scholarly journals Quantification of DNA damage induced γH2AX focus formation via super-resolution dSTORM localization microscopy

2019 ◽  
Author(s):  
Dániel Varga ◽  
Hajnalka Majoros ◽  
Zsuzsanna Újfaludi ◽  
Miklós Erdélyi ◽  
Tibor Pankotai
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Spatial Organization ◽  
Super Resolution ◽  
Quantitative Information ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Break Site ◽  
Nucleosome Density ◽  
Repair Mechanisms

SUMMARYIn eukaryotic cells, each process, in which DNA is involved, should take place in the context of chromatin structure. DNA double-strand breaks (DSBs) are one of the most deleterious damages often leading to chromosomal rearrangement. In response to environmental stresses, cells have developed repair mechanisms to eliminate the DSBs. Upon DSB induction, several factors play roles in chromatin relaxation by catalysing the appropriate histone posttranslational modification (PTM) steps, therefore promoting the access of the repair factors to the DSBs. Among these PTMs, the phosphorylation of the histone variant H2AX at its Ser139 residue (also known as γH2AX) could be observed at the break sites. The structure of γH2AX focus has to be organized during the repair as it contributes to accessibility of specific repair proteins to the damaged site. Our aim was to develop a quantitative approach to analyse the morphology of individual repair foci by super-resolution dSTORM microscopy to gain insight into genome organization in DNA repair. We have established a specific dSTORM measurement process by developing a new analytical algorithm for gaining quantitative information about chromatin morphology and repair foci topology at individual γH2AX enriched repair focus. By this method we quantified unique repair foci to show the average distribution of γH2AX clusters. By monitoring γH2AX signal, we could reach 20 nm spatial resolution and resolve a single DNA damage spot, which allow us to identify different chromatin sub-clusters around the break site. Additionally, based on our new analysis method, we were able to show the number of nucleosomes in each sub-cluster that could allow us to define the possible chromatin structure and the nucleosome density around the break sites. This method is the first demonstration of a single-cell based quantitative measurement of a discrete repair focus, which could provide new opportunities to categorize spatial organization of dot patterns by parametric determination of topological similarity.

scholarly journals Ubiquitylation, neddylation and the DNA damage response

Open Biology ◽  
2015 ◽  
Vol 5 (4) ◽  
pp. 150018 ◽  
Author(s):  
Jessica S. Brown ◽  
Stephen P. Jackson
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cellular Response ◽  
Dna Double Strand Breaks ◽  
Post Translational Modification ◽  
Strand Breaks ◽  
Damage Sensing ◽  
Damage Response ◽  
Repair Mechanisms ◽  
And Function

Failure of accurate DNA damage sensing and repair mechanisms manifests as a variety of human diseases, including neurodegenerative disorders, immunodeficiency, infertility and cancer. The accuracy and efficiency of DNA damage detection and repair, collectively termed the DNA damage response (DDR), requires the recruitment and subsequent post-translational modification (PTM) of a complex network of proteins. Ubiquitin and the ubiquitin-like protein (UBL) SUMO have established roles in regulating the cellular response to DNA double-strand breaks (DSBs). A role for other UBLs, such as NEDD8, is also now emerging. This article provides an overview of the DDR, discusses our current understanding of the process and function of PTM by ubiquitin and NEDD8, and reviews the literature surrounding the role of ubiquitylation and neddylation in DNA repair processes, focusing particularly on DNA DSB repair.

Heterochromatin and the DNA damage response: the need to relaxThis paper is one of a selection of papers in a Special Issue entitled 31st Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

2011 ◽  
Vol 89 (1) ◽  
pp. 45-60 ◽  
Author(s):  
Kendra L. Cann ◽  
Graham Dellaire
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Chromatin Structure ◽  
Peer Review Process ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Atm Kinase ◽  
Strand Breaks ◽  
Damage Response ◽  
Chromosomal Mutations

Higher order chromatin structure has an impact on all nuclear functions, including the DNA damage response. Over the past several years, it has become increasingly clear that heterochromatin and euchromatin represent separate entities with respect to both damage sensitivity and repair. The chromatin compaction present in heterochromatin helps to protect this DNA from damage; however, when lesions do occur, the compaction restricts the ability of DNA damage response proteins to access the site, as evidenced by its ability to block the expansion of H2AX phosphorylation. As such, DNA damage in heterochromatin is refractory to repair, which requires the surrounding chromatin structure to be decondensed. In the case of DNA double-strand breaks, this relaxation is at least partially mediated by the ATM kinase phosphorylating and inhibiting the function of the transcriptional repressor KAP1. This review will focus on the functions of KAP1 and other proteins involved in the maintenance or restriction of heterochromatin, including HP1 and TIP60, in the DNA damage response. As heterochromatin is important for maintaining genomic stability, cells must maintain a delicate balance between allowing repair factors access to these regions and ensuring that these regions retain their organization to prevent increased DNA damage and chromosomal mutations.

scholarly journals Characterizing Locus Specific Chromatin Structure and Dynamics with Correlative Conventional and Super Resolution imaging in living cells

2021 ◽  
Author(s):  
Dushyant Mehra ◽  
Santosh Adhikari ◽  
Chiranjib Banerjee ◽  
Elias M. Puchner
Keyword(s):  
Single Molecule ◽  
Chromatin Structure ◽  
Spatial Organization ◽  
Super Resolution ◽  
Living Cells ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Acquisition Time ◽  
Structure And Dynamics ◽  
Chromatin Structure And Dynamics

The dynamic rearrangement of chromatin is critical for gene regulation, but mapping both the spatial organization of chromatin and its dynamics remains a challenge. Many structural conformations are too small to be resolved via conventional fluorescence microscopy and the long acquisition time of super-resolution PALM imaging precludes the structural characterization of chromatin below the optical diffraction limit in living cells due to chromatin motion. Here we develop a correlative conventional fluorescence and PALM imaging approach to quantitatively map time-averaged chromatin structure and dynamics below the optical diffraction limit in living cells. By assigning localizations to a locus as it moves, we reliably discriminate between bound and searching dCas9 molecules, whose mobility overlap. Our approach accounts for changes in DNA mobility and relates local chromatin motion to larger scale domain movement. In our experimental system, we show that compacted telomeres have a higher density of bound dCas9 molecules, but the relative motion of those molecules is more restricted than in less compacted telomeres. Correlative conventional and PALM imaging therefore improves the ability to analyze the mobility and time-averaged nanoscopic structural features of locus specific chromatin with single molecule precision and yields unprecedented insights across length and time scales.

scholarly journals The BRCA2-Interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair

2005 ◽  
Vol 25 (5) ◽  
pp. 1949-1957 ◽  
Author(s):  
Huimei Lu ◽  
Xu Guo ◽  
Xiangbing Meng ◽  
Jingmei Liu ◽  
Chris Allen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Genome Stability ◽  
Recombinational Repair ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Interacting Protein ◽  
Strand Breaks ◽  
Homologous Recombinational Repair ◽  
Radiation Induced ◽  
Nuclear Foci

ABSTRACT Homologous recombinational repair (HRR) of DNA damage is critical for maintaining genome stability and tumor suppression. RAD51 and BRCA2 colocalization in nuclear foci is a hallmark of HRR. BRCA2 has important roles in RAD51 focus formation and HRR of DNA double-strand breaks (DSBs). We previously reported that BCCIPα interacts with BRCA2. We show that a second isoform, BCCIPβ, also interacts with BRCA2 and that this interaction occurs in a region shared by BCCIPα and BCCIPβ. We further show that chromatin-bound BRCA2 colocalizes with BCCIP nuclear foci and that most radiation-induced RAD51 foci colocalize with BCCIP. Reducing BCCIPα by 90% or BCCIPβ by 50% by RNA interference markedly reduces RAD51 and BRCA2 foci and reduces HRR of DSBs by 20- to 100-fold. Similarly, reducing BRCA2 by 50% reduces RAD51 and BCCIP foci. These data indicate that BCCIP is critical for BRCA2- and RAD51-dependent responses to DNA damage and HRR.

scholarly journals Nanostructure of Clustered DNA Damage in Leukocytes after In-Solution Irradiation with the Alpha Emitter Ra-223

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1877 ◽  
Author(s):  
Harry Scherthan ◽  
Jin-Ho Lee ◽  
Emanuel Maus ◽  
Sarah Schumann ◽  
Razan Muhtadi ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Super Resolution ◽  
Dna Lesions ◽  
Alpha Particles ◽  
Chromatin Domain ◽  
Dna Double Strand Breaks ◽  
Nano Structure ◽  
Alpha Emitter ◽  
Molecular Damage

Background: Cancer patients are increasingly treated with alpha-particle-emitting radiopharmaceuticals. At the subcellular level, alpha particles induce densely spaced ionizations and molecular damage. Induction of DNA lesions, especially clustered DNA double-strand breaks (DSBs), threatens a cell’s survival. Currently, it is under debate to what extent the spatial topology of the damaged chromatin regions and the repair protein arrangements are contributing. Methods: Super-resolution light microscopy (SMLM) in combination with cluster analysis of single molecule signal-point density regions of DSB repair markers was applied to investigate the nano-structure of DNA damage foci tracks of Ra-223 in-solution irradiated leukocytes. Results: Alpha-damaged chromatin tracks were efficiently outlined by γ-H2AX that formed large (super) foci composed of numerous 60–80 nm-sized nano-foci. Alpha damage tracks contained 60–70% of all γ-H2AX point signals in a nucleus, while less than 30% of 53BP1, MRE11 or p-ATM signals were located inside γ-H2AX damage tracks. MRE11 and p-ATM protein fluorescent tags formed focal nano-clusters of about 20 nm peak size. There were, on average, 12 (±9) MRE11 nanoclusters in a typical γ-H2AX-marked alpha track, suggesting a minimal number of MRE11-processed DSBs per track. Our SMLM data suggest regularly arranged nano-structures during DNA repair in the damaged chromatin domain.

scholarly journals DNA damage triggers increased mobility of chromosomes in G1-phase cells

2019 ◽  
Vol 30 (21) ◽  
pp. 2620-2625 ◽  
Author(s):  
Michael J. Smith ◽  
Eric E. Bryant ◽  
Fraulin J. Joseph ◽  
Rodney Rothstein
Keyword(s):  
Dna Damage ◽  
S Phase ◽  
Double Strand Breaks ◽  
Homology Search ◽  
G1 Phase ◽  
Dna Damage Checkpoint ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Global Mobility ◽  
Break Site

During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in response to DNA double-strand breaks both at the break site (local mobility) and throughout the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination machinery and the DNA damage checkpoint and is likely an important aspect of homology search. While mobility in response to DNA damage has been studied extensively in S phase, the response in interphase has not, and the question of whether homologous recombination proceeds to completion in G1 phase remains controversial. Here, we find that global mobility is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these observations indicate that the recombination and checkpoint machineries promote global mobility in G1 phase, supporting the notion that recombination can occur in interphase diploids.

scholarly journals Super-Resolution Radiation Biology: From Bio-Dosimetry towards Nano-Studies of DNA Repair Mechanisms

2021 ◽  
Author(s):  
Jin-Ho Lee ◽  
Michael Hausmann
Keyword(s):  
Dna Repair ◽  
Ionizing Radiation ◽  
Fluorescence Microscopy ◽  
Spatial Organization ◽  
Super Resolution ◽  
Biological Materials ◽  
Repair Processes ◽  
Repair Protein ◽  
Repair Mechanisms ◽  
Dna Strand

Past efforts in radiobiology, radio-biophysics, epidemiology and clinical research strongly contributed to the current understanding of ionizing radiation effects on biological materials like cells and tissues. It is well accepted that the most dangerous, radiation induced damages of DNA in the cell nucleus are double strand breaks, as their false rearrangements cause dysfunction and tumor cell proliferation. Therefore, cells have developed highly efficient and adapted ways to repair lesions of the DNA double strand. To better understand the mechanisms behind DNA strand repair, a variety of fluorescence microscopy based approaches are routinely used to study radiation responses at the organ, tissue and cellular level. Meanwhile, novel super-resolution fluorescence microscopy techniques have rapidly evolved and become powerful tools to study biological structures and bio-molecular (re-)arrangements at the nano-scale. In fact, recent investigations have increasingly demonstrated how super-resolution microscopy can be applied to the analysis of radiation damage induced chromatin arrangements and DNA repair protein recruitment in order to elucidate how spatial organization of damage sites and repair proteins contribute to the control of repair processes. In this chapter, we would like to start with some fundamental aspects of ionizing radiation, their impact on biological materials, and some standard radiobiology assays. We conclude by introducing the concept behind super-resolution radiobiology using single molecule localization microscopy (SMLM) and present promising results from recent studies that show an organized architecture of damage sites and their environment. Persistent homologies of repair clusters indicate a correlation between repair cluster topology and repair pathway at a given damage locus. This overview over recent investigations may motivate radiobiologists to consider chromatin architecture and spatial repair protein organization for the understanding of DNA repair processes.

scholarly journals DNA Damage and Repair Mechanisms Triggered by Exposure to Bioflavonoids and Natural Compounds

2021 ◽  
Author(s):  
Donna Goodenow ◽  
Kiran Lalwani ◽  
Christine Richardson
Keyword(s):  
Dna Damage ◽  
Cellular Response ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Environmental Agents ◽  
Repair Mechanisms ◽  
Alternative End Joining ◽  
Differentiation Status

Eukaryotic cells use homologous recombination (HR), classical end-joining (C-NHEJ), and alternative end-joining (Alt-EJ) to repair DNA double-strand breaks (DSBs). Repair pathway choice is controlled by the activation and activity of pathways specific proteins in eukaryotes. Activity may be regulated by cell cycle stage, tissue type, and differentiation status. Bioflavonoids and other environmental agents such as pesticides have been shown to biochemically act as inhibitors of topoisomerase II (Top2). In cells, bioflavonoids directly lead to DNA double-strand breaks through both Top2-dependent and independent mechanisms, as well as induce DNA damage response (DDR) signaling, and promote alternative end-joining and chromosome alterations. This chapter will present differences in expression and activity of proteins in major DNA repair pathways, findings of Top2 inhibition by bioflavonoids and cellular response, discuss how these compounds trigger alternative end-joining, and conclude with implications for genome instability and human disease.

scholarly journals Quantification of DNA damage induced repair focus formation via super-resolution dSTORM localization microscopy

Nanoscale ◽  
2019 ◽  
Vol 11 (30) ◽  
pp. 14226-14236 ◽  
Author(s):  
Dániel Varga ◽  
Hajnalka Majoros ◽  
Zsuzsanna Ujfaludi ◽  
Miklós Erdélyi ◽  
Tibor Pankotai
Keyword(s):  
Dna Damage ◽  
Super Resolution ◽  
Strand Break ◽  
Double Strand Break ◽  
Quantitative Approach ◽  
Focus Formation ◽  
Localization Microscopy ◽  
Dna Double Strand Break

A quantitative approach has been developed to analyse the morphology and distribution of DNA double-strand break induced single repair focus by super-resolution dSTORM microscopy.

scholarly journals A DNA Damage-Regulated BRCT-Containing Protein, TopBP1, Is Required for Cell Survival

2002 ◽  
Vol 22 (2) ◽  
pp. 555-566 ◽  
Author(s):  
Kazuhiko Yamane ◽  
Xianglin Wu ◽  
Junjie Chen
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Ataxia Telangiectasia ◽  
Topoisomerase Ii ◽  
Sequence Similarity ◽  
Dna Double Strand Breaks ◽  
Focus Formation ◽  
Replication Checkpoint ◽  
Strand Breaks

ABSTRACT BRCA1 carboxyl-terminal (BRCT) motifs are present in a number of proteins involved in DNA repair and/or DNA damage-signaling pathways. Human DNA topoisomerase II binding protein 1 (TopBP1) contains eight BRCT motifs and shares sequence similarity with the fission yeast Rad4/Cut5 protein and the budding yeast DPB11 protein, both of which are required for DNA damage and/or replication checkpoint controls. We report here that TopBP1 is phosphorylated in response to DNA double-strand breaks and replication blocks. TopBP1 forms nuclear foci and localizes to the sites of DNA damage or the arrested replication forks. In response to DNA strand breaks, TopBP1 phosphorylation depends on the ataxia telangiectasia mutated protein (ATM) in vivo. However, ATM-dependent phosphorylation of TopBP1 does not appear to be required for focus formation following DNA damage. Instead, focus formation relies on one of the BRCT motifs, BRCT5, in TopBP1. Antisense Morpholino oligomers against TopBP1 greatly reduced TopBP1 expression in vivo. Similar to that of ataxia telangiectasia-related protein (ATR), Chk1, or Hus1, downregulation of TopBP1 leads to reduced cell survival, probably due to increased apoptosis. Taken together, the data presented here suggest that, like its putative counterparts in yeast species, TopBP1 may be involved in DNA damage and replication checkpoint controls.

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