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

Mitokondriális eredetű nitrogén szabadgyökök és ATP-függő K-csatornák szerepe az organellum működésében = The involvement of mitochondrial-derived nitrogen radicals and mitoK-ATP channels in the regulation of organelle function

Elsőként a mitoKATP csatornák alegységeit terveztük azonosítani humán szív mintákon. A kísérletek során megbizonyosodtunk afelől, hogy a korábban ilyen csatornáknak gondolt fehérjék nincsenek jelen megfelelő számban és formában a mitokondriális membránokban, ezért a nem valószínű hogy a mitokondirális ATP-függő kálium áramokat valós mitoKATP csatornák folytatnák. A mitokondriális NO szintáz (mtNOS) enzimrendszer vizsgálata azonban nem várt új felfedezésekhez vezetett. Az előkísérletekben is bemutatott, a pályázat során több fajra és szövetre (ember, egér, patkány, malac, szív, agy és máj) kiterjesztett, több párhuzamos módszerrel nyert eredményeink szerint bizonítottuk, hogy a feltételezett mtNOS nem azonos egyik ismert NOS variánssal sem. Megállapítottuk hogy a mitokondriális légzési lánc ubiquinon ciklusának működése mentén keletkező nitrogén gyökök azok, amelyeket korábban a mtNOS-nak tulajdonítottak. A reakcióút pontosabb tisztázása végett mitokondriumokat oxidatív stressznek vetettünk alá, ezzel fehérje modifikációt indukáltunk. Proteomikai módszerekkel (2D gélelektroforzis és tömegspektrometria) azonosítottuk nitrált és poly-ADP-rbozilált (PAR-ált) fehérjéket, köztük a mitokondirális dihidro-lipoamid dehidrogenázt amely a poliADP-riboziláció folyamatot is katalizálhatja. Kísérleteink ezzel egy teljesen új, független mitokondriális reakcióutat tártak fel. További kísérleteink a mitokondriális nitrogén monoxid metabolizmus és a cukorbetegség kapcsolatát vizsgálták. | Our experimental findings indicated that there previously suspected potential mitoKATP channel subunits are not present in the required format and amount in human heart mitochondria, therefore, they cannot play a role in K fluxes. We extended our preliminary results about the nature of the putative mitochondrial NO synthase (mtNOS) to several species and tissues (human, mouse, rat, pig, heart, brain and liver) and concluded that mtNOS is not a variant of any known NOS enzymes. Mitochondria are capable of producing significant amounts of nitrogen radicals through the catalyzation of nitrosothiol release by the ubiquinon cycle of the respiratory chain. Subjecting mitochondria to oxidative stress resulted in the nitration and poly-ADPribosylation (PARylation) of proteins. Proteomic identification of the affected molecules revealed that dihydrolipoamide-dehydrogenase is PARylated and may act as a polyADP-ribose-polymerase (PARP). Thus, we uncovered a completely new and independent mechanism of mitochondrial nitrosative stress, which is catalyzed by enzymes of the respiratory chain. Then, we concentrated our efforts on the potential role of mitochondrial oxidants in pathological reactions such as diabetes

Use of a recombinant pseudorabies virus to analyze motor cortical reorganization after unilateral facial denervation

A unilateral facial nerve injury (n7x) was found to influence the transcallosal spread of the attenuated strain of pseudorabies virus (PRV Bartha) from the affected (left) primary motor cortex (MI) to the contralateral MI of rats. We used Ba-DupLac, a recombinant PRV strain, for the tracing experiments since this virus was demonstrated to exhibit much more restricted transportation kinetics than that of PRV Bartha, and is therefore more suitable for studies of neuronal plasticity. Ba-Duplac injection primarily infected several neurons around the penetration channel, but hardly any transcallosally infected neurons were observed in the contraleral MI. In contrast, after right facial nerve injury, Ba-DupLac was transported from the primarily infected neurons in the left MI to the contralateral side, and resulted in the labeling of several neurons due to a transneuronal infection. These results reveal that a peripheral nerve injury induces changes in the Ba-DupLac infection pattern in the related cortical areas. These findings and the literature data suggest that this phenomenon may be related to the changes in the expression or to the redistribution of cell-adhesion molecules, which are known to facilitate the entrance and/or transmission of PRV into neurons