DNA Double-strand Break Repair Research Articles

The Role of Microhomology-Mediated End Joining (MMEJ) at Dysfunctional Telomeres.

DNA double-strand break (DSB) repair pathways are crucial for maintaining genome stability and cell viability. However, these pathways can mistakenly recognize chromosome ends as DNA breaks, leading to adverse outcomes such as telomere fusions and malignant transformation. The shelterin complex protects telomeres from activation of DNA repair pathways by inhibiting nonhomologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). The focus of this paper is on MMEJ, an error-prone DSB repair pathway characterized by short insertions and deletions flanked by sequence homology. MMEJ is critical in mediating telomere fusions in cells lacking the shelterin complex and at critically short telomeres. Furthermore, studies suggest that MMEJ is the preferred pathway for repairing intratelomeric DSBs and facilitates escape from telomere crisis. Targeting MMEJ to prevent telomere fusions in hematologic malignancies is of potential therapeutic value.

TPX2 serves as a novel target for expanding the utility of PARPi in pancreatic cancer through conferring synthetic lethality

BackgroundPARP inhibitors (PARPi) have been licensed for the maintenance therapy of patients with metastatic pancreatic cancer carrying pathogenic germline BRCA1/2 mutations. However, mutations in BRCA1/2 are notably rare in pancreatic...

Senataxin Attenuates DNA Damage Response Activation and Suppresses Senescence

Oxidative stress, driven by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), induces DNA double-strand breaks (DSBs) that compromise genomic integrity. The DNA Damage Response (DDR), primarily mediated by ATM and ATR kinases, is crucial for recognizing and repairing DSBs. Senataxin (SETX), a DNA/RNA helicase, is critical in resolving R-loops, with mutations in SETX associated with neurodegenerative diseases. This study uncovers a novel function of senataxin in modulating DDR and its impact on cellular senescence. Senataxin is shown to be crucial not only for DSB repair but also for determining cell fate under oxidative stress. SETX knockout cells show impaired DSB repair and prolonged ATM/ATR signaling detected by Western blotting, leading to increased senescence, as indicated by elevated β-galactosidase activity following H2O2 exposure and I-PpoI-induced DSBs. Wild-type cells exhibit higher apoptosis levels compared to SETX knockout cells under H2O2 treatment, suggesting that senataxin promotes apoptosis over senescence in oxidative stress. This indicates that senataxin plays a protective role against the accumulation of senescent cells, potentially mitigating age-related cellular decline and neurodegenerative disease progression. These findings highlight senataxin as a critical mediator in DDR pathways and a potential therapeutic target for conditions where cellular senescence contributes to disease pathology.

Cohesin complex oligomerization maintains end-tethering at DNA double-strand breaks.

DNA double-strand breaks (DSBs) must be repaired to ensure genome stability. Crucially, DSB-ends must be kept together for timely repair. In Saccharomyces cerevisiae, two pathways mediate DSB end-tethering. One employs the Mre11-Rad50-Xrs2 (MRX) complex to physically bridge DSB-ends. Another requires the conversion of DSB-ends into single-strand DNA (ssDNA) by Exo1, but the bridging proteins are unknown. We uncover that cohesin, its loader and Smc5/6 act with Exo1 to tether DSB-ends. Remarkably, cohesin specifically impaired in oligomerization fails to tether DSB-ends, revealing a function for cohesin oligomerization. In addition to the known importance of sister chromatid cohesion, microscopy-based microfluidic experiments unveil a role for cohesin in repair by ensuring DSB end-tethering. Altogether, our findings demonstrate that oligomerization of cohesin prevents DSB end-separation and promotes DSB repair, revealing a previously undescribed mode of action and role for cohesin in safeguarding genome integrity.

Gold nanoparticles cause radiosensitization in 4T1 cells by inhibiting DNA double strand break repair: Single cell comparisons of DSB formation and γH2AX expression.

Metal nanoparticles sensitize cancers to radiotherapy however their mechanisms of action are complex. The conceptual inspiration arose from theories of physical dose deposition but various chemical and biological factors have also been identified. Interpretation of data has been limited by challenges in measuring true DNA damage compared to DNA damage repair factors. Here, we applied a new assay, STRIDE, for the first time to measure DNA double strand breaks (DSBs) in 4T1 cells as a model of triple negative breast cancer exposed to gold nanoparticles and radiation, and compared this to the common γH2AX assay for DSB repair. The STRIDE assay showed no increase in DSB detection 15 mins after irradiation for cells containing nanoparticles compared to cells without. Gold nanoparticles led to prolonged detection of DSBs after irradiation and delayed the DSB repair. The data show no evidence of increased radiation dose deposition with nanoparticles, but rather enhanced radiobiological effects resulting from nanoparticles which includes disruption of the recruitment of essential DDR machinery, thereby impairing DNA repair processes.

Tissue microarray analyses of the essential DNA repair factors ATM, DNA-PKcs and Ku80 in head and neck squamous cell carcinoma

BackgroundHead and neck squamous cell carcinoma (HNSCC) negative for Human Papillomavirus (HPV) has remained a difficult to treat entity, whereas tumors positive for HPV are characterized by radiosensitivity and favorable patient outcome. On the cellular level, radiosensitivity is largely governed by the tumor cells` ability to repair radiation-induced DNA double-strand breaks (DSBs), but no biomarker is established that could guide clinical decision making. Therefore, we tested the impact of the expression levels of ATM, the central kinase of the DNA damage response as well as DNA-PKcs and Ku80, two major factors in the main DSB repair pathway non-homologous end joining (NHEJ).MethodsA tissue microarray of a single center HNSCC cohort was stained for ATM, DNA-PKcs and Ku80 and the expression scored based on staining intensity and the percentages of tumor cells stained. Scores were correlated with clinicopathological parameters and survival.ResultsSamples from 427 HNSCC patients yielded interpretable stainings and were scored following an established algorithm. The majority of tumors showed strong expression of both NHEJ factors, whereas the expression of ATM varied more. The expression scores of ATM and DNA-PKcs were not associated with patient survival. For HPV-negative HNSCC, the minority of tumors without strong Ku80 expression trended towards superior survival when treatment included radiotherapy. Focusing stronger on staining intensity to define the subgroup with lowest and therefore potentially insufficient expression levels in the HPV-negative subgroup, we observed significantly better overall survival for patients treated with radiotherapy but not with surgery alone.ConclusionsOur data suggest that HPV-negative HNSCC with particularly low Ku80 expression represent a highly radiosensitive subpopulation. Confirmation in independent cohorts is required.

From Zebrafish To Humans: In Silico Comparative Study of RAD50 Sequences

DNA damage, particularly the occurrence of DNA double-strand breaks (DSBs), presents a significant hazard to the integrity and viability of cells. Improper repair of DSBs can result in chromosomal alterations, oncogenic changes, or cell demise. The MRE11-RAD50-NBS1 (MRN) complex plays a crucial role in DNA repair and signaling under the Ataxia Telangiectasia Mutated (ATM) kinase regulation. In this study, we employed comprehensive computational techniques to analyze the structure of RAD50 in Danio rerio (Zebrafish), utilized as a model organism. Additionally, we conducted in silico assessments of RAD50 from both Zebrafish and humans, comparing their characteristics. The substantial sequence resemblance between DrRAD50 and HsRAD50 suggests that DrRAD50 could potentially serve as a valuable model for HsRAD50. However, it is important to acknowledge that sequence similarity alone does not necessarily imply functional equivalence. Further functional studies are needed to confirm the extent of their functional similarities. By examining the secondary and tertiary protein structures of RAD50, we observed a notable likeness between Zebrafish and Human RAD50 proteins. In silico analysis demonstrated that the sequence of RAD50 in zebrafish shares 70% similarity with the human RAD50 protein.

RAD50 Deficiency and Its Effects on Zebrafish Embryonic Development and DNA Repair Mechanisms

The MRE11-RAD50-NBS1 (MRN) complex is essential in detecting, signaling, and repairing DNA double-strand breaks (DSBs), thus maintaining genomic integrity. Mutations in RAD50 are linked to severe conditions such as microcephaly, mental retardation, and growth retardation in humans. This study investigates the developmental impact of RAD50 protein disruption in zebrafish embryos. Zebrafish embryos were treated with MIRIN (35 µM) to inhibit RAD50 and subsequently exposed to gamma-ray irradiation (15 Gy) to analyze the role of RAD50 in managing DNA damage during embryogenesis. Time-point analysis indicated that inhibiting RAD50 and ATM proteins during early embryonic stages (at 1 hpf) leads to increased embryonic mortality and abnormalities. These adverse effects were exacerbated by irradiation, underscoring the critical role of RAD50 in DNA DSB repair. The study concludes that RAD50 deficiencies can lead to embryonic lethality and human deformities due to the inability of tissues to repair DNA DSBs effectively.

Non-classical action of Ku70 promotes Treg suppressive function through a FOXP3-dependent mechanism in lung adenocarcinoma.

Ku70, a DNA repair protein, binds to the damaged DNA ends and orchestrates the recruitment of other proteins to facilitate repair of DNA double-strand breaks. Besides its essential role in DNA repair, several studies have highlighted non-classical functions of Ku70 in cellular processes. However, its function in immune homeostasis and anti-tumor immunity remains unknown. Here, we discovered a marked association between elevated Ku70 expression and unfavorable prognosis in lung adenocarcinoma, focusing specifically on increased Ku70 levels in tumor-infiltrated Treg cells. Using a lung-colonizing tumor model of in mice with Treg-specific Ku70 deficiency, we demonstrated that deletion of Ku70 in Treg cells led to a stronger anti-tumor response and slower tumor growth due to impaired immune-suppressive capacity of Treg cells. Furthermore, we confirmed that Ku70 played a critical role in sustaining the suppressive function of human Treg cells. We found that Ku70 bound to FOXP3 and occupied FOXP3-bound genomic sites to support its transcriptional activities. These findings not only unveil a non-homologous end joining (NHEJ)-independent role of Ku70 crucial for Treg suppressive function, but also underscore the potential of targeting Ku70 as an effective strategy in cancer therapy, aiming to both restrain cancer cells and enhance pulmonary anti-tumor immunity.

Tetrameric INTS6-SOSS1 complex facilitates DNA:RNA hybrid autoregulation at double-strand breaks.

DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs)and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin(SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids andefficient DNA repair.

Endogenous neuronal DNA double-strand breaks are not sufficient to drive brain aging and neurodegeneration.

Loss of genomic information due to the accumulation of somatic DNA damage has been implicated in aging and neurodegeneration 1-3 . Somatic mutations in human neurons increase with age 4 , but it is unclear whether this is a cause or a consequence of brain aging. Here, we clarify the role of endogenous, neuronal DNA double-strand breaks (DSBs) in brain aging and neurodegeneration by generating mice with post-developmental inactivation of the classical non-homologous end-joining (C-NHEJ) core factor Xrcc4 in forebrain neurons. Xrcc4 is critical for the ligation step of C-NHEJ and has no known function outside of DSB repair 5,6 . We find that, unlike their wild-type counterparts, C-NHEJ-deficient neurons accumulate high levels of DSB foci with age, indicating that neurons undergo frequent DSBs that are typically efficiently repaired by C-NHEJ across their lifespan. Genome-wide mapping reveals that endogenous neuronal DSBs preferentially occur in promoter regions and other genic features. Analysis of 3-D genome organization shows intra-chromosomal clustering and loop extrusion of neuronal DSB regions. Strikingly, however, DSB accumulation caused by loss of C-NHEJ induces only minor epigenetic alterations and does not significantly affect gene expression, 3-D genome organization, or mutational outcomes at neuronal DSBs. Despite extensive aging-associated accumulation of neuronal DSBs, mice with neuronal Xrcc4 inactivation do not show neurodegeneration, neuroinflammation, reduced lifespan, or impaired memory and learning behavior. We conclude that the formation of spontaneous neuronal DSBs caused by normal cellular processes is insufficient to cause brain aging and neurodegeneration, even in the absence of C-NHEJ, the principal neuronal DSB repair pathway.

NBS1 dePARylation by NUDT16 is critical for DNA double-strand break repair.

NBS1, a protein linked to the autosomal recessive disorder Nijmegen breakage syndrome, plays an essential role in the DNA damage response and DNA repair. Despite its importance, the mechanisms regulating NBS1 and the impact of this regulation on DNA repair processes remain obscure. In this study, we discovered a new post-translational modification of NBS1, ADP-ribosylation. This modification can be removed by the NUDT16 hydrolase. The loss of NUDT16 results in a reduction of NBS1 protein levels due to NBS1 PARylation-dependent ubiquitination and degradation, which is mediated by the PAR-binding E3 ubiquitin ligase, RNF146. Importantly, ADP-ribosylation of NBS1 is crucial for its localization at DSBs and its involvement in homologous recombination (HR) repair. Additionally, the NUDT16-NBS1 interaction is regulated in response to DNA damage, providing further rationale for NBS1 regulation by NUDT16 hydrolase. In summary, our study unveils the critical role of NUDT16 in governing both the stability of NBS1 and recruitment of NBS1 to DNA double-strand breaks, providing novel insights into the regulation of NBS1 in the HR repair pathway.

NF-κB in Alzheimer’s Disease: Friend or Foe? Opposite Functions in Neurons and Glial Cells

Alzheimer’s disease (AD) is a devasting neurodegenerative disease afflicting mainly glutamatergic neurons together with a massive neuroinflammation mediated by the transcription factor NF-κB. A 65%-plus increase in Alzheimer’s patients by 2050 might be a major threat to society. Hallmarks of AD are neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau and amyloid beta (Aβ) plaques. Here, we review the potential involvement of transcription factor NF-κB by hereditary mutations of the tumor necrosis factor pathway in AD patients. One of the greatest genetic risk factors is APOE4. Recently, it was shown that the APOE4 allele functions as a null allele in human astrocytes not repressing NF-κB anymore. Moreover, NF-κB seems to be involved in the repair of DNA double-strand breaks during healthy learning and memory, a function blunted in AD. NF-κB could be a friend to healthy neurons by repressing apoptosis and necroptosis. But a loss of neuronal NF-κB and activation of glial NF-κB in AD makes it a foe of neuronal survival. Hopeful therapies include TNFR2 receptor bodies relieving the activation of glial NF-κB by TNFα.

Role of TRIP13 in human cancer development.

As an AAA + ATPase, thyroid hormone receptor interacting protein 13 (TRIP13) primarily functions in DNA double-strand break repair, chromosome recombination, and cell cycle checkpoint regulation; aberrant expression of TRIP13 can result in chromosomal instability (CIN). According to recent research, TRIP13 is aberrantly expressed in a variety of cancers, and a patient's poor prognosis and tumor stage are strongly correlated with high expression of TRIP13. Tumor cell and subcutaneous xenograft growth can be markedly inhibited by TRIP13 knockdown or TRIP13 inhibitor administration. In the initiation and advancement of human malignancies, TRIP13 seems to function as an oncogene. Based on available data, TRIP13 may function as a biological target and biomarker for cancer. The creation of inhibitors that specifically target TRIP13 may present novel approaches to treating cancer.

Ribosomal S6 kinase (RSK) plays a critical role in DNA damage response via the phosphorylation of histone lysine demethylase KDM4B

BackgroundEpigenetic dysregulation affecting oncogenic transcription and DNA damage response is a hallmark of cancer. The histone demethylase KDM4B, a factor regulating these processes, plays important roles in estrogen receptor-mediated transcription and DNA repair in breast cancer. However, how oncogenic phospho-signal transduction affects epigenetic regulation is not fully understood. Here we found that KDM4B phosphorylation by ribosomal S6 kinase (RSK), a downstream effector of the Ras/MAPK pathway, is critical for the function of KDM4B in response to DNA damage.MethodsKDM4B-knockout breast cancer cell lines were generated via CRISPR/Cas9-mediated gene editing. Re-expression of wild-type or phospho-site mutated KDM4B in knockout cells was performed by lentivirus-mediated gene transfer. Gene knockdown was achieved by RNA interference. DNA double-strand breaks (DSBs) were induced by ionizing radiation or laser-microirradiation. Protein accumulation at DSB sites was analyzed by immunofluorescence. KDM4B phosphorylation by RSK was assessed by in vitro and in vivo kinase assays. Gene and protein expression levels were analyzed by RT‒PCR and western blotting. The sensitivity of cells to ionizing radiation was examined by a clonogenic survival assay.ResultsRSK phosphorylated KDM4B at Ser666, and inhibition of the phosphorylation by RSK depletion or RSK inhibitors abrogated KDM4B accumulation at the sites of DNA double-strand breaks (DSBs). DSB repair was significantly delayed in KDM4B-knockout cells or cells treated with RSK inhibitors. The replacement of endogenous KDM4B with the phosphomimetic mutant S666D restored KDM4B accumulation and DSB repair that had been inhibited by RSK inhibitors, suggesting a critical role for RSK at the specific serine residue of KDM4B in the effect of RSK inhibitors on DSB repair. As a consequence of these aberrant responses, inhibition of KDM4B phosphorylation increased the sensitivity of the cells to ionizing radiation.ConclusionsOverall, the present study uncovered a novel function of RSK on the DNA damage response, which provides an additional role of its inhibitor in cancer therapy.

Double-strand breaks in facultative heterochromatin require specific movements and chromatin changes for efficient repair

DNA double-strand breaks (DSBs) must be properly repaired within diverse chromatin domains to maintain genome stability. Whereas euchromatin has an open structure and is associated with transcription, facultative heterochromatin is essential to silence developmental genes and forms compact nuclear condensates, called polycomb bodies. Whether the specific chromatin properties of facultative heterochromatin require distinct DSB repair mechanisms remains unknown. Here, we integrate single DSB systems in euchromatin and facultative heterochromatin in Drosophila melanogaster and find that heterochromatic DSBs rapidly move outside polycomb bodies. These DSB movements coincide with a break-proximal reduction in the canonical heterochromatin mark histone H3 Lysine 27 trimethylation (H3K27me3). We demonstrate that DSB movement and loss of H3K27me3 at heterochromatic DSBs depend on the histone demethylase dUtx. Moreover, loss of dUtx specifically disrupts completion of homologous recombination at heterochromatic DSBs. We conclude that DSBs in facultative heterochromatin require dUtx-mediated loss of H3K27me3 to promote DSB movement and repair.

The epistatic relationship of Drosophila melanogaster CtIP and Rif1 in homology-directed repair of DNA double-strand breaks.

Double-strand breaks (DSBs) are genotoxic DNA lesions that pose significant threats to genomic stability, necessitating precise and efficient repair mechanisms to prevent cell death or mutations. DSBs are repaired through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR), which includes homologous recombination (HR) and single-strand annealing (SSA). CtIP and Rif1 are conserved proteins implicated in DSB repair pathway choice, possibly through redundant roles in promoting DNA end-resection required for HDR. Although the roles of these proteins have been well-established in other organisms, the role of Rif1 and its potential redundancies with CtIP in Drosophila melanogaster remain elusive. To examine the roles of DmCtIP and DmRif1 in DSB repair, this study employed the direct repeat of white (DR-white) assay, tracking across indels by decomposition (TIDE) analysis, and P{wIw_2 kb 3'} assay to track repair outcomes in HR, NHEJ, and SSA, respectively. These experiments were performed in DmCtIPΔ/Δ single mutants, DmRif1Δ/Δ single mutants, and DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants. This work demonstrates significant defects in both HR and SSA repair in DmCtIPΔ/Δ and DmRif1Δ/Δ single mutants. However, experiments in DmRif1Δ/Δ; DmCtIPΔ/Δ double mutants reveal that DmCtIP is epistatic to DmRif1 in promoting HDR. Overall, this study concludes that DmRif1 and DmCtIP do not perform their activities in a redundant pathway, but rather DmCtIP is the main driver in promoting HR and SSA, most likely through its role in end resection.

RPS19 and RPL5, the most commonly mutated genes in Diamond Blackfan anemia, impact DNA double-strand break repair.

Diamond Blackfan anemia (DBA) is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in ribosomal protein (RP) genes, most commonly RPS19 and RPL5 . In addition to red cell aplasia, individuals with DBA are at increased risk of various cancers. Importantly, the mechanism(s) underlying cancer predisposition are poorly understood. We found that DBA patient-derived lymphoblastoid cells had persistent γ-H2AX foci following ionizing radiation (IR) treatment, suggesting DNA double-strand break (DSB) repair defects. RPS19- and RPL5-knocked down (KD) CD34+ cells had delayed repair of IR-induced DSBs, further implicating these RPs in DSB repair. Assessing the impact of RPS19- and RPL5-KD on specific DSB repair pathways, we found RPS19-KD decreased the efficiency of pathways requiring extensive end-resection, whereas RPL5-KD increased end-joining pathways. Additionally, RAD51 was reduced in RPS19- and RPL5-KD and RPS19- and RPL5-mutated DBA cells, whereas RPS19-deficient cells also had a reduction in PARP1 and BRCA2 proteins. RPS19-KD cells had an increase in nuclear RPA2 and a decrease in nuclear RAD51 foci post-IR, reflective of alterations in early, critical steps of homologous recombination. Notably, RPS19 and RPL5 interacted with poly(ADP)-ribose chains noncovalently, were recruited to DSBs in a poly(ADP)-ribose polymerase activity-dependent manner, and interacted with Ku70 and histone H2A. RPL5's recruitment, but not RPS19's, also required p53, suggesting that RPS19 and RPL5 directly participate in DSB repair via different pathways. We propose that defective DSB repair arising from haploinsufficiency of these RPs may underline the cancer predisposition in DBA.

GLTSCR1 deficiency promotes colorectal cancer development through regulating non-homologous end joining.

Non-homologous end joining (NHEJ), as one major pathway of DNA double-strand break (DSB) repair, could cause genomic instability, which plays pivotal roles in cancer development. While, chromatin remodeling complexes dictate the selection and orchestration of DSB repair pathways by regulating chromatin dynamics. However, the crosstalk between NHEJ and chromatin remodeling in cancer progress remains unclear. In this study, deficiency of GLTSCR1 causes resistance to DNA damage in colorectal cancer (CRC) cells by promoting NHEJ repair efficiency. Mechanistically, GLTSCR1 interacts with BRD9 to engage in the assembly of the non-canonical BAF complex (GBAF). However, GLTSCR1 deficiency disrupts GBAF and triggers the ubiquitination degradation of BRD9. Furthermore, GLTSCR1 deficiency causes aberrant opening in the promoter region of NHEJ repair-associated genes, which promotes CRC development. While, GLTSCR1 and its binding partner BRD9 are not directly involved in assembling NHEJ repair machinery; instead, they regulate the DNA accessibility of NHEJ repair-associated genes. Collectively, our findings confirm GLTSCR1 deficiency as a critical regulatory event of the NHEJ pathway in CRC development, which might require different therapeutic strategy for GLTSCR1 wild-type and mutant CRC.

Isolation and purification of DNA double-strand break repair intermediates for understanding complex molecular mechanisms.

Branched DNA molecules are key intermediates in the molecular pathways of DNA replication, repair and recombination. Understanding their structural details, therefore, helps to envisage the mechanisms underlying these processes. While the configurations of DNA molecules can be effectively analysed in bulk using gel electrophoresis techniques, direct visualization provides a complementary single-molecule approach to investigating branched DNA structures. However, for microscopic examination, the sample needs to be free from impurities that could obscure the molecules of interest, and free from the bulk of unwanted non-specific DNA molecules that would otherwise dominate the field of view. Additionally, in the case of recombination intermediates, the length of the DNA molecules becomes an important factor to consider since the structures can be spread over a large distance on the chromosome in vivo. As a result, apart from sample purity, efficient isolation of large-sized DNA fragments without damaging their branched structures is crucial for further analysis. These factors are illustrated by the example of DNA double-strand break repair in the bacterium E. coli. In E. coli recombination intermediates may be spread over a distance of 40 kb which constitutes less than 1% of the 4.6 Mb genome. This study reveals ways to overcome some of the technical challenges that are associated with the isolation and purification of large and complex branched DNA structures using E. coli DNA double-strand break repair intermediates. High-molecular weight and branched DNA molecules do not run into agarose gels subjected to electrophoresis. However, they can be extracted from the wells of the gels if they are agarose embedded, by using β-agarase digestion, filtration, and concentration. Furthermore, a second round of gel electrophoresis followed by purification is recommended to enhance the purity of the specific DNA samples. These preliminary findings may prove to be pioneering for various single-molecule analyses of large and complex DNA molecules of DNA replication, repair and recombination.