We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.
Cancer is a disease attributed to the accumulation of DNA damages due to incapacitation of DNA repair pathways resulting in genomic instability and a mutator phenotype. Among the DNA lesions, double stranded breaks (DSBs) are the most toxic forms of DNA damage which may arise as a result of extrinsic DNA damaging agents or intrinsic replication stress in fast proliferating cancer cells. Accurate repair of DSBs is therefore paramount to the cell survival, and several classes of proteins such as kinases, nucleases, helicases or core recombinational proteins have pre-defined jobs in precise execution of DSB repair pathways. On one hand, the proper functioning of these proteins ensures maintenance of genomic stability in normal cells, and on the other hand results in resistance to various drugs employed in cancer therapy and therefore presents a suitable opportunity for therapeutic targeting. Higher relapse and resistance in cancer patients due to non-specific, cytotoxic therapies is an alarming situation and it is becoming more evident to employ personalized treatment based on the genetic landscape of the cancer cells. For the success of personalized treatment, it is of immense importance to identify more suitable targetable proteins in DSB repair pathways and also to explore new synthetic lethal interactions with these pathways. Here we review the various alternative approaches to target the various protein classes termed as cancer TARGETases in DSB repair pathway to obtain more beneficial and selective therapy.
- MeSH
- Molecular Targeted Therapy methods MeSH
- DNA Breaks, Double-Stranded drug effects MeSH
- Humans MeSH
- Neoplasms drug therapy enzymology MeSH
- DNA Repair drug effects MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Double-strand breaks (DSBs), continuously introduced into DNA by cell metabolism, ionizing radiation and some chemicals, are the biologically most deleterious type of genome damage, and must be accurately repaired to protect genomic integrity, ensure cell survival, and prevent carcinogenesis. Although a huge amount of information has been published on the molecular basis and biological significance of DSB repair, our understanding of DSB repair and its spatiotemporal arrangement is still incomplete. In particular, the role of higher-order chromatin structure in DSB induction and repair, movement of DSBs and the mechanism giving rise to chromatin exchanges, and many other currently disputed questions are discussed in this review. Finally, a model explaining the formation of chromosome translocations is proposed.
- MeSH
- Models, Biological MeSH
- Chromatin radiation effects ultrastructure MeSH
- DNA Breaks, Double-Stranded MeSH
- Radiation, Ionizing MeSH
- Humans MeSH
- DNA Repair MeSH
- DNA Damage MeSH
- Translocation, Genetic MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
Mathematical analytical model of the processes running in individual radical clusters during the chemical phase (under the presence of radiomodifiers) proposed by us earlier has been further developed and improved. It has been applied to the data presented by Blok and Loman characterizing the oxygen effect in SSB and DSB formation (in water solution and at low-LET radiation) also in the region of very small oxygen concentrations, which cannot be studied with the help of experiments done with living cells. In this new analysis the values of all reaction rates and diffusion parameters known from literature have been made use of. The great increase of SSB and DSB at zero oxygen concentration may follow from the fact that at small oxygen concentrations the oxygen absorbs other radicals while at higher concentrations the formation of oxygen radicals prevails. It explains the double oxygen effect found already earlier by Ewing. The model may be easily extended to include also the effects of other radiomodifiers present in medium during irradiation.
- MeSH
- Bacteriophage phi X 174 genetics radiation effects MeSH
- Models, Biological * MeSH
- Chromosomes drug effects radiation effects MeSH
- Diffusion drug effects radiation effects MeSH
- DNA Breaks, Double-Stranded drug effects radiation effects MeSH
- Radiation, Ionizing * MeSH
- Oxygen pharmacology MeSH
- Publication type
- Journal Article MeSH
Homologous recombination (HR) protects replication forks (RFs) and repairs DNA double-strand breaks (DSBs). Within HR, BRCA2 regulates RAD51 via two interaction regions: the BRC repeats to form filaments on single-stranded DNA and exon 27 (Ex27) to stabilize the filament. Here, we identified a RAD51 S181P mutant that selectively disrupted the RAD51-Ex27 association while maintaining interaction with BRC repeat and proficiently forming filaments capable of DNA binding and strand invasion. Interestingly, RAD51 S181P was defective for RF protection/restart but proficient for DSB repair. Our data suggest that Ex27-mediated stabilization of RAD51 filaments is required for the protection of RFs, while it seems dispensable for the repair of DSBs.
- Publication type
- Journal Article MeSH
The proper repair of deleterious DNA lesions such as double strand breaks prevents genomic instability and carcinogenesis. In yeast, the Rad52 protein mediates DSB repair via homologous recombination. In mammalian cells, despite the presence of the RAD52 protein, the tumour suppressor protein BRCA2 acts as the predominant mediator during homologous recombination. For decades, it has been believed that the RAD52 protein played only a back-up role in the repair of DSBs performing an error-prone single strand annealing (SSA). Recent studies have identified several new functions of the RAD52 protein and have drawn attention to its important role in genome maintenance. Here, we show that RAD52 activities are enhanced by interacting with a small and highly acidic protein called DSS1. Binding of DSS1 to RAD52 changes the RAD52 oligomeric conformation, modulates its DNA binding properties, stimulates SSA activity and promotes strand invasion. Our work introduces for the first time RAD52 as another interacting partner of DSS1 and shows that both proteins are important players in the SSA and BIR pathways of DSB repair.
- MeSH
- Rad52 DNA Repair and Recombination Protein genetics MeSH
- DNA-Binding Proteins genetics MeSH
- DNA Breaks, Double-Stranded MeSH
- Genome, Human genetics MeSH
- Homologous Recombination genetics MeSH
- Carcinogenesis genetics MeSH
- Humans MeSH
- Genomic Instability genetics MeSH
- DNA Repair genetics MeSH
- Osteosarcoma genetics pathology MeSH
- Proteasome Endopeptidase Complex genetics MeSH
- BRCA2 Protein genetics MeSH
- Saccharomyces cerevisiae Proteins genetics MeSH
- Saccharomyces cerevisiae genetics MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
K novým poznatkům o vlivu různých druhů ionizujícího záření na buňky patří mikro- a nanodozimetrické aspekty poškození chromatinu. Fyzikální vlastnosti incidentního ionizujícího záření (fotonů gama, protonů a iontů s vysokým LET) souvisí s charakterem poškození chromatinu, možnostmi buňky opravit a přežít vytvořené léze DNA a rizikem genetických změn. Přestože výsledky jednoznačně potvrzují pozitivní korelaci mezi LET ionizujícího záření, komplexností indukovaných dvouřetězcových zlomů DNA (DSB) a biologickou účinností (RBE) záření, zároveň odhalují, že těmto vztahům ještě dostatečně nerozumíme. Příkladem budiž zjištění, že různé urychlené ionty s podobným LET mohou poškozovat DNA odlišným způsobem a zabíjet tak buňky s nestejnou účinností. Stále také neumíme vysvětlit mnoho aspektů reparace DSB, například co rozhoduje o aktivaci určité reparační dráhy v místě konkrétního DSB a jak je tento výběr ovlivněn použitým ionizujícím zářením a strukturou chromatinu. Diskutované výsledky mohou být mj. důležité z hlediska nově se rozvíjející hadronové terapie nádorových onemocnění a plánování pilotovaných meziplanetárních letů. Z metodického hlediska potom tato práce ilustruje obrovský pokrok, který se udál na poli optické mikroskopie a jejích výzkumných aplikací. Detailněji je představena metoda lokalizační mikroskopie s rozlišením jednotlivých molekul (SMLM – single-molecule localization microscopy).
The present work introduces new findings about the influence of different radiation types on the cells, with the concern on the micro- and nanodosimetric aspects of chromatin damage. Emphasized is the relationship between the physical parameters of the incident radiation (g-rays, protons and high-LET heavy ions), character of chromatin damage, ability of cells to repair and survive DNA damage, and risk of genetic changes. While confirming a positive correlation between the LET of ionizing radiation, complexity of induced DNA double-strand breaks (DSB), and biological effectiveness (RBE) of radiation, at the same time, we show that our understanding of this relationship is only incomplete. Our discovery that various accelerated ions with similar LET can damage DNA in different ways and kill cells with unequal efficiency, could serve as an example. In addition, many aspects of DSB repair remain to be explained, for instance, how the cell activates the particular repair pathway at sites of individual DSBs, and how it depends on the radiation used and the chromatin architecture. The discussed results may be important, above all, for newly developing hadron therapy and in the context of manned interstellar flights planning. From the methodological point of view, we point to a tremendous progress in the field of optical microscopy and its research applications. In more detail, we introduce single-molecule localization microscopy (SMLM).
- Keywords
- reparační ohniska indukovaná ionizujícím zářením (IRIF),
- MeSH
- Chromatin radiation effects MeSH
- Chromosome Aberrations radiation effects MeSH
- Phosphorylation MeSH
- Radiation, Ionizing * MeSH
- Humans MeSH
- Microscopy methods MeSH
- DNA Repair * radiation effects MeSH
- DNA Damage * radiation effects MeSH
- Radiation Exposure MeSH
- Single Molecule Imaging methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
The biological effect of ionizing particles is caused mainly by water radicals being formed by densely ionizing ends of primary or secondary charged particles during physical stage; only greater radical clusters being efficient in DNA molecule damaging. The given clusters diffuse after their formation and the radical concentration changes also by reactions running mutually or with other substances being present in corresponding clusters. The damage effect depends then on radical concentrations at a time when the cluster meets a DNA molecule. The influence of oxygen may be important (mainly in the case of low-LET radiation) because oxygen is always present in living cells. Oxygen may act then in two different directions: at small concentrations the interaction with hydrogen radicals prevails and final biological effect diminishes while at higher concentrations additional efficient oxygen radicals may be formed. The time evolution of changing radical concentrations during cluster diffusion may be modeled and analyzed well with the help of Continuous Petri nets.
- Keywords
- formace DSB, Petriho sítě, diferenciální rovnice, chemická fáze,
- MeSH
- Radiation, Ionizing * MeSH
- Radiobiology * MeSH
- Models, Theoretical MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
53BP1 is a key component of the genome surveillance network activated by DNA double strand breaks (DSBs). Despite its known accumulation at the DSB sites, the spatiotemporal aspects of 53BP1 interaction with DSBs and the role of other DSB regulators in this process remain unclear. Here, we used real-time microscopy to study the DSB-induced redistribution of 53BP1 in living cells. We show that within minutes after DNA damage, 53BP1 becomes progressively, yet transiently, immobilized around the DSB-flanking chromatin. Quantitative imaging of single cells revealed that the assembly of 53BP1 at DSBs significantly lagged behind Mdc1/NFBD1, another DSB-interacting checkpoint mediator. Furthermore, short interfering RNA-mediated ablation of Mdc1/NFBD1 drastically impaired 53BP1 redistribution to DSBs and triggered premature dissociation of 53BP1 from these regions. Collectively, these in vivo measurements identify Mdc1/NFBD1 as a key upstream determinant of 53BP1's interaction with DSBs from its dynamic assembly at the DSB sites through sustained retention within the DSB-flanking chromatin up to the recovery from the checkpoint.
- MeSH
- Chromatin physiology MeSH
- DNA-Binding Proteins physiology genetics MeSH
- Phosphoproteins physiology genetics MeSH
- Intracellular Signaling Peptides and Proteins physiology genetics MeSH
- Nuclear Proteins physiology genetics MeSH
- Microscopy, Confocal MeSH
- Humans MeSH
- RNA, Small Interfering physiology MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- DNA Damage * MeSH
- Recombinant Fusion Proteins genetics metabolism MeSH
- Trans-Activators physiology genetics MeSH
- Protein Transport MeSH
- Protein Binding MeSH
- Green Fluorescent Proteins genetics MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
