BACKGROUND: Chemical modifications in mRNAs, tRNAs, rRNAs, and non-coding RNAs stabilize these nucleic acids and regulate their function. In addition to regulating the translation of genetic information from mRNA to proteins, it has been revealed that modifications in RNAs regulate repair processes in the genome. METHODS: Using local laser microirradiation, confocal microscopy, dot blots, and mass spectrometry we studied the role of N7-methylguanosine (m7G), which is co-transcriptionally installed in RNA. RESULTS: Here, we show that after UVC and UVA irradiation, the level of m7G RNA is increased initially in the cytoplasm, and after local laser microirradiation, m7G RNA is highly abundant in UVA-damaged chromatin. This process is poly(ADP-ribose) polymerase (PARP)-dependent, but not accompanied by changes in the level of m7G-writers, including methyltransferases RNMT, METTL1, and WBSCR22. We also observed that METTL1 deficiency does not affect the recruitment of m7G RNA to microirradiated chromatin. Analyzing the levels of mRNA, let-7e, and miR-203a in both the cytoplasm and the cell nucleus, we revealed that UVC irradiation changed the level of mRNA, and significantly increased the pool of both let-7e and miR-203a, which correlated with radiation-induced m7G RNA increase in the cytoplasm. CONCLUSIONS: Irradiation by UV light increases the m7G RNA pool in the cytoplasm and in the microirradiated genome. Thus, epigenetically modified RNAslikely contribute to DNA damage responses or m7G signals the presence of RNA damage.

• Keywords
• DNA repair, RNA methylation, mRNA, miRNA, snRNA,
• Publication type
• Journal Article MeSH

RNA modifications have been known for many years, but their function has not been fully elucidated yet. For instance, the regulatory role of acetylation on N4-cytidine (ac4C) in RNA can be explored not only in terms of RNA stability and mRNA translation but also in DNA repair. Here, we observe a high level of ac4C RNA at DNA lesions in interphase cells and irradiated cells in telophase. Ac4C RNA appears in the damaged genome from 2 to 45 min after microirradiation. However, RNA cytidine acetyltransferase NAT10 did not accumulate to damaged sites, and NAT10 depletion did not affect the pronounced recruitment of ac4C RNA to DNA lesions. This process was not dependent on the G1, S, and G2 cell cycle phases. In addition, we observed that the PARP inhibitor, olaparib, prevents the recruitment of ac4C RNA to damaged chromatin. Our data imply that the acetylation of N4-cytidine, especially in small RNAs, has an important role in mediating DNA damage repair. Ac4C RNA likely causes de-condensation of chromatin in the vicinity of DNA lesions, making it accessible for other DNA repair factors involved in the DNA damage response. Alternatively, RNA modifications, including ac4C, could be direct markers of damaged RNAs.

• Keywords
• DNA repair, NAT10, PARP, RNA acetylation, RNA methylation,
• MeSH
• Acetylation MeSH
• Chromatin MeSH
• Cytidine * genetics   metabolism   MeSH
• Poly(ADP-ribose) Polymerase Inhibitors MeSH
• RNA * metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Chromatin MeSH
• Cytidine * MeSH
• Poly(ADP-ribose) Polymerase Inhibitors MeSH
• RNA * MeSH

RNA methylation, especially 6-methyladenosine (m6A)-modified RNAs, plays a specific role in DNA damage response (DDR). Here, we also observe that RNA modified at 8-methyladenosine (m8A) is recruited to UVA-damaged chromatin immediately after microirradiation. Interestingly, the level of m8A RNA at genomic lesions was reduced after inhibition of histone deacetylases and DNA methyltransferases. It appears in later phases of DNA damage response, accompanied by active DNA demethylation. Also, PARP inhibitor (PARPi), Olaparib, prevented adenosine methylation at microirradiated chromatin. PARPi abrogated not only m6A and m8A RNA positivity at genomic lesions, but also XRCC1, the factor of base excision repair (BER), did not recognize lesions in DNA. To this effect, Olaparib enhanced the genome-wide level of γH2AX. This histone modification interacted with m8A RNAs to a similar extent as m8A RNAs with DNA. Pronounced interaction properties we did not observe for m6A RNAs and DNA; however, m6A RNA interacted with XRCC1 with the highest efficiency, especially in microirradiated cells. Together, we show that the recruitment of m6A RNA and m8A RNA to DNA lesions is PARP dependent. We suggest that modified RNAs likely play a role in the BER mechanism accompanied by active DNA demethylation. In this process, γH2AX stabilizes m6A/m8A-positive RNA-DNA hybrid loops via its interaction with m8A RNAs. R-loops could represent basic three-stranded structures recognized by PARP-dependent non-canonical m6A/m8A-mediated DNA repair pathway.

• Keywords
• DNA demethylation, DNA repair, RNA methylation, base excision repair, epigenetics,
• MeSH
• Chromatin MeSH
• DNA Demethylation * MeSH
• DNA metabolism   MeSH
• DNA Methylation MeSH
• DNA Repair MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * pharmacology   MeSH
• DNA Damage MeSH
• RNA genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA MeSH
• Poly(ADP-ribose) Polymerase Inhibitors * MeSH
• RNA MeSH

The essential components of splicing are the splicing factors accumulated in nuclear speckles; thus, we studied how DNA damaging agents and A-type lamin depletion affect the properties of these regions, positive on the SC-35 protein. We observed that inhibitor of PARP (poly (ADP-ribose) polymerase), and more pronouncedly inhibitors of RNA polymerases, caused DNA damage and increased the SC35 protein level. Interestingly, nuclear blebs, induced by PARP inhibitor and observed in A-type lamin-depleted or senescent cells, were positive on both the SC-35 protein and another component of the spliceosome, SRRM2. In the interphase cell nuclei, SC-35 interacted with the phosphorylated form of RNAP II, which was A-type lamin-dependent. In mitotic cells, especially in telophase, the SC35 protein formed a well-visible ring in the cytoplasmic fraction and colocalized with β-catenin, associated with the plasma membrane. The antibody against the SRRM2 protein showed that nuclear speckles are already established in the cytoplasm of the late telophase and at the stage of early cytokinesis. In addition, we observed the occurrence of splicing factors in the nuclear blebs and micronuclei, which are also sites of both transcription and splicing. This conclusion supports the fact that splicing proceeds transcriptionally. According to our data, this process is A-type lamin-dependent. Lamin depletion also reduces the interaction between SC35 and β-catenin in mitotic cells.

• Keywords
• PARP inhibitor, RNA pol II, SC-35, splicing,
• MeSH
• HeLa Cells MeSH
• Lamins metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Poly(ADP-ribose) Polymerase Inhibitors therapeutic use   MeSH
• Poly (ADP-Ribose) Polymerase-1 MeSH
• RNA Polymerase II metabolism   MeSH
• RNA Splicing Factors metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lamins MeSH
• Poly(ADP-ribose) Polymerase Inhibitors MeSH
• PARP1 protein, human MeSH Browser
• Poly (ADP-Ribose) Polymerase-1 MeSH
• RNA Polymerase II MeSH
• RNA Splicing Factors MeSH

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.

• Keywords
• DNA repair, METTL-like enzymes, RNA methylation, epigenetics, histones,
• MeSH
• Adenosine analogs & derivatives   metabolism   MeSH
• Chromatin metabolism   MeSH
• DNA Demethylation radiation effects   MeSH
• Stress, Physiological radiation effects   MeSH
• Guanosine analogs & derivatives   metabolism   MeSH
• DNA Methylation genetics   radiation effects   MeSH
• Methylation radiation effects   MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• RNA, Untranslated metabolism   MeSH
• Genomic Instability radiation effects   MeSH
• DNA Damage MeSH
• RNA metabolism   MeSH
• Ultraviolet Rays * MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine MeSH
• Chromatin MeSH
• Guanosine MeSH
• N-methyladenosine MeSH Browser
• N(2),N(2),7-trimethylguanosine MeSH Browser
• RNA, Untranslated MeSH
• RNA MeSH

The family of heterochromatin protein 1 (HP1) isoforms is essential for chromatin packaging, regulation of gene expression, and repair of damaged DNA. Here we document that γ-radiation reduced the number of HP1α-positive foci, but not HP1β and HP1γ foci, located in the vicinity of the fibrillarin-positive region of the nucleolus. The additional analysis confirmed that γ-radiation has the ability to significantly decrease the level of HP1α in rDNA promoter and rDNA encoding 28S rRNA. By mass spectrometry, we showed that treatment by γ-rays enhanced the HP1β serine 88 phosphorylation (S88ph), but other analyzed modifications of HP1β, including S161ph/Y163ph, S171ph, and S174ph, were not changed in cells exposed to γ-rays or treated by the HDAC inhibitor (HDACi). Interestingly, a combination of HDACi and γ-radiation increased the level of HP1α and HP1γ. The level of HP1β remained identical before and after the HDACi/γ-rays treatment, but HDACi strengthened HP1β interaction with the KRAB-associated protein 1 (KAP1) protein. Conversely, HP1γ did not interact with KAP1, although approximately 40% of HP1γ foci co-localized with accumulated KAP1. Especially HP1γ foci at the periphery of nucleoli were mostly absent of KAP1. Together, DNA damage changed the morphology, levels, and interaction properties of HP1 isoforms. Also, γ-irradiation-induced hyperphosphorylation of the HP1β protein; thus, HP1β-S88ph could be considered as an important marker of DNA damage.

• Keywords
• FLIM-FRET, HP1, epigenetics, irradiation, mass spectrometry, nucleolus, phosphorylation,
• MeSH
• Cell Nucleolus metabolism   MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Phosphorylation MeSH
• HeLa Cells MeSH
• Chromobox Protein Homolog 5 MeSH
• Humans MeSH
• Tumor Cells, Cultured MeSH
• Optical Imaging MeSH
• DNA Damage MeSH
• Fluorescence Resonance Energy Transfer MeSH
• Serine metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CBX1 protein, human MeSH Browser
• CBX5 protein, human MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Chromobox Protein Homolog 5 MeSH
• Serine 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.

• Keywords
• DNA damage response, DNA repair, Nucleolus, UBF, UVA irradiation,
• MeSH
• Cell Nucleolus metabolism   MeSH
• Cell Nucleus metabolism   MeSH
• Chromatin Immunoprecipitation MeSH
• DNA-Binding Proteins MeSH
• Epigenesis, Genetic radiation effects   MeSH
• Fluorescent Antibody Technique MeSH
• Histones metabolism   MeSH
• Methylation MeSH
• Mice MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation radiation effects   MeSH
• DNA, Ribosomal genetics   MeSH
• Pol1 Transcription Initiation Complex Proteins metabolism   MeSH
• Ultraviolet Rays * MeSH
• Protein Binding MeSH
• High-Throughput Nucleotide Sequencing MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins MeSH
• Histones MeSH
• DNA, Ribosomal MeSH
• transcription factor UBF MeSH Browser
• Pol1 Transcription Initiation Complex Proteins MeSH

Methylation of histones H4 at lysine 20 position (H4K20me), which is functional in DNA repair, represents a binding site for the 53BP1 protein. Here, we show a radiation-induced increase in the level of H4K20me3 while the levels of H4K20me1 and H4K20me2 remained intact. H4K20me3 was significantly pronounced at DNA lesions in only the G1 phase of the cycle, while this histone mark was reduced in very late S and G2 phases when PCNA was recruited to locally micro-irradiated chromatin. H4K20me3 was diminished in locally irradiated Suv39h1/h2 double knockout (dn) fibroblasts, and the same phenomenon was observed for H3K9me3 and its binding partner, the HP1β protein. Immunoprecipitation showed the existence of an interaction between H3K9me3-53BP1 and H4K20me3-53BP1; however, HP1β did not interact with 53BP1. Together, H3K9me3 and H4K20me3 represent epigenetic markers that are important for the function of the 53BP1 protein in non-homologous end joining (NHEJ) repair. The very late S phase represents the cell cycle breakpoint when a DDR function of the H4K20me3-53BP1 complex is abrogated due to recruitment of the PCNA protein and other DNA repair factors of homologous recombination to DNA lesions.

• Keywords
• DNA damage, H3K9me3, H4K20me1/me2/me3, Suv39h1/h2, epigenetics,
• MeSH
• Tumor Suppressor p53-Binding Protein 1 genetics   metabolism   MeSH
• Cell Nucleus genetics   metabolism   radiation effects   MeSH
• Cell Line MeSH
• Cell Cycle MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Epigenesis, Genetic * radiation effects   MeSH
• Histones metabolism   MeSH
• Chromobox Protein Homolog 5 MeSH
• Humans MeSH
• DNA Methylation * radiation effects   MeSH
• Methylation MeSH
• Mice MeSH
• DNA End-Joining Repair * MeSH
• DNA Damage * MeSH
• Proliferating Cell Nuclear Antigen metabolism   MeSH
• Chromatin Assembly and Disassembly MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• CBX1 protein, human MeSH Browser
• Cbx1 protein, mouse MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Histones MeSH
• Chromobox Protein Homolog 5 MeSH
• PCNA protein, human MeSH Browser
• Proliferating Cell Nuclear Antigen MeSH
• TP53BP1 protein, human MeSH Browser
• Trp53bp1 protein, mouse MeSH Browser

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
• Genes, p53 * MeSH
• Protein Interaction Domains and Motifs * MeSH
• Kinetics MeSH
• Microscopy, Confocal methods   MeSH
• DNA Damage * MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH

53BP1 is a very well-known protein that is recruited to DNA lesions. The focal accumulation of p53 binding protein, 53BP1, is a main feature indicating the repair of spontaneous or irradiation-induced foci (IRIF). Thus, here, we addressed the question of whether mutations in the TP53 gene, which often affect the level of p53 protein, can change the recruitment of 53BP1 to γ- or UVA-irradiated chromatin. In various TP53 mutants, we observed a distinct accumulation of 53BP1 protein to UV-induced DNA lesions: in R273C mutants, 53BP1 appeared transiently at DNA lesions, during 10-30 min after irradiation; the mutation R282W was responsible for accumulation of 53BP1 immediately after UVA-damage; and in L194F mutants, the first appearance of 53BP1 protein at the lesions occurred during 60-70 min. These results showed that specific mutations in the TP53 gene stand behind not only different levels of p53 protein, but also affect the localized kinetics of 53BP1 protein in UVA-damaged chromatin. However, after γ-irradiation, only G245S mutation in TP53 gene was associated with surprisingly decreased level of 53BP1 protein. In other mutant cell lines, levels of 53BP1 were not affected by γ-rays. To these effects, we conversely found a distinct number of 53BP1-positive irradiation-induced foci in various TP53 mutants. The R280K, G245S, L194F mutations, or TP53 deletion were also characterized by radiation-induced depletion in MDC1 protein. Moreover, in mutant cells, an interaction between MDC1 and 53BP1 proteins was abrogated when compared with wild-type counterpart. Together, the kinetics of 53BP1 accumulation at UV-induced DNA lesions is different in various TP53 mutant cells. After γ-irradiation, despite changes in a number and a volume of 53BP1-positive foci, levels of 53BP1 protein were relatively stable. Here, we showed a link between the status of MDC1 protein and TP53 gene, which specific mutations caused radiation-induced MDC1 down-regulation. This observation is significant, especially with regard to radiotherapy of tumors with abrogated function of TP53 gene.

• Keywords
• 53BP1 protein, DNA repair, Histone γH2AX, MDC1 protein, TP53 gene,
• MeSH
• Tumor Suppressor p53-Binding Protein 1 metabolism   MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Down-Regulation MeSH
• Nuclear Proteins deficiency   metabolism   MeSH
• Humans MeSH
• Mutation * MeSH
• Tumor Cells, Cultured MeSH
• Tumor Suppressor Protein p53 deficiency   genetics   metabolism   MeSH
• DNA Damage * MeSH
• Cell Cycle Proteins MeSH
• Trans-Activators deficiency   metabolism   MeSH
• Ultraviolet Rays * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Tumor Suppressor p53-Binding Protein 1 MeSH
• Adaptor Proteins, Signal Transducing MeSH
• Nuclear Proteins MeSH
• MDC1 protein, human MeSH Browser
• Tumor Suppressor Protein p53 MeSH
• Cell Cycle Proteins MeSH
• TP53 protein, human MeSH Browser
• TP53BP1 protein, human MeSH Browser
• Trans-Activators MeSH