The EMBO | EMBL Symposium ‘DNA replication: from basic biology to disease‘ took place last month at EMBL Heidelberg and virtually.

In this edition, the symposium highlighted how cutting-edge research can help to answer pivotal questions about DNA replication at multiple levels. From the biochemical intricacies of the replication machinery to the three-dimensional structures of the proteins involved, the field is making strides toward a comprehensive understanding.

With 175 on-site participants, 64 virtual participants, and 13 fellowships provided by the EMBL Corporate Partnership Programme and EMBO, we had the opportunity to gather researchers, PhD students, and experts in the field form different parts of the world. With a total of 111 posters available on Helix A and B in the Advanced Training Center, we held two poster sessions during which the presenters could discuss their research — their work was then voted by the participants. There were three poster prizes awarded during the event.

Please give a well-deserved round of applause to these brilliant minds for their contributions and interesting discussions.

Replication stress in cortical organoids reveals potential common fragile sites involved in uronal disorders

Presenter: Aurélie Masson

Authors: Aurélie Masson, Théo Baret, Kamal Bouhali, Mikaelle Bocel, Stéphane Koundrioukoff, Michelle Debatisse, Marie-Noëlle Prioleau

Abstract:

During human brain development, neural precursors divide thousands of times to produce about 80 billion neurons. The rapid divisions can cause DNA breaks and genomic damage that can lead to the initiation of brain cancer or its abnormal development. Experiments in mice have shown that neuronal precursors are indeed the site of high genetic instability, with recurrent DNA break sites (RDCs)1. They correspond to long genes that are specifically expressed during neurogenesis and replicate at the end of S phase, three general features also observed in common fragile sites (CFSs). We purpose to determine whether the previously mapped recurrent DNA break sites correspond to regions that, following replicative stress such as a slowing down of replication fork progression, are likely to fail to complete their duplication in time before entry into mitosis2. We investigated this by differentiating human induced pluripotent stem cells (hiPSCs) into cortical organoids. Neural precursors were exposed to a low dose of aphidicolin (APH), which slows down replication forks, at an early stage of active division. We determined 181 long, transcribed and late replicated genes that are under-replicated under replicative stress conditions and defined them as potential CFSs (pCFSs). 26.5% of these are true CFSs, because they have been identified as broken in previous studies. These pCFSs are involved in neuronal disorders, such as autism, schizophrenia and bipolarity. Finally, we observed that genes up-regulated under replicative stress are involved in innate immune response pathways.

View poster.

Delayed lagging strand synthesis drives asymmetric histone incorporation and promotes progenitor cell reprogramming in the Drosophila male germline

Presenter: Brendon Davis

Authors: Brendon Davis, Xin Chen, Jonathan Snedeker, Rajesh Ranjan, Matthew Wooten

Abstract:

In the Drosophila male germline lineage, stem cells display asymmetric histone inheritance while non-stem progenitor cells exhibit symmetric patterns. We report that an essential molecular mechanism underlying this cellular specificity is delayed lagging strand synthesis, which drives old histone incorporation into the leading strand and is sufficient to generate asymmetric histone incorporation in non-stem cells. A candidate screen identified that proteins involved in lagging-strand synthesis, such as DNA Polymerase α (Polα) and DNA Polymerase δ (Polδ), are expressed at reduced levels in stem cells compared to non-stem cells in the same germline lineage. Genetically compromising Polα induces the replication-coupled histone incorporation pattern in non-stem cells to be indistinguishable from that in stem cells, and this is recapitulated using a Polα inhibitor in a concentration-dependent manner. Furthermore, stem cell-derived chromatin fibers display a spatiotemporal difference in the replication of both DNA strands and show a significantly higher degree of old histone recycling to the leading strand than in progenitor cell-derived fibers. However, upon reducing Polα levels in non-stem cells, the progenitor cell chromatin fibers now display asymmetric old histone recycling just like stem cell-derived fibers. Importantly, these altered chromatin features in progenitor cells allow them to act like stem cells under both pathological and physiological conditions in which bona fide stem cells are lost. We have found no evidence of increased DNA damage in progenitor cells following the compromise of lagging strand synthesis, likely in part due to increased levels of Replication Protein A (RPA) in the germline. Together, these results indicate that developmentally programmed expression of key DNA replication components underlies asymmetric histone incorporation and could lead to differential cell fates following mitosis. Manipulating even a single DNA replication component can induce replication-coupled histone dynamics in non-stem cells that resemble the dynamics observed in stem cells.

View poster.

CFAP20 salvages arrested RNAPII from the path of co-directional replisomes

Presenter: Sidrit Uruci

Authors: Sidrit Uruci , Daphne E.C. Boer , Paul W. Chrystal , Ibrahim Ilic , Andreas Panagopoulos , George Yakoub , Annelotte P. Wondergem , Klaas de Lint1, Tiemen J. Wendel , Sem J. Brussee , Nila K. van Overbeek , Román González-Prieto , Jolanthe Lingeman , Mats Ljungman , Haico van Attikum , Alfred C.O. Vertegaal , Sylvie Noordermeer, Rob Wolthuis1, Vincent Tropepe, Matthias Altmeyer , Tugce Aktas , Diana van den Heuvel , Martijn Luijsterburg

Abstract:

Fine-tuning DNA replication and transcription is vital to avoid collisions between their machineries. These conflicts can occur in a head-on or co-directional orientation, and while the former are considered more damaging to the genome integrity, the latter are more common as highly transcribed genes are evolutionarily oriented in a co-directional manner with respect to replication directionality. Mechanisms to avoid conflicts are especially relevant near promoters, where RNA polymerase II (RNAPII) initiates transcription and often pauses or arrests, leading to the formation and accumulation of R-loops. Arrested RNAPII engaged with an R-loop poses as a roadblock for DNA replication, which evolutionarily initiates near promoters, thus making co-directional conflicts likely to occur. While considerable focus has been devoted in understanding pathways preventing and/or resolving these conflicts, the specific mechanism that salvages the arrested RNAPII during elongation to avoid conflicts with the incoming co-directional replisomes is unknown. Through genomic and proteomics approaches, we identified and characterized CFAP20, a small, highly conserved and understudied protein previously known only as a ciliary protein. We found that CFAP20, in addition to its localization at the primary cilium, also localizes to the cell nucleus. Here, CFAP20 is a part of a protective pathway that rescues arrested RNAPII in promoter-proximal regions, diverting it from the path of co-directional replisomes. CFAP20-deficient cells accumulate R-loops specifically near promoters, displaying defects in origin firing and replication fork progression. Separation-of-function mutants in cells and zebrafish larvae demonstrate that the nuclear function of CFAP20 is distinct from its ciliary role. Using CRISPR genome-wide screening, we identified the Mediator transcription complex as the source of the phenotypes. Co-depletion of Mediator coactivator complex or removal of RNAPII engaged with R-loops rescues replication phenotypes observed in CFAP20-deficient cells. Therefore, we propose that CFAP20 salvages arrested RNAPII under conditions of high Mediator-driven transcription to fine-tune RNAPII elongation and prevent collisions with co-directional replisomes.

Due to the confidentiality of the unpublished data, we cannot share the poster.

EMBO | EMBL Symposium ‘DNA replication: from basic biology to disease’ took place from 5 – 8 November 2024 at EMBL Heidelberg and virtually.