The DNA damage response is mediated by both DNA repair proteins and epigenetic markers. Here, we observe that N6-methyladenosine (m6A), a mark of the epitranscriptome, was common in RNAs accumulated at UV-damaged chromatin; however, inhibitors of RNA polymerases I and II did not affect the m6A RNA level at the irradiated genomic regions. After genome injury, m6A RNAs either diffused to the damaged chromatin or appeared at the lesions enzymatically. DNA damage did not change the levels of METTL3 and METTL14 methyltransferases. In a subset of irradiated cells, only the METTL16 enzyme, responsible for m6A in non-coding RNAs as well as for splicing regulation, was recruited to microirradiated sites. Importantly, the levels of the studied splicing factors were not changed by UVA light. Overall, if the appearance of m6A RNAs at DNA lesions is regulated enzymatically, this process must be mediated via the coregulatory function of METTL-like enzymes. This event is additionally accompanied by radiation-induced depletion of 2,2,7-methylguanosine (m3G/TMG) in RNA. Moreover, UV-irradiation also decreases the global cellular level of N1-methyladenosine (m1A) in RNAs. Based on these results, we prefer a model in which m6A RNAs rapidly respond to radiation-induced stress and diffuse to the damaged sites. The level of both (m1A) RNAs and m3G/TMG in RNAs is reduced as a consequence of DNA damage, recognized by the nucleotide excision repair mechanism.

• Klíčová slova
• DNA repair, METTL-like enzymes, RNA methylation, epigenetics, histones,
• MeSH
• adenosin analogy a deriváty   metabolismus   MeSH
• chromatin metabolismus   MeSH
• demetylace DNA účinky záření   MeSH
• fyziologický stres účinky záření   MeSH
• guanosin analogy a deriváty   metabolismus   MeSH
• metylace DNA genetika   účinky záření   MeSH
• metylace účinky záření   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nekódující RNA metabolismus   MeSH
• nestabilita genomu účinky záření   MeSH
• poškození DNA MeSH
• RNA metabolismus   MeSH
• ultrafialové záření * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosin MeSH
• chromatin MeSH
• guanosin MeSH
• N-methyladenosine MeSH Prohlížeč
• N(2),N(2),7-trimethylguanosine MeSH Prohlížeč
• nekódující RNA MeSH
• RNA MeSH

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

Repair of ribosomal DNA (rDNA) is a very important nuclear process due to the most active transcription of ribosomal genes. Proper repair of rDNA is required for physiological biogenesis of ribosomes. Here, we analyzed the epigenetics of the DNA damage response in a nucleolar compartment, thus in the ribosomal genes studied in nonirradiated and UVA-irradiated mouse embryonic fibroblasts (MEFs). We found that the promoter of ribosomal genes is not abundant on H4K20me2, but it is densely occupied by H4K20me3. Ribosomal genes, regulated via UBF1/2 proteins, were characterized by an interaction between UBF1/2 and H4K20me2/me3. This interaction was strengthened by UVA irradiation that additionally causes a focal accumulation of H4K20me3 in the nucleolus. No interaction has been found between UBF1/2 and H3K9me3. Interestingly, UVA irradiation decreases the levels of H3K9me3 and H4K20me3 at 28S rDNA. Altogether, the UVA light affects the epigenetic status of ribosomal genes at 28S rDNA and strengthens an interaction between UBF1/2 proteins and H4K20me2/me3.

• Klíčová slova
• DNA damage response, DNA repair, Nucleolus, UBF, UVA irradiation,
• MeSH
• buněčné jadérko metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• chromatinová imunoprecipitace MeSH
• DNA vazebné proteiny MeSH
• epigeneze genetická účinky záření   MeSH
• fluorescenční protilátková technika MeSH
• histony metabolismus   MeSH
• metylace MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese účinky záření   MeSH
• ribozomální DNA genetika   MeSH
• transkripční iniciační komplex Pol1 - proteiny metabolismus   MeSH
• ultrafialové záření * MeSH
• vazba proteinů MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• histony MeSH
• ribozomální DNA MeSH
• transcription factor UBF MeSH Prohlížeč
• transkripční iniciační komplex Pol1 - proteiny 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

DNA damage response (DDR) in ribosomal genes and mechanisms of DNA repair in embryonic stem cells (ESCs) are less explored nuclear events. DDR in ESCs should be unique due to their high proliferation rate, expression of pluripotency factors, and specific chromatin signature. Given short population doubling time and fast progress through G1 phase, ESCs require a sustained production of rRNA, which leads to the formation of large and prominent nucleoli. Although transcription of rRNA in the nucleolus is relatively well understood, little is known about DDR in this nuclear compartment. Here, we directed formation of double-strand breaks in rRNA genes with I- PpoI endonuclease, and we studied nucleolar morphology, DDR, and chromatin modifications. We observed a pronounced formation of I- PpoI-induced nucleolar caps, positive on BRCA1, NBS1, MDC1, γH2AX, and UBF1 proteins. We showed interaction of nucleolar protein TCOF1 with HDAC1 and TCOF1 with CARM1 after DNA injury. Moreover, H3R17me2a modification mediated by CARM1 was found in I- PpoI-induced nucleolar caps. Finally, we report that heterochromatin protein 1 is not involved in DNA repair of nucleolar caps.

• Klíčová slova
• CARM1, DNA repair, HDAC1, NBS1, PpoI, chromatin, nucleolus,
• MeSH
• acetylace MeSH
• arginin metabolismus   MeSH
• buněčné jadérko genetika   ultrastruktura   MeSH
• buněčné linie MeSH
• dvouřetězcové zlomy DNA * MeSH
• embryonální kmenové buňky metabolismus   ultrastruktura   MeSH
• fosfoproteiny metabolismus   MeSH
• geny rRNA MeSH
• histondeacetylasa 1 metabolismus   MeSH
• histony metabolismus   MeSH
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny metabolismus   MeSH
• metylace MeSH
• myši MeSH
• oprava DNA MeSH
• proteinarginin-N-methyltransferasy metabolismus   MeSH
• RNA ribozomální genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• arginin MeSH
• coactivator-associated arginine methyltransferase 1 MeSH Prohlížeč
• fosfoproteiny MeSH
• Hdac1 protein, mouse MeSH Prohlížeč
• histondeacetylasa 1 MeSH
• histony MeSH
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny MeSH
• proteinarginin-N-methyltransferasy MeSH
• RNA ribozomální MeSH
• Tcof1 protein, mouse MeSH Prohlížeč