell cycle checkpoint and apoptosis signaling. Importantly, recent progress in delineating the effects of specific defects in repair and checkpoint control has helped to explain several disease phenotypes, including cancer and premature aging. Improved understanding of these pathways has also sparked development of novel chemotherapeutic strategies that kill tumors with increased specificity and efficacy. This review aims to provide a foundational understanding of how the homologous recombination and non-homologous end joining pathways operate, and to demonstrate how a better understanding of these processes has advanced both our understanding of the underlying causes of cancer and our ability to innovate novel cancer treatment strategies.     

Repair of ionizing radiation damage in mammalian cells. Alternative pathways and their fidelity

Ionizing radiation causes a variety of types of damage to DNA in cells, requiring the concerted action of a number of DNA repair enzymes to restore genomic integrity. The DNA base-excision repair and DNA double-strand break repair pathways are particularly important. While single base damages are rapidly excised and repaired using the opposite (undamaged) strand as a template, the correct repair of DNA double-strand breaks may present more difficulties to cellular enzymes owing to the loss of template. In the last few years evidence in support of several enzymatic pathways for the repair of such double-stranded damage has been found. At present we may distinguish at least three pathways: homologous recombination repair, non-homologous (DNA-PK-dependent) end joining, and repeat-driven end joining. This paper focuses on evidence for the first and third of these pathways, and considers in particular their relative importance in mammalian cells and implications for the fidelity of 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.     

Mechanism of DNA double-strand break repair by non-homologous end joining

The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Although the non-homologous end joining (NHEJ) pathway frequently results in minor changes in DNA sequence at the break site and occasionally the joining of previously unlinked DNA molecules, it is a major contributor to cell survival following exposure of mammalian cells to agents that cause DSBs. This repair mechanism is conserved in lower eukaryotes and in some prokaryotes although the majority of DSBs are repaired by recombinational repair pathways in these organisms. Here we will describe the biochemical properties of NHEJ factors from bacteria, Saccharomyces cerevisiae and mammals, and how physical and functional interactions among these factors co-ordinate the repair of DSBs.     

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.     

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.     

Main steps in DNA double-strand break repair: an introduction to homologous recombination and related processes

DNA double-strand breaks arise accidentally upon exposure of DNA to radiation and chemicals or result from faulty DNA metabolic processes. DNA breaks can also be introduced in a programmed manner, such as during the maturation of the immune system, meiosis, or cancer chemo- or radiotherapy. Cells have developed a variety of repair pathways, which are fine-tuned to the specific needs of a cell. Accordingly, vegetative cells employ mechanisms that restore the integrity of broken DNA with the highest efficiency at the lowest cost of mutagenesis. In contrast, meiotic cells or developing lymphocytes exploit DNA breakage to generate diversity. Here, we review the main pathways of eukaryotic DNA double-strand break repair with the focus on homologous recombination and its various subpathways. We highlight the differences between homologous recombination and end-joining mechanisms including non-homologous end-joining and microhomology-mediated end-joining and offer insights into how these pathways are regulated. Finally, we introduce noncanonical functions of the recombination proteins, in particular during DNA replication stress.     

The role of double-strand break repair - insights from human genetics

The efficient repair of DNA double-strand breaks is crucial in safeguarding the genomic integrity of organisms. Responses to double-strand breaks include complex signal-transduction, cell-cycle-checkpoint and repair pathways. Defects in these pathways lead to several human disorders with pleiotropic clinical features. Dissection of the molecular basis that underlies the diverse clinical features is enhancing our understanding of the damage-response mechanisms and their role in development, and might ultimately facilitate treatment.     

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.     

Chromosomal fragmentation is the major consequence of the rdgB defect in Escherichia coli

The rdgB mutants depend on recombinational repair of double-strand breaks. To assess other consequences of rdgB inactivation in Escherichia coli, we isolated RdgB-dependent mutants. All transposon inserts making cells dependent on RdgB inactivate genes of double-strand break repair, indicating that chromosomal fragmentation is the major consequence of RdgB inactivation.     

Reduced repair of DNA double-strand breaks by homologous recombination in a DNA ligase I-deficient human cell line

Genetic and biochemical studies of mammalian DNA ligase I indicate that this multifunctional enzyme plays a key role in the completion of DNA replication and certain DNA excision repair pathways. However, the involvement of DNA ligase I in DNA double-strand break repair has not been examined. Here we have determined the effect of DNA ligase I-deficiency on the frequency of homologous recombination initiated by a site-specific DNA double-strand break. We found that expression of wild-type DNA ligase I in a human DNA ligase I mutant cell line significantly increased the frequency of homologous recombination. Notably, the ability of DNA ligase I to promote the recombinational repair of DNA double-strand breaks was dependent upon its interaction with proliferating cell nuclear antigen. Thus, our results demonstrate that DNA ligase I-deficiency reduces recombinational repair of DNA double-strand breaks.     

The mechanism of human nonhomologous DNA end joining

Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S or G(2), whereas nonhomologous DNA end joining (NHEJ) can occur throughout the cell cycle and is the major pathway for the repair of double-strand breaks in multicellular eukaryotes. NHEJ is distinctive for the flexibility of the nuclease, polymerase, and ligase activities that are used. This flexibility permits NHEJ to function on the wide range of possible substrate configurations that can arise when double-strand breaks occur, particularly at sites of oxidative damage or ionizing radiation. NHEJ does not return the local DNA to its original sequence, thus accounting for the wide range of end results. Part of this heterogeneity arises from the diversity of the DNA ends, but much of it arises from the many alternative ways in which the nuclease, polymerases, and ligase can act during NHEJ. Physiologic double-strand break processes make use of the imprecision of NHEJ in generating antigen receptor diversity. Pathologically, the imprecision of NHEJ contributes to genome mutations that arise over time.     

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.