Caffeine-induced radiosensitization is independent of nonhomologous end joining of DNA double-strand breaks

After exposure to ionizing radiation, proliferating cells actively slow down progression through the cell cycle through the activation of checkpoints to provide time for repair. Two major complementary DNA double-strand break (DSB) repair pathways exist in mammalian cells, homologous recombination repair (HRR) and nonhomologous end joining (NHEJ). The relationship between checkpoint activation and these two types of DNA DSB repair pathways is not clear. Caffeine, as a nonspecific inhibitor of ATM and ATR, abolishes multi-checkpoint responses and sensitizes cells to radiation-induced killing. However, it remains unknown which DNA repair process, NHEJ or HRR, or both, is affected by caffeine-abolished checkpoint responses. We report here that caffeine abolishes the radiation-induced G(2)-phase checkpoint and efficiently sensitizes both NHEJ-proficient and NHEJ-deficient mammalian cells to radiation-induced killing without affecting NHEJ. Our results indicate that caffeine-induced radiosensitization occurs by affecting an NHEJ-independent process, possibly HRR.     

Dynamic Dependence on ATR and ATM for Double-Strand Break Repair in Human Embryonic Stem Cells and Neural Descendants

The DNA double-strand break (DSB) is the most toxic form of DNA damage. Studies aimed at characterizing DNA repair during development suggest that homologous recombination repair (HRR) is more critical in pluripotent cells compared to differentiated somatic cells in which nonhomologous end joining (NHEJ) is dominant. We have characterized the DNA damage response (DDR) and quality of DNA double-strand break (DSB) repair in human embryonic stem cells (hESCs), and in vitro-derived neural cells. Resolution of ionizing radiation-induced foci (IRIF) was used as a surrogate for DSB repair. The resolution of γ-H2AX foci occurred at a slower rate in hESCs compared to neural progenitors (NPs) and astrocytes perhaps reflective of more complex DSB repair in hESCs. In addition, the resolution of RAD51 foci, indicative of active homologous recombination repair (HRR), showed that hESCs as well as NPs have high capacity for HRR, whereas astrocytes do not. Importantly, the ATM kinase was shown to be critical for foci formation in astrocytes, but not in hESCs, suggesting that the DDR is different in these cells. Blocking the ATM kinase in astrocytes not only prevented the formation but also completely disassembled preformed repair foci. The ability of hESCs to form IRIF was abrogated with caffeine and siRNAs targeted against ATR, implicating that hESCs rely on ATR, rather than ATM for regulating DSB repair. This relationship dynamically changed as cells differentiated. Interestingly, while the inhibition of the DNA-PKcs kinase (and presumably non-homologous endjoining [NHEJ]) in astrocytes slowed IRIF resolution it did not in hESCs, suggesting that repair in hESCs does not utilize DNA-PKcs. Altogether, our results show that hESCs have efficient DSB repair that is largely ATR-dependent HRR, whereas astrocytes critically depend on ATM for NHEJ, which, in part, is DNA-PKcs-independent.     

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.     

Ionizing radiation and genetic risks XIV. Potential research directions in the post-genome era based on knowledge of repair of radiation-induced DNA double-strand breaks in mammalian somatic cells and the origin of deletions associated with human genomic disorders

Recent estimates of genetic risks from exposure of human populations to ionizing radiation are those presented in the 2001 report of the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR). These estimates incorporate two important concepts, namely, the following: (1) most radiation-induced mutations are DNA deletions, often encompassing multiple genes, but only a small proportion of the induced deletions is compatible with offspring viability; and (2) the viability-compatible deletions induced in germ cells are more likely to manifest themselves as multi-system developmental anomalies rather than as single gene disorders. This paper: (a) pursues these concepts further in the light of knowledge of mechanisms of origin of deletions and other rearrangements from two fields of contemporary research: repair of radiation-induced DNA double-strand breaks (DSBs) in mammalian somatic cells and human molecular genetics; and (b) extends them to deletions induced in the germ cell stages of importance for radiation risk estimation, namely, stem cell spermatogonia in males and oocytes in females. DSB repair studies in somatic cells have elucidated the roles of two mechanistically distinct pathways, namely, homologous recombination repair (HRR) that utilizes extensive sequence homology and non-homologous end-joining (NHEJ) that requires little or no homology at the junctions. A third process, single-strand annealing (SSA), which utilizes short direct repeat sequences, is considered a variant of HRR. HRR is most efficient in late S and G2 phases of the cell cycle and is a high fidelity mechanism. NHEJ operates in all cell cycle phases, but is especially important in G1. In the context of radiation-induced DSBs, NHEJ is error-prone. SSA is also an error-prone mechanism and its role is presumably similar to that of HRR. Studies in human molecular genetics have demonstrated that the occurrence of large deletions, duplications or other rearrangements in certain regions of the genome is related to the presence of large segments of repetitive DNA called segmental duplications (also called duplicons or low copy repeats, LCRs) in such regions. The mechanism that is envisaged for the origin of deletions and other rearrangements involves misalignment of region-specific LCRs of homologous chromosomes in meiosis followed by unequal crossing-over (i.e., non-allelic homologous recombination, NAHR). We hypothesize that: (a) in spermatogonial stem cells, NHEJ is probably the principal mechanism underlying the origin of radiation-induced deletions, although SSA and NAHR may also be involved to some extent, especially at low doses; and (b) in irradiated oocytes, NAHR is likely to be the main mechanism for generating deletions. We suggest future research possibilities, including the development of models for identifying regions of the genome that are susceptible to radiation-induced deletions. Such efforts may have particular significance in the context of the estimation of genetic risks of radiation exposure of human females, a problem that is still with us.     

P element excision and repair by non-homologous end joining occurs in both G1 and G2 of the cell cycle

P element excision generates a DNA double-strand break at the transposon donor site. Genetic studies have demonstrated a strong bias toward repair of P element-induced DNA breaks by homologous recombination with the sister chromatid, suggesting that P element excision occurs after DNA replication, in G2 of the cell cycle. We developed methods to arrest Drosophila tissue culture cells and assay P element excision in either G1- or G2-arrested cells. Dacapo or tribbles transgene expression arrests cells in either G2 or G2, respectively. RNA-mediated gene interference (RNAi) directed against cyclin E or cyclin A arrests cells in G1 or G2, respectively. P element excision occurs efficiently in both G1- and G2-arrested cells, suggesting that cell cycle regulation of P element transposase does not occur in our somatic cell system. DNA double-strand break repair occurs by two predominant mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is thought to be restricted to the post-replicative, G2, phase of the cell cycle, while NHEJ may occur throughout the cell cycle. Our results indicate that NHEJ repair of an extrachromasomal plasmid substrate occurs at least as efficiently in G2-arrested cells as in asynchronous cells or in G1-arrested cells.     

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.     

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.     

DNA Ligase IV and Artemis Act Cooperatively to Suppress Homologous Recombination in Human Cells: Implications for DNA Double-Strand Break Repair

Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are two major pathways for repairing DNA double-strand breaks (DSBs); however, their respective roles in human somatic cells remain to be elucidated. Here we show using a series of human gene-knockout cell lines that NHEJ repairs nearly all of the topoisomerase II- and low-dose radiation-induced DNA damage, while it negatively affects survival of cells harbouring replication-associated DSBs. Intriguingly, we find that loss of DNA ligase IV, a critical NHEJ ligase, and Artemis, an NHEJ factor with endonuclease activity, independently contribute to increased resistance to replication-associated DSBs. We also show that loss of Artemis alleviates hypersensitivity of DNA ligase IV-null cells to low-dose radiation- and topoisomerase II-induced DSBs. Finally, we demonstrate that Artemis-null human cells display increased gene-targeting efficiencies, particularly in the absence of DNA ligase IV. Collectively, these data suggest that DNA ligase IV and Artemis act cooperatively to promote NHEJ, thereby suppressing HR. Our results point to the possibility that HR can only operate on accidental DSBs when NHEJ is missing or abortive, and Artemis may be involved in pathway switching from incomplete NHEJ to HR.     

Homologous and non-homologous recombination differentially affect DNA damage repair in mice

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents are modulated by the ability of cells to repair the inflicted DNA damage. Here we demonstrate that homologous recombination-deficient mRAD54(-/-) mice are hypersensitive to ionizing radiation at the embryonic but, unexpectedly, not at the adult stage. However, at the adult stage mRAD54 deficiency dramatically aggravates the ionizing radiation sensitivity of severe combined immune deficiency (scid) mice that are impaired in DNA double-strand break repair through DNA end-joining. In contrast, regardless of developmental stage, mRAD54(-/-) mice are hypersensitive to the interstrand DNA crosslinking compound mitomycin C. These results demonstrate that the two major DNA double-strand break repair pathways in mammals have overlapping as well as specialized roles, and that the relative contribution of these pathways towards repair of ionizing radiation-induced DNA damage changes during development of the animal.     

RAD-51-dependent and -independent roles of a Caenorhabditis elegans BRCA2-related protein during DNA double-strand break repair

The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.     

Nonhomologous end joining of complementary and noncomplementary DNA termini in mouse testicular extracts

Mammalian somatic cells are known to repair DNA double-strand breaks (DSBs) by nonhomologous end joining (NHEJ) and homologous recombination (HR); however, how male germ cells repair DSBs is not yet characterized. We have previously reported the highly efficient and mostly precise DSB joining ability of mouse testicular germ cell extracts for cohesive and blunt ends, with only a minor fraction undergoing terminal deletion [Mutat. Res. 433 (1999) 1]; however, the precise mechanism of joining was not established. In the present study, we therefore tested the ability of testicular extracts to join noncomplementary ends; we have also sequenced the junctions of both complementary and noncomplementary termini and established the joining mechanisms. While a major proportion of complementary and blunt ends were joined by simple ligation, the small fraction having noncleavable junctions predominantly utilized short stretches of direct repeat homology with limited end processing. For noncomplementary ends, the major mechanism was "blunt-end ligation" subsequent to "fill-in" or "blunting", with no insertions or large deletions; the microhomology-dependent joining with end deletion was less frequent. This is the first functional study of the NHEJ mechanism in mammalian male germ cell extracts. Our results demonstrate that testicular germ cell extracts promote predominantly accurate NHEJ for cohesive ends and very efficient blunt-end ligation, perhaps to preserve the genomic sequence with minimum possible alteration. Further, we demonstrate the ability of the extracts to catalyze in vitro plasmid homologous recombination, which suggests the existence of both NHEJ and HR pathways in germ cells.     

To trim or not to trim: Progression and control of DSB end resection

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5′-3′ nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.     

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.     

A Robust Network of Double-Strand Break Repair Pathways Governs Genome Integrity during C. elegans Development

To preserve genomic integrity, various mechanisms have evolved to repair DNA double-strand breaks (DSBs) [1]. Depending on cell type or cell cycle phase, DSBs can be repaired error-free, by homologous recombination, or with concomitant loss of sequence information, via nonhomologous end-joining (NHEJ) or single-strand annealing (SSA) [2]. Here, we created a transgenic reporter system in C. elegans to investigate the relative contribution of these pathways in somatic cells during animal development. Although all three canonical pathways contribute to repair in the soma, in their combined absence, animals develop without growth delay and chromosomal breaks are still efficiently repaired. This residual repair, which we call alternative end-joining, dominates DSB repair only in the absence of NHEJ and resembles SSA, but acts independent of the SSA nuclease XPF and repair proteins from other pathways. The dynamic interplay between repair pathways might be developmentally regulated, because it was lost from terminally differentiated cells in adult animals. Our results demonstrate profound versatility in DSB repair pathways for somatic cells of C. elegans, which are thus extremely fit to deal with chromosomal breaks.     

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.     

DNA双链断裂的非同源末端连接修复

细胞内普遍存在的DNA双链断裂(DSB)可通过同源重组(HR)或非同源末端连接(NHEJ)修复。由于HR仅在存在相同染色体作为模板的时候进行,因此,NHEJ通常为主要的修复方式。在NHEJ中,DSB末端首先由Ku识别,接着由核酸酶、聚合酶在Ku与DNA-PKcs协助下加工,并由连接酶IVXRCC4-XLF连接。NHEJ底物类型多样,末端的修复常包含反复加工的过程,导致修复产物通常无法复原损伤前的序列。虽然无法确保准确修复DNA,NHEJ仍对维持基因组的稳定性具有重要的意义。对NHEJ的研究有助于理解癌症的发生机制并将促进癌症的治疗。     

NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must first be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells.     

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

Requirement for the SRS2 DNA helicase gene in non-homologous end joining in yeast

Mitotic cells experience double-strand breaks (DSBs) from both exogenous and endogenous sources. Since unrepaired DSBs can result in genome rearrangements or cell death, cells mobilize multiple pathways to repair the DNA damage. In the yeast Saccharomyces cerevisiae, mitotic cells preferentially use a homologous recombination repair pathway. However, when no significant homology to the DSB ends is available, cells utilize a repair process called non-homologous end joining (NHEJ), which can join ends with no homology through resection to uncover microhomologies of a few nucleotides. Although components of the homologous recombination repair system are also involved in NHEJ, the rejoining does not involve all of the homologous recombination repair genes. The SRS2 DNA helicase has been shown to be required for DSB repair when the homologous single-stranded regions are short. Here it is shown that SRS2 is also required for NHEJ, regardless of the cell mating type. Efficient NHEJ of sticky ends requires the Ku70 and Ku80 proteins and the silencing genes SIR2, SIR3 and SIR4. However, NHEJ of blunt ends, while very inefficient, is not further reduced by mutations in YKU70, SIR2, SIR3, SIR4 or SRS2, suggesting that this rejoining process occurs by a different mechanism.     

BRCA1-Ku80 Protein Interaction Enhances End-joining Fidelity of Chromosomal Double-strand Breaks in the G1 Phase of the Cell Cycle

Quality control of DNA double-strand break (DSB) repair is vital in preventing mutagenesis. Non-homologous end-joining (NHEJ), a repair process predominant in the G₁ phase of the cell cycle, rejoins DSBs either accurately or with errors, but the mechanisms controlling its fidelity are poorly understood. Here we show that BRCA1, a tumor suppressor, enhances the fidelity of NHEJ-mediated DSB repair and prevents mutagenic deletional end-joining through interaction with canonical NHEJ machinery during G₁. BRCA1 binds and stabilizes Ku80 at DSBs through its N-terminal region, promotes precise DSB rejoining, and increases cellular resistance to radiation-induced DNA damage in a G₁ phase-specific manner. These results suggest that BRCA1, as a central player in genome integrity maintenance, ensures high fidelity repair of DSBs by not only promoting homologous recombination repair in G₂/M phase but also facilitating fidelity of Ku80-dependent NHEJ repair, thus preventing deletional end-joining of chromosomal DSBs during G₁.