Podcasts about rad52

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.12.536536v1?rss=1 Authors: Di Biagi, L., Malacaria, E., Aiello, F. A., Valenzisi, P., Marozzi, G., Franchitto, A., Pichierri, P. Abstract: Replication gaps can arise as a consequence of perturbed DNA replication and their accumulation might undermine the stability of the genome. Loss of RAD52, a protein involved in the regulation of fork reversal, promotes accumulation of parental ssDNA gaps during replication perturbation. Here, we demonstrate that this is due to the engagement of Polalpha downstream of the extensive degradation of perturbed replication forks after their reversal, and is not dependent on PrimPol. Polalpha is hyper-recruited at parental ssDNA in the absence of RAD52, and this recruitment is dependent on fork reversal enzymes and RAD51. Of note, we report that the interaction between Polalpha and RAD51 is stimulated by RAD52 inhibition, and Polalpha-dependent gap accumulation requires nucleation of RAD51 suggesting that it occurs downstream strand invasion. Altogether, our data indicate that RAD51-Polalpha-dependent repriming is essential to promote fork restart and limit DNA damage accumulation when RAD52 function is disabled. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

In this podcast, I admittedly nerd out a bit on genetics and how our cells repair our genes when exposed to mutagens (things that cause mutations). However, with good reason, because a new paper has added some clarity to a part of the process and man is it cool! Beyond that, we will go into heart rate variability, as well as if caffeine is associated with migraines? Check it out and share! Rad DNA Repair, 1:03 Roxanne Oshidari, Richard Huang, Maryam Medghalchi, Elizabeth Y. W. Tse, Nasser Ashgriz, Hyun O. Lee, Haley Wyatt, Karim Mekhail. DNA repair by Rad52 liquid droplets. Nature Communications, 2020; 11 (1) DOI: 10.1038/s41467-020-14546-z Heart Rate Variability, 26:20 Giorgio Quer, Pishoy Gouda, Michael Galarnyk, Eric J. Topol, Steven R. Steinhubl. Inter- and intraindividual variability in daily resting heart rate and its associations with age, sex, sleep, BMI, and time of year: Retrospective, longitudinal cohort study of 92,457 adults. PLOS ONE, 2020; 15 (2): e0227709 DOI: 10.1371/journal.pone.0227709 Caffeine on Migraines, 31:28 https://www.sciencedaily.com/releases/2019/08/190808091422.htm YouTube: bit.ly/2JUjXVt Facebook: bit.ly/2PlIOaB Instagram: bit.ly/2OBFe7i Email List: bit.ly/2AXIzK6 Patreon: bit.ly/2OBBna0

The recruitment kinetics of double-strand break (DSB) signaling and repair proteins Mdc1, 53BP1 and Rad52 into radiation-induced foci was studied by live-cell fluorescence microscopy after ion microirradiation. To investigate the influence of damage density and complexity on recruitment kinetics, which cannot be done by UV laser irradiation used in former studies, we utilized 43 MeV carbon ions with high linear energy transfer per ion (LET = 370 keV/µm) to create a large fraction of clustered DSBs, thus forming complex DNA damage, and 20 MeV protons with low LET (LET = 2.6 keV/µm) to create mainly isolated DSBs. Kinetics for all three proteins was characterized by a time lag period T(0) after irradiation, during which no foci are formed. Subsequently, the proteins accumulate into foci with characteristic mean recruitment times τ(1). Mdc1 accumulates faster (T(0) = 17 ± 2 s, τ(1) = 98 ± 11 s) than 53BP1 (T(0) = 77 ± 7 s, τ(1) = 310 ± 60 s) after high LET irradiation. However, recruitment of Mdc1 slows down (T(0) = 73 ± 16 s, τ(1) = 1050 ± 270 s) after low LET irradiation. The recruitment kinetics of Rad52 is slower than that of Mdc1, but exhibits the same dependence on LET. In contrast, the mean recruitment time τ(1) of 53BP1 remains almost constant when varying LET. Comparison to literature data on Mdc1 recruitment after UV laser irradiation shows that this rather resembles recruitment after high than low LET ionizing radiation. So this work shows that damage quality has a large influence on repair processes and has to be considered when comparing different studies.

Nuclear architecture is a biological field of research that studies the spatio-temporal organization of the components within cell nuclei. Since nuclei are the organelles that harbor the genome and epigenome, they are the place where most of the genetic processes like replication, transcription, splicing, gene-regulation, DNA repair, re- combination etc. are carried out. In the presented doctoral thesis modern 4D live-cell microscopy in combination with laser or ion microbeam irradiation (to label or damage chromatin, respectively) was used to study nuclear architecture in living cells over extended periods of time at the single cell level. The results presented in this thesis can be partitioned into three main parts: (a) chromatin dynamics in cycling cells, (b) adaptation of the ion micro beam facil- ity SNAKE to the needs of live-cell observation (including first experiments) and (c) exploring spatio-temporal dynamics of DNA repair proteins after laser micro ir- radiation. (A) Chromatin dynamics in cycling cells Distribution of interphase chromosomes within cell nuclei has been found to be non- random with respect to gene density and chromosome size. Changes in nuclear orga- nization have been reported in several disorders and diseases. To which extent relative chromosome positioning is conserved through mitosis in cycling cells and whether certain chromatin domains are able change their relative position dramatically in the interphase nucleus has been the subject of various mechanistic models and contro- versial discussions. In 1909 German biologist theodor Boveri was the first one to comment on this topic in his publication: “Die Blastomerenkerne von Ascaris mega- locephala und die Theorie der Chromosomenindividualität” (included as an appendix to this thesis). In order to test Boveri’s hypotheses, 4D live-cell observations were carried out on a modern spinning disc confocal microscope using a human cell line that possesses photoactivatable chromatin. In experiments that used photoactivation and photobleaching of chromatin, it could be demonstrated that – as stated by Boveri – chromatin proximity relationships are in general not conserved through mitosis but destroyed during early prometaphase by the mechanics of mitosis. Other experiments showed that nuclear rotations in a conveyer-belt-like manner are able to bring initially distant chromatin domains into close proximity in a matter of a few minutes. (B) Adaptation of the SNAKE micro beam facility to the needs of live- cell microscopy (including first experiments) Since ordinary irradiation sources lack the ability to perform targeted micro irradia- tion at the micrometer scale and laser micro irradiation produces an artificial mix of various DNA damages, the ion microbeam SNAKE represents an interesting tool to explore the dynamics of repair proteins in a spatio-temporal context. In the course of a collaboration project the ion microbeam was adapted to the needs of long-term live-cell microscopy. These adaptations and first live-cell experiments performed at the refurbished ion micro beam are described in this part of the results. (C) Exploring spatio-temporal dynamics of DNA repair proteins after laser micro irradiation. Mutation of genetic information can cause serious harm to a cell or even a whole or- ganism. DNA repair serves to protect and clean the genome from undirected poten- tially hazardous changes. Compared to the wealth of information which is available about DNA repair at the molecular level only little attention has been payed to it in context of nuclear architecture. In the last part of the results cells stably expressing GFP tagged versions of the repair proteins MDC1, Rad52 and 53BP1 were damaged by laser micro irradiation and imaged over extended periods of time. It could be de- monstrated that at the used damage induction conditions most of the cells show only minor changes with respect to localization of damage signals, kinetochores and nu- cleoli pattern over time. Furthermore, disappearance of spontaneous 53BP1-GFP foci in favor of protein recruitment to damaged chromatin and mutual exclusion between kinetochore signals and Rad52-GFP damage foci could be observed. In a few U2OS Rad52-GFP nuclei DNA damage foci disappeared simultaneously after a dramatic phase in which the total number of foci drastically increased – even adjacent to the laser damaged chromatin.

Gene expression is highly regulated and interconnected to processes like mRNP processing, mRNA export as well as to DNA repair and replication. The first step of gene expression is the transcription of protein coding genes by RNA polymerase II. Transcription is controlled by general transcription factors, the phosphorylation of the C-terminal domain of Rpb1, the largest subunit of RNA polymerase II, and chromatin modifications that allow proper accessibility of the DNA. A major player in these coupling processes is the TREX complex, coupling transcription elongation to the nucleo-cytoplasmic export of the mRNP via the nuclear pore complex. Particularly, the THO subcomplex of TREX has functions in hyperrecombination, nucleotide excision repair and transcription coupled repair. A genetic screen with TREX components, performed to identify genes involved in these processes, lead to the identification of the cyclin dependent kinase Bur1. Bur1 and its cyclin Bur2 are needed for efficient transcription elongation by RNA polymerase II by regulating the methylation of histone tails. Interestingly, Bur1 interacts in vivo with RPA, a single strand DNA binding protein essential for genome stability. This biochemical interaction raised the idea of a novel interconnection between transcription, chromatin modification and genome maintenance. Mutations in the BUR1 as well as in the RFA1 gene lead to sensitivity to drugs that cause DNA damage and replication or transcription stress. Deletion of the C-terminus of Bur1, which is sufficient for the binding to RPA, also renders cells sensitive to those agents. This shows the functional significance of this protein-protein interaction in the cell upon stress induction. However, attempts to identify the DNA repair pathway Bur1 is involved in showed that mutations in BUR1 do not behave epistatic with deletions of specific pathways. This result points to a more general, maybe regulatory role of Bur1 in the response to DNA damage. It is interesting to note that mutations in BUR1 lead to increased genomic instability as they show the appearance of a higher amount and longer persistence of nuclear foci, DNA repair “factories” that contain, among other proteins, Rfa1 and Rad52. Furthermore, RFA1 mutants show decreased levels of histone H3 alone as well as lower levels of histone H3 Lysine 4 trimethylation, a mark for transcription elongation, when combined with a mutation in BUR1. The RFA1 mutant is also impaired in the expression of a β-galactosidase reporter gene, pointing to a function of RPA in transcription. Interestingly, combining BUR1 and RFA1 mutants leads to a lower susceptibility of cells to stress than one of the mutations alone. On the one hand, this could be elucidated by better growth of the double mutant strains upon stress compared to the single mutants. On the other hand, whole genome expression analysis shows that the double mutant strain clusters with the bur1 mutant whereas the rfa1 mutant does not, showing that its expression pattern is closer to the bur1 mutant. Both results show that the protein complexes have antagonistic roles as the combination of both mutations leads to a suppression phenotype based on differential gene expression. Taken together, a function of Bur1 in genome maintenance could be established, as well as an effect of RPA on transcription elongation and chromatin modification. The results provide a possibility to speculate about a coupling of transcription and genome stability mediated by the interaction of Bur1-2 with RPA.

Rad5 is a decisive protein in S. cerevisiae due to its role in the Post-replication repair (PRR) pathway, in which Rad5 is necessary for at least one error-free and one error-prone repair subpathway. In addition, Rad5 plays a role in other repair pathways; for instance, Rad5 regulates the balance between the double strand break (DSB) repair pathways, favoring the Rad52-dependent Homologous Recombination (HR) over the yKu70-dependent Non-Homologous-End Joining (NHEJ). Furthermore, since UV-induced damages are substrates for Rad5 but also for Base Excision Repair (BER) proteins, Rad5 is possibly involved directly or indirectly in the BER pathway. To get a deeper insight into the interaction of Rad5 with HR, NHEJ and BER proteins, survival curves, plasmid assays, and mutagenicity experiments were carried out in this work. In addition, a new software tool has been developed for the quantification of DSB. This software, called Geltool, allows the quantification of DSB in haploid cells from PFGE gels, even if the number of DSB is small. This represents a decisive advantage in comparison with previous programs. The sensitivity of Geltool has permitted the quantification of DSB repair during the stationary growth phase in haploid cells, detecting a repair of 46 %- 57 % of the gamma-induced DSB in HR proficient strains against 6 % - 16 % in HR deficient strains. Studies of the functional interactions of Rad5 with HR and NHEJ proteins revealed a synergistic effect between Rad5 and Rad52 proteins for the repair of DSB at chromosomal and plasmidial level. Differences in the repair of plasmids from the rad52 and the rad5 mutants revealed different end joining mechanisms for gap repair. Severe degradations found in plasmids from rad52 and rad52rad5 mutants could indicate an end protection function for Rad52 and also for Rad5, when Rad52 is absent. Moreover, the regulatory role of the Rad5 protein is confirmed, since the additional deletion of YKU70 suppresses the rad5 phenotype and forces the yku70rad5 mutant to repair by HR. The further study of the interplay of Rad5 with BER proteins shows that while BER only plays a minor role for the repair of gamma-induced damage, the rad5 phenotype is suppressed in the BER deficient apn1ntg1ntg2rad5 mutant. The same phenotype of suppression is also found for survival after UV irradiation. An enhanced mutagenicity of the apn1ntg1ntg2rad5 mutant indicates a possible repair through the REV3-dependent Translesion Synthesis Repair (TLS) pathway, suggesting that an error-prone tolerance of UV-induced damage can be very effective for survival.

Der Erhalt der genomischen Integrität ist für das Überleben von Organismen notwendig, jedoch können verschiedene DNA-Läsionen das genetische Material gefährden. DNA-Doppelstrangbrüche (DSB) stellen dabei eine besonders toxische DNA-Läsion dar, und schon ein einzelner DSB kann bei ausbleibender oder fehlerhafter Reparatur zum Absterben der Zelle führen. In höheren Eukaryonten gibt es zwei Mechanismen für die Reparatur eines DSB: nicht-homologe Endverknüpfung und homologe Rekombination. Bei der homologen Rekombination spielt der Rekombinationsfaktor Rad52 eine zentrale Rolle und wurde zu Beginn dieser Arbeit als ein Substrat für eine posttranslationale Modifikation mit SUMO identifiziert. Daraufhin wurde die Regulation von Rad52 durch die Modifikation mit SUMO untersucht. So konnte im Rahmen dieser Arbeit gezeigt werden, dass die SUMOylierung von Rad52 in Saccharomyces cerevisiae hauptsächlich an zwei nicht konservierten Lysinresten außerhalb der hoch konservierten Rad52-Domäne erfolgt und eng an Rekombinations- und DNA-Reparaturereignisse gekoppelt ist. So wird die Rad52-SUMOylierung durch enzymatische DSB während der Meiose und durch chemisch induzierte DSB in mitotischen Zellen ausgelöst. Hierfür ist der MRX-Komplex (bestehend aus Mre11, Rad50 und Xrs2) notwendig, der vor Rad52 im Rekombinationsprozess aktiv ist. Des Weiteren zeigt die vorliegende Arbeit, dass Zellen mit einer Rad52-Mutante, die nicht mehr mit SUMO modifiziert werden kann, keine auffälligen Wachstumsdefekte aufweisen, beispielsweise weder in Gegenwart DNA-schädigender Agenzien noch in der Meiose. Allerdings hat die SUMOylierung einen pro-rekombinatorischen Einfluss auf Rad52. Denn zum einen können Zellen, in denen zwei der Helikasen Rrm3, Sgs1 oder Srs2 deletiert sind, in Gegenwart von SUMOylierungsdefizientem Rad52 wachsen, da vermutlich keine toxischen Rekombinationsintermediate mehr entstehen wie in Gegenwart von Wildtyp Rad52. Zum anderen weisen Zellen mit SUMOylierungsdefizientem Rad52 Defekte bei speziellen Rekombinationsreaktionen auf. Die SUMOylierung schützt Rad52 zudem vor dem Abbau durch das Proteasom und ist besonders für die Rad52-Moleküle relevant, die am Rekombinationsgeschehen beteiligt sind. Diese Arbeit zeigt somit, dass die SUMOylierung von Rad52 die Aktivität des Rekombinationsfaktors dadurch reguliert, dass die im Rekombinationsprozess involvierten Rad52-Moleküle vor einem vorzeitigen Abbau geschützt werden.