Project description:Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.
Project description:The dynamic organization of chromatin inside the cell nucleus plays a key role in gene regulation and genome replication, as well as maintaining genome integrity. Although the static folded state of the genome has been extensively studied, dynamical signatures of processes such as transcription or DNA repair remain an open question. Here, we investigate the interphase chromatin dynamics in human cells in response to local DNA damage, specifically, DNA double-strand breaks (DSBs). Using simultaneous two-color spinning-disk confocal microscopy, we monitor the DSB dynamics and the compaction of the surrounding chromatin, visualized by fluorescently labeled 53BP1 and histone H2B, respectively. Our study reveals a surprising difference between the mobility of DSBs located in the nuclear interior versus periphery (less than 1 μm from the nuclear envelope), with the interior DSBs being almost twice as mobile as the periphery DSBs. Remarkably, we find that the DSB sites possess a robust structural signature in a form of a unique chromatin compaction profile. Moreover, our data show that the DSB motion is subdiffusive and ATP-dependent and exhibits unique dynamical signatures, different from those of undamaged chromatin. Our findings reveal that the DSB mobility follows a universal relationship defined solely by the physical parameters describing the DSBs and their local environment, such as the DSB focus size (represented by the local accumulation of 53BP1), DSB density, and the local chromatin compaction. This suggests that the DSB-related repair processes are robust and likely deterministic because the observed dynamical signatures (DSB mobility) can be explained solely by their structural features (DSB focus size, local chromatin compaction). Such knowledge might help in detecting local DNA damage in live cells, as well as in aiding our biophysical understanding of genome integrity in health and disease.
Project description:Many cancer therapies operate by inducing double-strand breaks (DSBs) in cancer cells, however treatment-resistant cells rapidly initiate mechanisms to repair damage enabling survival. While the DNA repair mechanisms responsible for cancer cell survival following DNA damaging treatments are becoming better understood, less is known about the role of the epigenome in this process. Using prostate cancer cell lines with differing sensitivities to radiation treatment, we analysed the DNA methylation profiles prior to and following a single dose of radiotherapy (RT) using the Illumina Infinium HumanMethylation450 BeadChip platform. DSB formation and repair, in the absence and presence of the DNA hypomethylating agent, 5-azacytidine (5-AzaC), were also investigated using γH2A.X immunofluorescence staining. Here we demonstrate that DNA methylation is generally stable following a single dose of RT; however, a small number of CpG sites are stably altered up to 14 d following exposure. While the radioresistant and radiosensitive cells displayed distinct basal DNA methylation profiles, their susceptibility to DNA damage appeared similar demonstrating that basal DNA methylation has a limited influence on DSB induction at the regions examined. Recovery from DSB induction was also similar between these cells. Treatment with 5-AzaC did not sensitize resistant cells to DNA damage, but rather delayed recruitment of phosphorylated BRCA1 (S1423) and repair of DSBs. These results highlight that stable epigenetic changes are possible following a single dose of RT and may have significant clinical implications for cancer treatment involving recurrent or fractionated dosing regimens.
Project description:Investigations into the spatiotemporal dynamics of DNA repair using live-cell imaging are aided by the ability to generate well defined regions of ultravioletlike photolesions in an optical microscope. We demonstrate that multiphoton excitation of DNA in live cells with visible femtosecond pulses produces thymine cyclopyrimidine dimers (CPDs), the primary ultraviolet DNA photoproduct. The CPDs are produced with a cubic to supercubic power dependence using pulses in the wavelength range from at least 400 to 525 nm. We show that the CPDs are confined in all three spatial dimensions, making multiphoton excitation of DNA with visible light an ideal technique for generating localized DNA photolesions in a wide variety of samples, from cultured cells to thicker tissues. We demonstrate the utility of this method by applying it to investigate the spatiotemporal recruitment of GFP-tagged topoisomerase I (TopI) to sites of localized DNA damage in polytene chromosomes within live cells of optically thick Drosophila salivary glands.
Project description:Recent studies have suggested that human RNA helicase, DDX3X, is important for DNA repair, but little is known about the nuclear activity of this protein. In vitro analysis of nuclear DDX3X interactions and localization with DNA damage pointed to a direct role for DDX3X in the DNA damage response. We aimed to investigate whether DDX3X plays a direct role in the DNA damage response in live cells. In order to track nuclear DDX3X, we generated a nuclear-export deficient DDX3X mutant construct and performed microirradiation in live cells. We found that DDX3X accumulates at sites of microirradiation shortly after DNA damage induction. We further found DDX3X recruitment to be mediated by its intrinsically disordered domains, similar to other RNA binding proteins that are recruited to sites of DNA damage. Inhibition of liquid-liquid phase separation also reduced DDX3X recruitment. CRISPR/Cas9-mediated knockout of PARP1 ablated DDX3X recruitment, which was restored upon transgenic expression of wild-type PARP1 but not catalytically inactive PARP1, suggesting that DDX3X recruitment is PARP1-dependent.
Project description:Nuclear functions including gene expression, DNA replication and genome maintenance intimately rely on dynamic changes in chromatin organization. The movements of chromatin fibers might play important roles in the regulation of these fundamental processes, yet the mechanisms controlling chromatin mobility are poorly understood owing to methodological limitations for the assessment of chromatin movements. Here, we present a facile and quantitative technique that relies on photoactivation of GFP-tagged histones and paired-particle tracking to measure chromatin mobility in live cells. We validate the method by comparing live cells to ATP-depleted cells and show that chromatin movements in mammalian cells are predominantly energy dependent. We also find that chromatin diffusion decreases in response to DNA breaks induced by a genotoxic drug or by the ISceI meganuclease. Timecourse analysis after cell exposure to ionizing radiation indicates that the decrease in chromatin mobility is transient and precedes subsequent increased mobility. Future applications of the method in the DNA repair field and beyond are discussed.
Project description:Cellular senescence is a major barricade on the path of cancer development, yet proteins secreted from senescent cells exert complex and often discordant effects on subsequent cancer evolution. Somatic genome alternations driving the formation of nevi and melanoma are efficient inducers of cellular senescence. Melanocyte and melanoma cell senescence is likely to come into play as a key factor affecting the course of tumorigenesis and responsiveness to therapy; little mechanistic information has been generated, however, that substantiates this idea and facilitates its clinical translation. Here, we established and characterized a model of melanoma cell senescence in which pharmacologically induced DNA damage triggered divergent ATM kinase- and STING-dependent intracellular signaling cascades and resulted in cell cycle arrest, cytomorphologic remodeling, and drastic secretome changes. Targeted proteome profiling revealed that senescent melanoma cells in this model secreted a panoply of proteins shaping the tumor immune microenvironment. CRISPR-mediated genetic ablation of the p38α and IKKβ signaling modules downstream of the ATM kinase severed the link between DNA damage and this secretory phenotype without restoring proliferative capacity. A similar genetic dissection showed that loss of STING signaling prevented type I interferon induction in DNA-damaged melanoma cells but otherwise left the senescence-associated processes in our model intact. Actionable proteins secreted from senescent melanoma cells or involved in senescence-associated intracellular signaling hold potential as markers for melanoma characterization and targets for melanoma treatment.
Project description:Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1. The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones, forming foci within 5-15 minutes. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis. In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.
Project description:H3K9me3 (methylation of lysine 9 of histone H3) is an epigenetic modification that acts as a repressor mark. Several diseases, including cancers and neurological disorders, have been associated with aberrant changes in H3K9me3 levels. Different tools have been developed to enable detection and quantification of H3K9me3 levels in cells. Most techniques, however, lack live cell compatibility. To address this concern, we have engineered recombinant protein sensors for probing H3K9me3 in situ. A heterodimeric sensor containing a chromodomain and chromo shadow domain from HP1a was found to be optimal in recognizing H3K9me3 and exhibited similar spatial resolution to commercial antibodies. Our sensor offers similar quantitative accuracy in characterizing changes in H3K9me3 compared to antibodies but claims single cell resolution. The sensor was applied to evaluate changes in H3K9me3 responding to environmental chemical atrazine (ATZ). ATZ was found to result in significant reductions in H3K9me3 levels after 24 h of exposure. Its impact on the distribution of H3K9me3 among cell populations was also assessed and found to be distinctive. We foresee the application of our sensors in multiple toxicity and drug-screening applications.
Project description:DNA modifications can be used to monitor pathological processes. We have previously shown that estimating the amount of the main DNA epigenetic mark, 5-methylcytosine (m5C), is an efficient and reliable way to diagnose brain tumors, hypertension, and other diseases. Abnormal increases of reactive oxygen species (ROS) are a driving factor for mutations that lead to changes in m5C levels and cancer evolution. 8-oxo-deoxyguanosine (8-oxo-dG) is a specific marker of ROS-driven DNA-damage, and its accumulation makes m5C a hotspot for mutations. It is unknown how m5C and 8-oxo-dG correlate with the malignancy of gliomas. We analyzed the total contents of m5C and 8-oxo-dG in DNA from tumor tissue and peripheral blood samples from brain glioma patients. We found an opposite relationship in the amounts of m5C and 8-oxo-dG, which correlated with glioma grade in the way that low level of m5C and high level of 8-oxo-dG indicated increased glioma malignancy grade. Our results could be directly applied to patient monitoring and treatment protocols for gliomas, as well as bolster previous findings, suggesting that spontaneously generated ROS react with m5C. Because of the similar mechanisms of m5C and guanosine oxidation, we concluded that 8-oxo-dG could also predict glioma malignancy grade and global DNA demethylation in cancer cells.