Nuclear architecture plays a significant role in DNA repair mechanisms. It is evident that proteins involved in DNA repair are compartmentalized in not only spontaneously occurring DNA lesions or ionizing radiation-induced foci (IRIF), but a specific clustering of these proteins can also be observed within the whole cell nucleus. For example, 53BP1-positive and BRCA1-positive DNA repair foci decorate chromocenters and can appear close to nuclear speckles. Both 53BP1 and BRCA1 are well-described factors that play an essential role in double-strand break (DSB) repair. These proteins are members of two protein complexes: 53BP1-RIF1-PTIP and BRCA1-CtIP, which make a "decision" determining whether canonical nonhomologous end joining (NHEJ) or homology-directed repair (HDR) is activated. It is generally accepted that 53BP1 mediates the NHEJ mechanism, while HDR is activated via a BRCA1-dependent signaling pathway. Interestingly, the 53BP1 protein appears relatively quickly at DSB sites, while BRCA1 is functional at later stages of DNA repair, as soon as the Mre11-Rad50-Nbs1 complex is recruited to the DNA lesions. A function of the 53BP1 protein is also linked to a specific histone signature, including phosphorylation of histone H2AX (γH2AX) or methylation of histone H4 at the lysine 20 position (H4K20me); therefore, we also discuss an epigenetic landscape of 53BP1-positive DNA lesions.

• Klíčová slova
• 53BP1, BRCA1, DNA damage, epigenetics, histone modifications,
• MeSH
• 53BP1 genetika   metabolismus   MeSH
• buněčné jádro genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• oprava DNA * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• 53BP1 MeSH

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

• MeSH
• geny p53 * MeSH
• interakční proteinové domény a motivy * MeSH
• kinetika MeSH
• konfokální mikroskopie metody   MeSH
• poškození DNA * MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH

This review focuses on the function of heterochromatin protein HP1 in response to DNA damage. We specifically outline the regulatory mechanisms in which HP1 and its interacting partners are involved. HP1 protein subtypes (HP1α, HP1β, and HP1γ) are the main components of constitutive heterochromatin, and HP1α and HP1β in particular are responsible for heterochromatin maintenance. The recruitment of these proteins to DNA lesions is also important from the perspective of proper DNA repair mechanisms. For example, HP1α is necessary for the binding of the main DNA damage-related protein 53BP1 at DNA repair foci, which are positive not only for the HP1α protein but also for the RAD51 protein, a component of DNA repair machinery. The HP1β protein also appears in monomeric form in DNA lesions together with the evolutionarily well-conserved protein called proliferating cell nuclear antigen (PCNA). The role of HP1 in DNA lesions is also mediated via the Kap1 transcription repressor. Taken together, these results indicate that the function of HP1 after DNA injury depends strongly on the kinetics of other DNA repair-related factors and their post-translational modifications, such as the phosphorylation of Kap-1.

• Klíčová slova
• DNA damage response, DNA repair, HP1 protein,
• MeSH
• chromatin metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• homolog proteinu s chromoboxem 5 MeSH
• lidé MeSH
• oprava DNA genetika   MeSH
• poškození DNA genetika   MeSH
• posttranslační úpravy proteinů MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• protein TRIM28 MeSH
• represorové proteiny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• CBX1 protein, human MeSH Prohlížeč
• CBX5 protein, human MeSH Prohlížeč
• chromatin MeSH
• chromozomální proteiny, nehistonové MeSH
• homolog proteinu s chromoboxem 5 MeSH
• proliferační antigen buněčného jádra MeSH
• protein TRIM28 MeSH
• represorové proteiny MeSH
• TRIM28 protein, human MeSH Prohlížeč

The nucleolus is a well-organized site of ribosomal gene transcription. Moreover, many DNA repair pathway proteins, including ATM, ATR kinases, MRE11, PARP1 and Ku70/80, localize to the nucleolus (Moore et al., 2011 ). We analyzed the consequences of DNA damage in nucleoli following ultraviolet A (UVA), C (UVC), or γ-irradiation in order to test whether and how radiation-mediated genome injury affects local motion and morphology of nucleoli. Because exposure to radiation sources can induce changes in the pattern of UBF1-positive nucleolar regions, we visualized nucleoli in living cells by GFP-UBF1 expression for subsequent morphological analyses and local motion studies. UVA radiation, but not 5 Gy of γ-rays, induced apoptosis as analyzed by an advanced computational method. In non-apoptotic cells, we observed that γ-radiation caused nucleolar re-positioning over time and changed several morphological parameters, including the size of the nucleolus and the area of individual UBF1-positive foci. Radiation-induced nucleoli re-arrangement was observed particularly in G2 phase of the cell cycle, indicating repair of ribosomal genes in G2 phase and implying that nucleoli are less stable, thus sensitive to radiation, in G2 phase.

• Klíčová slova
• DNA damage, UBF1, live cells, nucleolus, nuncleoli tracking,
• MeSH
• apoptóza účinky záření   MeSH
• buněčné jadérko účinky záření   MeSH
• buněčné linie MeSH
• buněčný cyklus účinky záření   MeSH
• G2 fáze účinky záření   MeSH
• genetická transkripce MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• poškození DNA účinky záření   MeSH
• transkripční iniciační komplex Pol1 - proteiny genetika   metabolismus   MeSH
• ultrafialové záření MeSH
• výpočetní biologie MeSH
• záření gama škodlivé účinky   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny MeSH

BACKGROUND: The repair of spontaneous and induced DNA lesions is a multistep process. Depending on the type of injury, damaged DNA is recognized by many proteins specifically involved in distinct DNA repair pathways. RESULTS: We analyzed the DNA-damage response after ultraviolet A (UVA) and γ irradiation of mouse embryonic fibroblasts and focused on upstream binding factor 1 (UBF1), a key protein in the regulation of ribosomal gene transcription. We found that UBF1, but not nucleolar proteins RPA194, TCOF, or fibrillarin, was recruited to UVA-irradiated chromatin concurrently with an increase in heterochromatin protein 1β (HP1β) level. Moreover, Förster Resonance Energy Transfer (FRET) confirmed interaction between UBF1 and HP1β that was dependent on a functional chromo shadow domain of HP1β. Thus, overexpression of HP1β with a deleted chromo shadow domain had a dominant-negative effect on UBF1 recruitment to UVA-damaged chromatin. Transcription factor UBF1 also interacted directly with DNA inside the nucleolus but no interaction of UBF1 and DNA was confirmed outside the nucleolus, where UBF1 recruitment to DNA lesions appeared simultaneously with cyclobutane pyrimidine dimers; this occurrence was cell-cycle-independent. CONCLUSIONS: We propose that the simultaneous presence and interaction of UBF1 and HP1β at DNA lesions is activated by the presence of cyclobutane pyrimidine dimers and mediated by the chromo shadow domain of HP1β. This might have functional significance for nucleotide excision repair.

• Klíčová slova
• DNA repair, DNA-damage response, Irradiation, Live-cell studies, Nucleolus, UBF1,
• Publikační typ
• časopisecké články MeSH