DNA repair patch-mediated double strand DNA break formation in human cells

To investigate the mechanism of double strand DNA break formation in mammalian cells, an in vitro assay was established using closed circular DNA containing two uracils on opposite DNA strands (18 and 30 base pairs apart) and extracts prepared from human cells. In this assay, formation of double strand breaks was detected by the conversion of circular DNA to linear DNA. Approximately 4-fold more double strand DNA breaks were produced by extracts from cells deficient in DNA ligase I (46BR) relative to those produced by extracts from control cells (MRC5, derived from a clinically normal individual). In parallel with the amount of double strand DNA breaks, extracts from 46BR cells produced longer repair patches (up to 24 bases in length) than those from MRC5 cells (typically <5 bases long). When purified DNA ligase I was added to 46BR extracts to complement the DNA ligase deficiency, only a negligible difference was found between the amount of doublestrand DNA breaks or the repair patch size generated in the assay relative to MRC5 extracts. Together, our data demonstrate that double strand DNA breaks are produced through formation of DNA repair patches. We refer to this process of double strand break formation as the "DNA repair patch-mediated pathway."     

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Roles of chromatin remodellers in DNA double strand break repair

Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.     

X-ray induced DNA double strand break production and repair in mammalian cells as measured by neutral filter elution.

This work presents a neutral filter elution method for detecting DNA double strand breaks in mouse L1210 cells after X-ray. The assay will detect the number of double strand breaks induced by as little as 1000 rad of X-ray. The rate of DNA elution through the filters under neutral conditions increases with X-ray dose. Certain conditions for deproteinization, pH, and filter type are shown to increase the assay's sensitivity. Hydrogen peroxide and Bleomycin also induce apparent DNA double strand breaks, although the ratios of double to single strand breaks vary from those produced by X-ray. The introduction of double strand cuts by HpA I restriction endonuclease in DNA lysed on filters results in a rapid rate of elution under neutral conditions, implying that the method can detect double strand breaks if they exist in the DNA. The eluted DNA bands with a double stranded DNA marker in cesium chloride. This evidence suggests that the assay detects DNA double strand breaks. L1210 cells are shown to rejoin most of the DNA double strand breaks induced by 5-10 krad of X-ray with a half-time of about 40 minutes.     

Chromatin structure in double strand break repair

Cells are under constant assault by endogenous and environmental DNA damaging agents. DNA double strand breaks (DSBs) sever entire chromosomes and pose a major threat to genome integrity as a result of chromosomal fragment loss or chromosomal rearrangements. Exogenous factors such as ionizing radiation, crosslinking agents, and topoisomerase poisons, contribute to break formation. DSBs are associated with oxidative metabolism, form during the normal S phase, when replication forks collapse and are generated during physiological processes such as V(D)J recombination, yeast mating type switching and meiosis. It is estimated that in mammalian cells ∼10 DSBs per cell are formed daily. If left unrepaired DSBs can lead to cell death or deregulated growth, and cancer development. Cellular response to DSB damage includes mechanisms to halt the progression of the cell cycle and to restore the structure of the broken chromosome. Changes in chromatin adjacent to DNA break sites are instrumental to the DNA damage response (DDR) with two apparent ends: to control compaction and to bind repair and signaling molecules to the lesion. Here, we review the key findings related to each of these functions and examine their cross-talk.     

Targeting the DNA double strand break repair machinery in prostate cancer

Regardless of the achievable remissions with first line hormone therapy in patients with prostate cancer (CaP), the disease escapes the hormone dependent stage to a more aggressive status where chemotherapy is the only effective treatment and no treatment is curative. This makes it very important to identify new targets that can improve the outcome of treatment. ATM and DNA-PK are the two kinases responsible for signalling and repairing double strand breaks (DSB). Thus, both kinases are pertinent targets in CaP treatment to enhance the activity of the numerous DNA DSB inducing agents used in CaP treatment such as ionizing radiation (IR). Colony formation assay was used to assess the sensitivity of hormone dependent, p53 wt (LNCaP) and hormone independent p53 mutant (PC3) CaP cell lines to the cytotoxic effect of IR and Doxorubicin in the presence or absence of Ku55933 and NU7441 which are small molecule inhibitors of ATM and DNA-PK, respectively. Flow cytometry based methods were used to assess the effect of the two inhibitors on cell cycle, apoptosis and H2AX foci formation. Neutral comet assay was used to assess the induction of DNA DSBs. Ku55933 or NU7441 alone increased the sensitivity of CaP cell lines to the DNA damaging agents, however combining both inhibitors together resulted in further enhancement of sensitivity. The cell cycle profile of both cell lines was altered with increased cell death, DNA DSBs and H2AX foci formation. This study justifies further evaluation of the ATM and DNA-PK inhibitors for clinical application in CaP patients. Additionally, the augmented effect resulting from combining both inhibitors may have a significant implication for the treatment of CaP patients who have a defect in one of the two DSB repair pathways.     

A DNA double strand break repair defect in Fanconi anemia fibroblasts

Fanconi anemia (FA) is a heterogeneous autosomal recessive disease characterized by congenital abnormalities, pancytopenia, and an increased incidence of cancer. Cells cultured from FA patients display elevated spontaneous chromosomal breaks and deletions and are hypersensitive to bifunctional cross-linking agents. Thus, it has been hypothesized that FA is a DNA repair disorder. We analyzed plasmid end-joining in intact diploid fibroblast cells derived from FA patients. FA fibroblasts from complementation groups A, C, D2, and G rejoined linearized plasmids with a significantly decreased efficiency compared with non-FA fibroblasts. Retrovirus-mediated expression of the respective FA cDNAs in FA cells restored their end-joining efficiency to wild type levels. Human FA fibroblasts and fibroblasts from FA rodent models were also significantly more sensitive to restriction enzyme-induced chromosomal DNA double strand breaks than were their retrovirally corrected counterparts. Taken together, these data show that FA fibroblasts have a deficiency in both extra-chromosomal and chromosomal DNA double strand break repair, a defect that could provide an attractive explanation for some of the pathologies associated with FA.     

DNA double strand break repair: Zooming in on the focus

Comment on: Pseudo-DNA damage response in senescent cells. Pospelova T, et al. Cell Cycle 2009; 8:In press.     

RecQ helicases in DNA double strand break repair and telomere maintenance

Organisms are constantly exposed to various environmental insults which could adversely affect the stability of their genome. To protect their genomes against the harmful effect of these environmental insults, organisms have evolved highly diverse and efficient repair mechanisms. Defective DNA repair processes can lead to various kinds of chromosomal and developmental abnormalities. RecQ helicases are a family of evolutionarily conserved, DNA unwinding proteins which are actively engaged in various DNA metabolic processes, telomere maintenance and genome stability. Bacteria and lower eukaryotes, like yeast, have only one RecQ homolog, whereas higher eukaryotes including humans possess multiple RecQ helicases. These multiple RecQ helicases have redundant and/or non-redundant functions depending on the types of DNA damage and DNA repair pathways. Humans have five different RecQ helicases and defects in three of them cause autosomal recessive diseases leading to various kinds of cancer predisposition and/or aging phenotypes. Emerging evidence also suggests that the RecQ helicases have important roles in telomere maintenance. This review mainly focuses on recent knowledge about the roles of RecQ helicases in DNA double strand break repair and telomere maintenance which are important in preserving genome integrity.     

Dynamic formation of RecA filaments at DNA double strand break repair centers in live cells

We show that RecN protein is recruited to a defined DNA double strand break (DSB) in Bacillus subtilis cells at an early time point during repair. Because RecO and RecF are successively recruited to DSBs, it is now clear that dynamic DSB repair centers (RCs) exist in prokaryotes. RecA protein was also recruited to RCs and formed highly dynamic filamentous structures, which we term threads, across the nucleoids. Formation of RecA threads commenced approximately 30 min after the induction of DSBs, after RecN recruitment to RCs, and disassembled after 2 h. Time-lapse microscopy showed that the threads rapidly changed in length, shape, and orientation within minutes and can extend at 1.02 microm/min. The formation of RecA threads was abolished in recJ addAB mutant cells but not in each of the single mutants, suggesting that RecA filaments can be initiated via two pathways. Contrary to proteins forming RCs, DNA polymerase I did not form foci but was present throughout the nucleoids (even after induction of DSBs or after UV irradiation), suggesting that it continuously scans the chromosome for DNA lesions.     

Mechanisms of DNA double strand break repair and chromosome aberration formation

It is widely accepted that unrepaired or misrepaired DNA double strand breaks (DSBs) lead to the formation of chromosome aberrations. DSBs induced in the DNA of higher eukaryotes by endogenous processes or exogenous agents can in principle be repaired either by non-homologous endjoining (NHEJ), or homology directed repair (HDR). The basis on which the selection of the DSB repair pathway is made remains unknown but may depend on the inducing agent, or process. Evaluation of the relative contribution of NHEJ and HDR specifically to the repair of ionizing radiation (IR) induced DSBs is important for our understanding of the mechanisms leading to chromosome aberration formation. Here, we review recent work from our laboratories contributing to this line of inquiry. Analysis of DSB rejoining in irradiated cells using pulsed-field gel electrophoresis reveals a fast component operating with half times of 10-30 min. This component of DSB rejoining is severely compromised in cells with mutations in DNA-PKcs, Ku, DNA ligase IV, or XRCC4, as well as after chemical inhibition of DNA-PK, indicating that it reflects classical NHEJ; we termed this form of DSB rejoining D-NHEJ to signify its dependence on DNA-PK. Although chemical inhibition, or mutation, in any of these factors delays processing, cells ultimately remove the majority of DSBs using an alternative pathway operating with slower kinetics (half time 2-10 h). This alternative, slow pathway of DSB rejoining remains unaffected in mutants deficient in several genes of the RAD52 epistasis group, suggesting that it may not reflect HDR. We proposed that it reflects an alternative form of NHEJ that operates as a backup (B-NHEJ) to the DNA-PK-dependent (D-NHEJ) pathway. Biochemical studies confirm the presence in cell extracts of DNA end joining activities operating in the absence of DNA-PK and indicate the dominant role for D-NHEJ, when active. These observations in aggregate suggest that NHEJ, operating via two complementary pathways, B-NHEJ and D-NHEJ, is the main mechanism through which IR-induced DSBs are removed from the DNA of higher eukaryotes. HDR is considered to either act on a small fraction of IR induced DSBs, or to engage in the repair process at a step after the initial end joining. We propose that high speed D-NHEJ is an evolutionary development in higher eukaryotes orchestrated around the newly evolved DNA-PKcs and pre-existing factors. It achieves within a few minutes restoration of chromosome integrity through an optimized synapsis mechanism operating by a sequence of protein-protein interactions in the context of chromatin and the nuclear matrix. As a consequence D-NHEJ mostly joins the correct DNA ends and suppresses the formation of chromosome aberrations, albeit, without ensuring restoration of DNA sequence around the break. B-NHEJ is likely to be an evolutionarily older pathway with less optimized synapsis mechanisms that rejoins DNA ends with kinetics of several hours. The slow kinetics and suboptimal synapsis mechanisms of B-NHEJ allow more time for exchanges through the joining of incorrect ends and cause the formation of chromosome aberrations in wild type and D-NHEJ mutant cells.     

Characterization of mammalian RAD51 double strand break repair using non-lethal dominant-negative forms

In contrast to yeast RAD51, mammalian mRAD51 is an essential gene. Its role in double strand break (DSB) repair and its consequences on cell viability remain to be characterized precisely. Here, we used a hamster cell line carrying tandem repeat sequences with an I-SCE:I cleavage site. We characterized conservative recombination after I-SCE:I cleavage as gene conversion or intrachromatid crossing over associated with random reintegration of the excised reciprocal product. We identified two dominant-negative RAD51 forms that specifically inhibit conservative recombination: the yeast ScRAD51 or the yeast-mouse chimera SMRAD51. In contrast, the mouse MmRAD51 stimulates conservative recombination. None of these RAD51 forms affects non-conservative recombination or global DSB healing. Consistently, although resistance to gamma-rays remains unaffected, MmRAD51 stimulates whereas ScRAD51 or SMRAD51 prevents radiation-induced recombination. This suggests that mRAD51 does not significantly affect the global DSB repair efficiency but controls the classes of recombination events. Finally, both ScRAD51 and SMRAD51 drastically inhibit spontaneous recombination but not cell proliferation, showing that RAD51-dependent spontaneous and DSB-induced conservative recombination can be impaired significantly without affecting cell viability.     

DNA strand break repair and neurodegeneration

A number of DNA repair disorders are known to cause neurological problems. These disorders can be broadly characterised into early developmental, mid-to-late developmental or progressive. The exact developmental processes that are affected can influence disease pathology, with symptoms ranging from early embryonic lethality to late-onset ataxia. The category these diseases belong to depends on the frequency of lesions arising in the brain, the role of the defective repair pathway, and the nature of the mutation within the patient. Using observations from patients and transgenic mice, we discuss the importance of double strand break repair during neuroprogenitor proliferation and brain development and the repair of single stranded lesions in neuronal function and maintenance.     

Nijmegen breakage syndrome: consequences of defective DNA double strand break repair

The autosomal recessive genetic disorder, Nijmegen Breakage Syndrome, is characterised by an excessively high risk for the development of lymphatic tumours and an extreme sensitivity towards ionising radiation. The most likely explanation for these characteristics, a deficiency in the repair of DNA lesions, has been greatly substantiated by the recent cloning of the gene mutated in Nijmegen Breakage Syndrome patients and the analysis of its protein product, nibrin. The direct involvement of this protein in the processing of DNA double strand breaks caused by ionising radiation and those also necessary for normal DNA metabolism can be correlated with many of the cellular and clinical aspects of the disease, including the cancer predisposition of patients and their heterozygous relatives. BioEssays 21:649–656, 1999. © 1999 John Wiley & Sons, Inc.     

A PIAS-ed view of DNA double strand break repair focuses on SUMO

Through the action of multiple sensors, mediators, and effectors, the DNA damage response (DDR) orchestrates the repair of DNA damage to ensure maintenance of genomic integrity. Recently, in addition to phosphorylation, other post-translational modifications such as ubiquitylation and SUMOylation have emerged as important regulators of the DDR network. Two recent papers highlight the importance of SUMO modifications of proteins that execute the response to DNA damage.     

Molecular mechanisms of double strand break repair in eukaryotes

Genome stability is of primary importance for the survival and for the proper functioning of all organisms. Double strand breaks (DSBs) arise spontaneously during growth, or can be created by external insults. In response to even a single DSB, organisms must trigger series of events to promote repair of the DNA damage in order to survive and restore chromosome integrity. In doing so, cells must regulate a fine balance between potentially competing DSB repair pathways. Much of what we know today on the mechanisms of repair in eukaryotes come from studies carried out in budding yeast. In this review, the main attention is focused on exciting new work eminating from yeast research that provides fresh insights into the DSB repair process.     

XRCC1 and DNA strand break repair

DNA single-strand breaks can arise indirectly, as normal intermediates of DNA base excision repair, or directly from damage to deoxyribose. Because single-strand breaks are induced by endogenous reactive molecules such as reactive oxygen species, these lesions pose a continuous threat to genetic integrity. XRCC1 protein plays a major role in facilitating the repair of single-strand breaks in mammalian cells, via an ability to interact with multiple enzymatic components of repair reactions. Here, the protein-protein interactions facilitated by XRCC1, and the repair processes in which these interactions operate, are reviewed. Models for the repair of single-strand breaks during base excision repair and at direct breaks are presented.     

Co-ordination of DNA single strand break repair

DNA damaging agents generated as a consequence of endogenous metabolism or via exogenous factors can produce a wide variety of lesions in DNA. These include base damage, sites of base loss (abasic sites) and single strand breaks (SSBs). Moreover, reactive oxygen species (ROS) create more diversity by generating SSBs containing modified 3'-ends, such as those containing phosphate, phosphoglycolate and oxidative base damage. Ionising radiation also generates DNA base lesions in close proximity to SSBs. The majority of these non-bulky lesions in DNA are repaired by proteins involved in the base excision repair (BER) pathway. It is apparent that due to the complexity of these lesions, they may require individual subsets of BER proteins for repair. However, the mechanism unravelling the required enzymes and directing damage-specific repair of SSBs is unclear. In this review we will discuss recent studies that identify new enzymes and activities involved in the repair of SSBs containing modified ends and in particular outline the possible mechanisms involved in the co-ordinated repair of "damaged" SSBs that can not be resealed directly and require preliminary processing.     

Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice

DNA double strand breaks are the most cytotoxic lesions that can occur on the DNA. They can be repaired by different mechanisms and optimal survival requires a tight control between them. Here we uncover protein deneddylation as a major controller of repair pathway choice. Neddylation inhibition changes the normal repair profile toward an increase on homologous recombination. Indeed, RNF111/UBE2M-mediated neddylation acts as an inhibitor of BRCA1 and CtIP-mediated DNA end resection, a key process in repair pathway choice. By controlling the length of ssDNA produced during DNA resection, protein neddylation not only affects the choice between NHEJ and homologous recombination but also controls the balance between different recombination subpathways. Thus, protein neddylation status has a great impact in the way cells respond to DNA breaks.