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.     

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.     

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.     

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

Background  

Efficient 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.     

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.     

Choosing the right path: does DNA-PK help make the decision?

DNA double-strand breaks are extremely harmful lesions that can lead to genomic instability and cell death if not properly repaired. There are at least three pathways that are responsible for repairing DNA double-strand breaks in mammalian cells: non-homologous end joining, homologous recombination and alternative non-homologous end joining. Here we review each of these three pathways with an emphasis on the role of the DNA-dependent protein kinase, a critical component of the non-homologous end joining pathway, in influencing which pathway is ultimately utilized for repair.     

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.     

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.     

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.     

53BP1与DNA双链断裂损伤修复

DNA的精确复制和遗传对维持基因组稳定性有重要作用。DNA双链断裂损伤可能诱导细胞凋亡和染色质重排,在肿瘤的发生发展过程中发挥作用。53BP1是DNA双链断裂修复中的重要调节蛋白质之一,对调控损伤修复平衡和维持基因组稳定性起着重要作用。本文主要对53BP1的结构、生物学功能、信号通路、分子机制和翻译后修饰做一浅显的总结和展望,希望能为53BP1的深入研究提供一些理论基础。     

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.     

Control of alternative end joining by the chromatin remodeler p400 ATPase

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.     

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.     

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.     

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.     

Induction of CAF-1 expression in response to DNA strand breaks in quiescent human cells

Genome stability in eukaryotic cells is maintained through efficient DNA damage repair pathways, which have to access and utilize chromatin as their natural template. Here we investigate the role of chromatin assembly factor 1 (CAF-1) and its interacting protein, PCNA, in the response of quiescent human cells to DNA double-strand breaks (DSBs). The expression of CAF-1 and PCNA is dramatically induced in quiescent cells upon the generation of DSBs by the radiomimetic drug bleocin (a bleomycin compound) or by ionizing radiation. This induction depends on DNA-PK. CAF-1 and PCNA are recruited to damaged chromatin undergoing DNA repair of single- and double-strand DNA breaks by the base excision repair and nonhomologous end-joining pathways, respectively, in the absence of extensive DNA synthesis. CAF-1 prepared from repair-proficient quiescent cells after induction by bleocin mediates nucleosome assembly in vitro. Depletion of CAF-1 by RNA interference in bleocin-treated quiescent cells in vivo results in a significant loss of cell viability and an accumulation of DSBs. These results support a novel and essential role for CAF-1 in the response of quiescent human cells to DSBs, possibly by reassembling chromatin following repair of DNA strand breaks.     

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.     

spn-A/rad51 mutant exhibits enhanced genomic damage,cell death and low temperature sensitivity in somatic tissues

Chromosoma - Homologous recombination (HR) is one of the key pathways to repair double-strand breaks (DSBs). Rad51 serves an important function of catalysing strand exchange between two homologous...     

The role of RecQ helicases in non-homologous end-joining

Abstract

DNA double-strand breaks are highly toxic DNA lesions that cause genomic instability, if not efficiently repaired. RecQ helicases are a family of highly conserved proteins that maintain genomic stability through their important roles in several DNA repair pathways, including DNA double-strand break repair. Double-strand breaks can be repaired by homologous recombination (HR) using sister chromatids as templates to facilitate precise DNA repair, or by an HR-independent mechanism known as non-homologous end-joining (NHEJ) (error-prone). NHEJ is a non-templated DNA repair process, in which DNA termini are directly ligated. Canonical NHEJ requires DNA-PKcs and Ku70/80, while alternative NHEJ pathways are DNA-PKcs and Ku70/80 independent. This review discusses the role of RecQ helicases in NHEJ, alternative (or back-up) NHEJ (B-NHEJ) and microhomology-mediated end-joining (MMEJ) in V(D)J recombination, class switch recombination and telomere maintenance.     

Development of a high-content high-throughput screening assay for the discovery of ATM signaling inhibitors

The genome is constantly exposed to DNA damage agents, leading up to as many as 1 million individual lesions per cell per day. Cells have developed a variety of DNA damage repair (DDR) mechanisms to respond to harmful effects of DNA damage. Failure to repair the damaged DNA causes genomic instability and, as a result, leads to cellular transformation. Indeed, deficiencies of DDR frequently occur in human cancers, thus providing a great opportunity for cancer therapy by developing anticancer agents that work by synthetic lethality-based mechanisms or enhancing the clinical efficacy of radiotherapy and existing chemotherapies. Ataxia-telangiectasia mutated (ATM) plays a key role in regulating the cellular response to DNA double-strand breaks. Ionizing radiation causes double-strand breaks and induces rapid ATM autophosphorylation on serine 1981 that initiates ATM kinase activity. Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage-response pathways, most notably cell-cycle checkpoints. We describe here the development and validation of a high-throughput imaging assay measuring levels of phospho-ATM Ser1981 in HT29 cells after exposure to ionizing radiation. We also examined activation of downstream ATM effectors and checked specificity of the endpoint using known inhibitors of DNA repair pathways.