Mechanistic insights into the transcriptional arrest in the presence of Double Strand Breaks

Double-strand breaks (DSBs) occur frequently in the genome during genome replication or by DNA damaging agents. DNA lesions affect fundamental DNA-dependent nuclear processes such as replication and transcription. We have developed an experimental system where DSBs are induced at coding regions of RNA polymerase II transcribing genes. We have started to study the kinetics of RNA polymerase II transcription inhibition in the presence of DNA breaks. We observed that induction of the break led to transcription inhibition and the restoration of transcription closely followed the dynamics of the repair of breaks. We confirmed by chromatin-immunoprecipitation that the break induction led to displacement of RNA polymerase II affecting both the elongation and the initiation of transcription. Our results show that this is dependent on one of the major kinase in DNA damage repair called DNAPKcs. We also investigated the downstream steps of RNA polymerase II removal and we claimed that it was a multistep process involving additional kinases and ubiquitin ligases NEDD4 and CUL3. At the last step of break dependent transcriptional silencing the RNA polymerase II is targeted for proteasome dependent degradation. These data demonstrate that the DNA damage repair complexes and proteasomal system have a synergistic and active role in transcriptional silencing during the DSB repair by removing the RNA pol II from the transcribing region. We show here that DNA lesions occurring at transcribed regions cause a transient repression until the lesion is repaired. This is probably a cell defense mechanism to avoid production of truncated or mutated transcripts in essential genes whose alterations in their gene expression would endanger cell viability. Understudying the role of DNAPKcs, in preventing RNA pol II bypassing a DSB might be a key in avoiding the production of mutated transcripts that could lead to cancerous phenotypes

Cloning of an engenieered histone cluster in Drosophila melanogaster

Unrepaired DNA damages could lead to cancer formation. To allow repair of these damages, the repair proteins must assess the damage site, so the condensed DNA needs to be looser. Our group is interested in to understand chromatin changes during DNA repair processes by using novel experimental system. In eukaryotic cells the nucleus contains highly condensed DNA. The base of the chromatin structure is the evolutionarily conserved histone protein family. The histone proteins necessary for nucleosome assembly by forming a heterooctamer with two copies of H2A, H2B, H3 and H4. Finally the linker histone H1 requires for the proper chromatin condensation. All of the histone proteins can be post-translationally modified. These PTMs are the main rulers of epigenetic regulation. Processes using DNA as a template (transcription, replication and DNA repair) are greatly affected by the chromatin structure. Unimproved breaks lead genome instability or translocations which easily results tumor formation. Our aim is to understand how does the chromatin structure change around the break during the DNA repair and what is the link between unique histone PTMs and the mechanisms of the repair. We plan to set up an experimental system by which we will be able to study how do unique histone modifications affect the DSB repair. The advantage of this new experimental system is the clone of the histone cluster which contains all of the canonic (H2A, H2B, H3, H4) and linker (H1) histone genes in Drosophila melanogaster

Developing new plasmid for studying histone PTM during DNA repair

Cells are threatened by different damaging effects, which cause many errors, so the cell has to defend itself. Repair pathways are responsible for defence and they also keep the cells in healthy state. In order to study the DNA repair mechanism, we developed new plasmid based method, which could help us. Our cells are constantly attacked by diversity of DNA damaging agents, which are able to induce different structural changes in the DNA. For protecting genome integrity, the cell must eliminate the damage and restore the original sequence of DNA. Most of the cases, accumulation of mutations lead to the formation of cancer. During the DNA repair numerous repair factors are responsible for the integrity of the genetic information. On the other side for DNA repair many histone post-translational modification are also requires. The histone PTMs are indispensable, because they ensure the accessibility of DNA and help the communication between DNA repair factors and enzymes. The center of our research is double strand DNA breaks and their repair pathways: Non-homologous End Joining and Homologous Recombination Repair. For the better overview the histone PTMs during the repair we have developed histone cloning vectors (donor and acceptor vectors). With these vectors we could create transgenic animals, and using of an inducible system we can examine the orchestrated protein recruitment at the repair foci around the DNA breaks

Emerging Roles of Post-Translational Modifications in Nucleotide Excision Repair

Nucleotide excision repair (NER) is a versatile DNA repair pathway which can be activated in response to a broad spectrum of UV-induced DNA damage, such as bulky adducts, including cyclobutane-pyrimidine dimers (CPDs) and 6–4 photoproducts (6–4PPs). Based on the genomic position of the lesion, two sub-pathways can be defined: (I) global genomic NER (GG-NER), involved in the ablation of damage throughout the whole genome regardless of the transcription activity of the damaged DNA locus, and (II) transcription-coupled NER (TC-NER), activated at DNA regions where RNAPII-mediated transcription takes place. These processes are tightly regulated by coordinated mechanisms, including post-translational modifications (PTMs). The fine-tuning modulation of the balance between the proteins, responsible for PTMs, is essential to maintain genome integrity and to prevent tumorigenesis. In this review, apart from the other substantial PTMs (SUMOylation, PARylation) related to NER, we principally focus on reversible ubiquitylation, which involves E3 ubiquitin ligase and deubiquitylase (DUB) enzymes responsible for the spatiotemporally precise regulation of NER

Beyond initiation: Human P53 plays role in transcription elongation

The P53 tumor suppressor regulates the transcrip-tion initiation of selected genes by binding to specif-ic DNA sequences at their promoters. Here we report a novel role of P53 in transcription elongation in human cells. Upon transcription elongation block-age P53 is associated with genes, which have not been reported earlier as its direct targets. P53 could be co-immunoprecipitated with active forms of RPB1, highlighting its association with the elongat-ing RNAPII. During normal transcription cycle, P53 and RPB1 localized at distinct regions of selected non-canonical P53-target genes and this pattern of localization was changed upon transcription elonga-tion block. Additionally, transcription elongation block induced the ubiquitylation and the proteo-somal degradation of RPB1. Finally, we showed that the transcription block induced RPB1 degradation is mediated by P53. Our results reveal a novel role of P53 in human cells during transcription elongation blockage that might serve to facilitate the removal of RNAPII from DNA