RESUMEN

The repair of DNA double-strand breaks is a crucial yet delicate process which is affected by a multitude of factors. In this study, our goal is to analyse the influence of the linear energy transfer (LET) on the DNA repair kinetics. By utilizing the database of repair of DNA and aggregating the results of 84 experiments, we conduct various model fits to evaluate and compare different hypothesis regarding the effect of LET on the rejoining of DNA ends. Despite the considerable research efforts dedicated to this topic over the past decades, our findings underscore the complexity of the relationship between LET and DNA repair kinetics. This study leverages big data analysis to capture overall trends that single experimental studies might miss, providing a valuable model for understanding how radiation quality impacts DNA damage and subsequent biological effects. Our results highlight the gaps in our current understanding, emphasizing the pressing need for further investigation into this phenomenon.

RESUMEN

Based on classical karyotyping, structural genome variations (SVs) have generally been considered to be either "simple" (with one or two breakpoints) or "complex" (with more than two breakpoints). Studying the breakpoints of SVs at nucleotide resolution revealed additional, subtle structural variations, such that even "simple" SVs turned out to be "complex." Genome-wide sequencing methods, such as fosmid and paired-end mapping, short-read and long-read whole genome sequencing, and single-molecule optical mapping, also indicated that the number of SVs per individual was considerably larger than expected from karyotyping and high-resolution chromosomal array-based studies. Interestingly, SVs were detected in studies of cohorts of individuals without clinical phenotypes. The common denominator of all SVs appears to be a failure to accurately repair DNA double-strand breaks (DSBs) or to halt cell cycle progression if DSBs persist. This review discusses the various DSB response mechanisms during the mitotic cell cycle and during meiosis and their regulation. Emphasis is given to the molecular mechanisms involved in the formation of translocations, deletions, duplications, and inversions during or shortly after meiosis I. Recently, CRISPR-Cas9 studies have provided unexpected insights into the formation of translocations and chromothripsis by both breakage-fusion-bridge and micronucleus-dependent mechanisms.

Asunto(s)

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complimentary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 hr treatment of 10µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS affected neurons.

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects the motor neuron. One aspect of the neuropathology involved in ALS includes increased genomic damage and impaired DNA repair capability. The TAR-DNA binding protein 43 (TDP43) has been associated with both sporadic and familial forms of ALS, and is typically observed as cytosolic mislocalization of protein aggregates, termed TDP43 proteinopathy. TDP43 is a ubiquitous RNA/DNA binding protein with functional implications in a wide range of disease processes, including the repair of DNA double-strand breaks (DSBs). While TDP43 is widely known to regulate RNA metabolism, our lab has reported it also functions directly at the protein level to facilitate DNA repair. Here, we show that the TDP43 protein interacts with DNA mismatch repair (MMR) proteins MLH1 and MSH6 in a DNA damage-inducible manner. We utilized differentiated SH-SY5Y neuronal cultures to identify this inducible relationship using complementary approaches of proximity ligation assay (PLA) and co-immunoprecipitation (CoIP) assay. We observed that signals of TDP43 interaction with MLH1 and MSH6 increased significantly following a 2 h treatment of 10 µM methylmethanesulfonate (MMS), a DNA alkylating agent used to induce MMR repair. Likewise, we observed this effect was abolished in cell lines treated with siRNA directed against TDP43. Finally, we demonstrated these protein interactions were significantly increased in lumbar spinal cord samples of ALS-affected patients compared to age-matched controls. These results will inform our future studies to understand the mechanisms and consequences of this TDP43-MMR interaction in the context of ALS-affected neurons.

Asunto(s)

RESUMEN

Advances in chromatin mapping have exposed the complex chromatin hierarchical organization in mammals, including topologically associating domains (TADs) and their substructures, yet the functional implications of this hierarchy in gene regulation and disease progression are not fully elucidated. Our study delves into the phenomenon of shared TAD boundaries, which are pivotal in maintaining the hierarchical chromatin structure and regulating gene activity. By integrating high-resolution Hi-C data, chromatin accessibility, and DNA double-strand breaks (DSBs) data from various cell lines, we systematically explore the complex regulatory landscape at high-level TAD boundaries. Our findings indicate that these boundaries are not only key architectural elements but also vibrant hubs, enriched with functionally crucial genes and complex transcription factor binding site-clustered regions. Moreover, they exhibit a pronounced enrichment of DSBs, suggesting a nuanced interplay between transcriptional regulation and genomic stability. Our research provides novel insights into the intricate relationship between the 3D genome structure, gene regulation, and DNA repair mechanisms, highlighting the role of shared TAD boundaries in maintaining genomic integrity and resilience against perturbations. The implications of our findings extend to understanding the complexities of genomic diseases and open new avenues for therapeutic interventions targeting the structural and functional integrity of TAD boundaries.

Asunto(s)

RESUMEN

TDP43 is an RNA/DNA binding protein increasingly recognized for its role in neurodegenerative conditions including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). As characterized by its aberrant nuclear export and cytoplasmic aggregation, TDP43 proteinopathy is a hallmark feature in over 95% of ALS/FTD cases, leading to the formation of detrimental cytosolic aggregates and a reduction in nuclear functionality within neurons. Building on our prior work linking TDP43 proteinopathy to the accumulation of DNA double-strand breaks (DSBs) in neurons, the present investigation uncovers a novel regulatory relationship between TDP43 and DNA mismatch repair (MMR) gene expressions. Here, we show that TDP43 depletion or overexpression directly affects the expression of key MMR genes. Alterations include MLH1, MSH2, MSH3, MSH6, and PMS2 levels across various primary cell lines, independent of their proliferative status. Our results specifically establish that TDP43 selectively influences the expression of MLH1 and MSH6 by influencing their alternative transcript splicing patterns and stability. We furthermore find aberrant MMR gene expression is linked to TDP43 proteinopathy in two distinct ALS mouse models and post-mortem brain and spinal cord tissues of ALS patients. Notably, MMR depletion resulted in the partial rescue of TDP43 proteinopathy-induced DNA damage and signaling. Moreover, bioinformatics analysis of the TCGA cancer database reveals significant associations between TDP43 expression, MMR gene expression, and mutational burden across multiple cancers. Collectively, our findings implicate TDP43 as a critical regulator of the MMR pathway and unveil its broad impact on the etiology of both neurodegenerative and neoplastic pathologies.

RESUMEN

Sex chromosome aneuploidies are among the most common variations in human whole chromosome copy numbers, with an estimated prevalence in the general population of 1:400 to 1:1400 live births. Unlike whole-chromosome aneuploidies of autosomes, those of sex chromosomes, such as the 47, XXY aneuploidy that causes Klinefelter Syndrome (KS), often originate from the paternal side, caused by a lack of crossover (CO) formation between the X and Y chromosomes. COs must form between all chromosome pairs to pass meiotic checkpoints and are the product of meiotic recombination that occurs between homologous sequences of parental chromosomes. Recombination between male sex chromosomes is more challenging compared to both autosomes and sex chromosomes in females, as it is restricted within a short region of homology between X and Y, called the pseudo-autosomal region (PAR). However, in normal individuals, CO formation occurs in PAR with a higher frequency than in any other region, indicating the presence of mechanisms that promote the initiation and processing of recombination in each meiotic division. In recent years, research has made great strides in identifying genes and mechanisms that facilitate CO formation in the PAR. Here, we outline the most recent and relevant findings in this field. XY chromosome aneuploidy in humans has broad-reaching effects, contributing significantly also to Turner syndrome, spontaneous abortions, oligospermia, and even infertility. Thus, in the years to come, the identification of genes and mechanisms beyond XY aneuploidy is expected to have an impact on the genetic counseling of a wide number of families and adults affected by these disorders.

Asunto(s)

RESUMEN

Several meiotic events reshape the genome prior to its transfer (via gametes) to the next generation. The occurrence of new meiotic mutations is tightly linked to homologous recombination (HR) and firmly depends on Spo11-induced DNA breaks. To gain insight into the molecular mechanisms governing mutagenicity during meiosis, we examined the timing of mutation and recombination events in cells deficient in various DNA HR-repair genes, which represent distinct functions along the meiotic recombination process. Despite sequence similarities and overlapping activities of the two DNA translocases, Rad54 and Tid1, we observed essential differences in their roles in meiotic mutation occurrence: in the absence of Rad54, meiotic mutagenicity was elevated 8-fold compared to the wild type (WT), while in the tid1Δ mutant, there were few meiotic mutations, nine percent compared to the WT. We propose that the presence of Rad54 channels recombinational repair to a less mutagenic pathway, whereas repair assisted by Tid1 is more mutagenic. A 3.5-fold increase in mutation level was observed in dmc1∆ cells, suggesting that single-stranded DNA (ssDNA) may be a potential source for mutagenicity during meiosis. Taken together, we suggest that the introduction of de novo mutations also contributes to the diversification role of meiotic recombination. These rare meiotic mutations revise genomic sequences and may contribute to long-term evolutionary changes.

Asunto(s)

RESUMEN

BACKGROUND: Heavy ion irradiation (IR) with high-linear energy transfer (LET) is characterized by a unique depth dose distribution and increased biological effectiveness. Following high-LET IR, localized energy deposition along the particle trajectories induces clustered DNA lesions, leading to low electron density domains (LEDDs). To investigate the spatiotemporal dynamics of DNA repair and chromatin remodeling, we established the automated image analysis of transmission electron micrographs. METHODS: Human fibroblasts were irradiated with high-LET carbon ions or low-LET photons. At 0.1 h, 0.5 h, 5 h, and 24 h post-IR, nanoparticle-labeled repair factors (53BP1, pKu70, pKu80, DNA-PKcs) were visualized using transmission electron microscopy in interphase nuclei to monitor the formation and repair of DNA damage in the chromatin ultrastructure. Using AI-based software tools, advanced image analysis techniques were established to assess the DNA damage pattern following low-LET versus high-LET IR. RESULTS: Low-LET IR induced single DNA lesions throughout the nucleus, and most DNA double-strand breaks (DSBs) were efficiently rejoined with no visible chromatin decondensation. High-LET IR induced clustered DNA damage concentrated along the particle trajectories, resulting in circumscribed LEDDs. Automated image analysis was used to determine the exact number of differently sized nanoparticles, their distance from one another, and their precise location within the micrographs (based on size, shape, and density). Chromatin densities were determined from grayscale features, and nanoparticles were automatically assigned to euchromatin or heterochromatin. High-LET IR-induced LEDDs were delineated using automated segmentation, and the spatial distribution of nanoparticles in relation to segmented LEDDs was determined. CONCLUSIONS: The results of our image analysis suggest that high-LET IR induces chromatin relaxation along particle trajectories, enabling the critical repair of successive DNA damage. Following exposure to different radiation qualities, automated image analysis of nanoparticle-labeled DNA repair proteins in the chromatin ultrastructure enables precise characterization of specific DNA damage patterns.

Asunto(s)

RESUMEN

Radiation therapy is an essential component of present-day cancer management, utilizing ionizing radiation (IR) of different modalities to mitigate cancer progression. IR functions by generating ionizations in cells that induce a plethora of DNA lesions. The most detrimental among them are the DNA double strand breaks (DSBs). In the course of evolution, cells of higher eukaryotes have evolved four major DSB repair pathways: classical non-homologous end joining (c-NHEJ), homologous recombination (HR), alternative end-joining (alt-EJ), and single strand annealing (SSA). These mechanistically distinct repair pathways have different cell cycle- and homology-dependencies but, surprisingly, they operate with widely different fidelity and kinetics and therefore contribute unequally to cell survival and genome maintenance. It is therefore reasonable to anticipate tight regulation and coordination in the engagement of these DSB repair pathway to achieve the maximum possible genomic stability. Here, we provide a state-of-the-art review of the accumulated knowledge on the molecular mechanisms underpinning these repair pathways, with emphasis on c-NHEJ and HR. We discuss factors and processes that have recently come to the fore. We outline mechanisms steering DSB repair pathway choice throughout the cell cycle, and highlight the critical role of DNA end resection in this process. Most importantly, however, we point out the strong preference for HR at low DSB loads, and thus low IR doses, for cells irradiated in the G2-phase of the cell cycle. We further explore the molecular underpinnings of transitions from high fidelity to low fidelity error-prone repair pathways and analyze the coordination and consequences of this transition on cell viability and genomic stability. Finally, we elaborate on how these advances may help in the development of improved cancer treatment protocols in radiation therapy.

Asunto(s)

RESUMEN

BACKGROUND: The human population living in high level natural radiation areas (HLNRAs) of Kerala coast provide unique opportunities to study the biological effects of low dose and low dose rate ionizing radiation below 100 mGy. The level of radiation in this area varies from < 1.0 to 45 mGy/year. The areas with ≤ 1.50 mGy/year are considered as normal level natural radiation areas (NLNRA) and > 1.50 mGy/year, as high level natural radiation areas (HLNRA). The present study evaluated dose response relationship between DNA double strand breaks (DSBs) and background radiation dose in individuals residing in Kerala coast. Venous blood samples were collected from 200 individuals belonging to NLNRA (n = 50) and four dose groups of HLNRA; 1.51-5.0 mGy/year (n = 50), 5.01-10.0 mGy/year (n = 30), 10.01-15.0 mGy/year (n = 33), > 15.0 mGy/year (n = 37) with written informed consent. The mean dose of NLNRA and four HLNRA dose groups studied are 1.21 ± 0.21 (range: 0.57-1.49), 3.02 ± 0.95 (range: 1.57-4.93), 7.43 ± 1.48 (range: 5.01-9.75), 12.22 ± 1.47 (range: 10.21-14.99), 21.64 ± 6.28 (range: 15.26-39.88) mGy/year, respectively. DNA DSBs were quantified using γH2AX as a marker, where foci were counted per cell using fluorescence microscopy. RESULTS: Our results revealed that the frequency of γH2AX foci per cell was 0.090 ± 0.051 and 0.096 ± 0.051, respectively in NLNRA and HLNRA individuals, which were not significantly different (t198 = 0.33; P = 0.739). The frequency of γH2AX foci was observed to be 0.090 ± 0.051, 0.096 ± 0.051, 0.076 ± 0.036, 0.087 ± 0.042, 0.108 ± 0.046 per cell, respectively in different dose groups of ≤ 1.50, 1.51-5.0, 5.01-10.0, 10.01-15.0, > 15.0mGy/year (ANOVA, F4,195 = 2.18, P = 0.072) and suggested non-linearity in dose response. The frequency of γH2AX foci was observed to be 0.098 ± 0.042, 0.078 ± 0.037, 0.084 ± 0.042, 0.099 ± 0.058, 0.097 ± 0.06 and 0.114 ± 0.033 per cell in the age groups of ≤ 29, 30-34, 35-39, 40-44, 45-49 and ≥ 50 years, respectively (ANOVA, F5,194 = 2.17, P = 0.059), which suggested marginal influence of age on the baseline of DSBs. Personal habits such as smoking (No v/s Yes: 0.092 ± 0.047 v/s 0.093 ± 0.048, t198 = 0.13; P = 0.895) and drinking alcohol (No v/s Yes: 0.096 ± 0.052 v/s 0.091 ± 0.045, t198 = 0.62; P = 0.538) did not show any influence on DSBs in the population. CONCLUSION: The present study did not show any increase in DSBs in different dose groups of HLNRA compared to NLNRA, however, it suggested a non-linear dose response between DNA DSBs and chronic low dose radiation.

RESUMEN

Daunomycin (DM), an anthracycline antibiotic, is frequently used to treat various cancers, but the direct effects of DM on gene expression and DNA structure are unclear. We used an in vitro cell-free system, optimized with spermine (SP), to study the effect of DM on gene expression. A bimodal effect of DM on gene expression, weak promotion followed by inhibition, was observed with increasing concentration of DM. We also performed atomic force microscopy observation to measure how DM affects the higher-order structure of DNA induced with SP. DM destroyed SP-induced flower-like conformations of DNA by generating double-strand breaks, and this destructive conformational change of DNA corresponded to the inhibitory effect on gene expression. Interestingly, the weakly enhanced cell-free gene expression occurred as DNA conformations were elongated or relaxed at lower DM concentrations. We expect these newly unveiled DM effects on gene expression and the higher-order structure of DNA will contribute further to the development and refinement of useful anticancer therapy chemicals.

Asunto(s)

RESUMEN

CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.

Asunto(s)

RESUMEN

People exposed to ionizing radiation (IR) both for diagnostic and therapeutic purposes is constantly increasing. Since the use of IR involves a risk of harmful effects, such as the DNA DSB induction, an accurate determination of this induced DNA damage and a correct evaluation of the risk-benefit ratio in the clinical field are of key relevance. γH2AX (the phosphorylated form of the histone variant H2AX) is a very early marker of DSBs that can be induced both in physiological conditions, such as in the absence of specific external agents, and by external factors such as smoking, heat, background environmental radiation, and drugs. All these internal and external conditions result in a basal level of γH2AX which must be considered for the correct assessment of the DSBs after IR exposure. In this review we analyze the most common conditions that induce H2AX phosphorylation, including specific exogenous stimuli, cellular states, basic environmental factors, and lifestyles. Moreover, we discuss the most widely used methods for γH2AX determination and describe the principal applications of γH2AX scoring, paying particular attention to clinical studies. This knowledge will help us optimize the use of available methods in order to discern the specific γH2AX following IR-induced DSBs from the basal level of γH2AX in the cells.

RESUMEN

Boron Neutron Capture Therapy (BNCT) is a tumor cell selective high LET (linear energy transfer) particle beam therapy. The patient is administrated a boron (10B) compound via intravenous injection or infusion, and when 10B is sufficiently accumulated in the tumor, neutron beams containing epithermal neutrons as the main component are irradiated. Epithermal neutrons lose energy in the body and become thermal neutrons. The captured 10B undergoes a (n, α) reaction with thermal neutrons, and the resulting α particles and 7Li nuclei have short ranges of 9-10µm and 4-5µm, respectively, and do not reach the surrounding cells in normal tissues. Therefore, these high LET-heavy charged particles can selectively kill cancer cells. The cell-killing effect of these heavy charged particles is thought to be triggered by DNA damage. It is known that DNA damage caused by heavy charged particles is more serious and difficult to repair than DNA damage caused by Low LET radiation such as X-rays and γ-rays. This review focuses on DNA damage, e.g., DNA strand breaks and DNA damage repair caused by BNCT and describes the resulting biological response.

Asunto(s)

RESUMEN

Cancer development is often connected to impaired DNA repair and DNA damage signaling pathways. The presence of DNA damage in cells activates DNA damage response, which is a complex cellular signaling network that includes DNA repair, activation of the cell cycle checkpoints, cellular senescence, and apoptosis. DNA double-strand breaks (DSBs) are toxic lesions that are mainly repaired by the non-homologous end joining and homologous recombination repair (HRR) pathways. Estrogen-dependent cancers, like breast and ovarian cancers, are frequently associated with mutations in genes that play a role in HRR. The female sex hormone estrogen binds and activates the estrogen receptors (ERs), ERα, ERß and G-protein-coupled ER 1 (GPER1). ERα drives proliferation, while ERß inhibits cell growth. Estrogen regulates the transcription, stability and activity of numerus DDR factors and DDR factors in turn modulate ERα expression, stability and transcriptional activity. Additionally, estrogen stimulates DSB formation in cells as part of its metabolism and proliferative effect. In this review, we will present an overview on the crosstalk between estrogen and the cellular response to DSBs. We will discuss how estrogen regulates DSB signaling and repair, and how DDR factors modulate the expression, stability and activity of estrogen. We will also discuss how the regulation of HRR genes by estrogen promotes the development of estrogen-dependent cancers.

Asunto(s)

RESUMEN

When DNA repair is inadequate it increases the chances of the genome becoming unstable and it undergoes a malignant mutation. The deficiency of DNA repair PARP proteins may be leveraged for cancer therapy by increasing genomic instability and causing massive DNA damage in cancer cells. DNA repair components are under increased demand in cancer cells because of the continuous replication of DNA. The oncogenic loss of BRCA and an inefficient DNA repair led to cancer cells being dependent on particular DNA repair pathways, like the Poly (ADP-ribose) polymerase pathway. Breast cancer gene 1 and 2 plays a crucial role in DNA repair and genome integrity explaining how BRCA1 and BRCA2 mutations raise the menace of cancer. PARP inhibitors inhibit the base exclusion repair pathway, resulting in the buildup of unrepaired single strand breaks, which cause inflated replication forks in the S phase and subsequently the development of damaging double stranded breaks. Cells having BRCA mutations are unable to repair DNA breaks, leading to apoptosis and eventually death of cancer cells. Numerous indicators, such as a lack of homologous recombination and a high degree of replication pressure, indicate that this therapy will be very effective. Combining PARP inhibitors with chemotherapy, an immune checkpoint inhibitor, and a targeted drug is an effective strategy for combating PARP inhibitors resistance. Several PARP-based combination approaches are in preclinical and clinical development. Various clinical trials are successfully completed and some are undergoing to evaluate the efficacy of these molecules. This review will describe the current views and clinical updates on PARP inhibitors.

Asunto(s)

RESUMEN

Double-strand DNA breakes (DSBs) are the most deleterious and widespread examples of DNA damage. They inevitably originate from endogenous mechanisms in the course of transcription, replication, and recombination, as well as from different exogenous factors. If not properly repaired, DSBs result in cell death or diseases. Genome-wide analysis of DSBs has revealed the numerous endogenous DSBs in human chromosomes. However, until now, it has not been clear what kind of genes are preferentially subjected to breakage. We performed a genetic and epigenetic analysis of the most frequent DSBs in HEK293T cells. Here, we show that they predominantly occur in the active genes controlling differentiation, development, and morphogenesis. These genes are highly associated with cancers and other diseases. About one-third of the genes possessing frequent DSBs correspond to rDNA-contacting genes. Our data suggest that a specific set of active genes controlling morphogenesis are the main targets of DNA breakage in human cells, although there is a specific set of silent genes controlling metabolism that also are enriched in DSBs. We detected this enrichment by different activators and repressors of transcription at DSB target sites, as well breakage at promoters. We propose that both active transcription and silencing of genes give a propensity for DNA breakage. These results have implications for medicine and gene therapy.

Asunto(s)

RESUMEN

Cancer genomes are characterized by the accumulation of small-scale somatic mutations as well as large-scale chromosomal deletions, amplifications, and complex structural rearrangements. This characteristic is at least partially dependent on the ability of cancer cells to undergo recurrent chromosome breakage. In order to address the extent to which chromosomal structural rearrangement breakpoints correlate with recurrent DNA double-strand breaks (DSBs), we simultaneously mapped chromosome structural variation breakpoints (using whole-genome DNA-seq) and spontaneous DSB formation (using Break-seq) in the estrogen receptor (ER)-positive breast cancer cell line MCF-7 and a non-cancer control breast epithelium cell line MCF-10A. We identified concurrent DSBs and structural variation breakpoints almost exclusively in the pericentromeric region of chromosome 16q in MCF-7 cells. We fine-tuned the identification of copy number variation breakpoints on 16q. In addition, we detected recurrent DSBs that occurred in both MCF-7 and MCF-10A. We propose a model for DSB-driven chromosome rearrangements that lead to the translocation of 16q, likely with 10q, and the eventual 16q loss that does not involve the pericentromere of 16q. We present evidence from RNA-seq data that select genes, including SHCBP1, ORC6, and MYLK3, which are immediately downstream from the 16q pericentromere, show heightened expression in MCF-7 cell line compared to the control. Data published by The Cancer Genome Atlas show that all three genes have increased expression in breast tumor samples. We found that SHCBP1 and ORC6 are both strong poor prognosis and treatment outcome markers in the ER-positive breast cancer cohort. We suggest that these genes are potential oncogenes for breast cancer progression. The search for tumor suppressor loss that accompanies the 16q loss ought to be augmented by the identification of potential oncogenes that gained expression during chromosomal rearrangements.

Asunto(s)

RESUMEN

Loss or rearrangement of genetic information can result from incorrect responses to DNA double strand breaks (DSBs). The cellular responses to DSBs encompass a range of highly coordinated events designed to detect and respond appropriately to the damage, thereby preserving genomic integrity. In analogy with events occurring during viral infection, we appropriate the terms Immediate-Early, Early, and Late to describe the pre-repair responses to DSBs. A distinguishing feature of the Immediate-Early response is that the large protein condensates that form during the Early and Late response and are resolved upon repair, termed foci, are not visible. The Immediate-Early response encompasses initial lesion sensing, involving poly (ADP-ribose) polymerases (PARPs), KU70/80, and MRN, as well as rapid repair by so-called 'fast-kinetic' canonical non-homologous end joining (cNHEJ). Initial binding of PARPs and the KU70/80 complex to breaks appears to be mutually exclusive at easily ligatable DSBs that are repaired efficiently by fast-kinetic cNHEJ; a process that is PARP-, ATM-, 53BP1-, Artemis-, and resection-independent. However, at more complex breaks requiring processing, the Immediate-Early response involving PARPs and the ensuing highly dynamic PARylation (polyADP ribosylation) of many substrates may aid recruitment of both KU70/80 and MRN to DSBs. Complex DSBs rely upon the Early response, largely defined by ATM-dependent focal recruitment of many signalling molecules into large condensates, and regulated by complex chromatin dynamics. Finally, the Late response integrates information from cell cycle phase, chromatin context, and type of DSB to determine appropriate pathway choice. Critical to pathway choice is the recruitment of p53 binding protein 1 (53BP1) and breast cancer associated 1 (BRCA1). However, additional factors recruited throughout the DSB response also impact upon pathway choice, although these remain to be fully characterised. The Late response somehow channels DSBs into the appropriate high-fidelity repair pathway, typically either 'slow-kinetic' cNHEJ or homologous recombination (HR). Loss of specific components of the DSB repair machinery results in cells utilising remaining factors to effect repair, but often at the cost of increased mutagenesis. Here we discuss the complex regulation of the Immediate-Early, Early, and Late responses to DSBs proceeding repair itself.