Periodic Reporting for period 4 - G4DSB (G-quadruplex DNA Structures and Genome Stability) Periodo di rendicontazione: 2019-07-01 al 2021-08-31 Sintesi del contesto e degli obiettivi generali del progetto To this date, cancer is still one of the most common causes of death. In most cases cancer starts with a mutation or a deletion in a gene. This mutation/deletion leads to increased genome instability, enhanced cell growth and consequently to tumor formation. Obviously, many biological processes are altered in such cancer cells. To understand the causes and consequences of tumor formation the complete understanding of healthy cells or the "normal" biological state is essential.It has been shown that DNA can form in addition to a double helix alternative structures, which have a great impact on various biological processes. In the last years I have focused on understanding the biological impact of DNA secondary structures on genome stability. My lab focuses on a particular DNA structure called G-quadruplex (G4). These structures bear a specific sequence motif, which can be mapped genome-wide using computational methods. G4 motifs are found at very specific regions within the genome. Due to the specific locations, the evolutionary conservation and the strong stability of G4 structures they have been proposed to impact biological processes such as transcription, replication, recombination and telomere maintenance. The current idea is that those DNA structures serve as a regulatory tool in the cell to fine tune biological processes (positively and negatively) and by this change or alter the fate of the cell. It is still not clear when, where and why these structures form. If the hypothesis that G4 structures are fine tuning biological processes is correct it stands to reason that proteins are needed to form and unfold these structures in a timely manner. So far only a handful of such proteins were identified and characterized.Due to the stability of G4 structures many helicases have been identified to disrupt G4 structures in vitro and in vivo. In the absence of these helicases mutations, deletions and increased recombination events accumulate. Interestingly, most of the helicases that have been described to regulate G4 structures have been implicated in human health. The direct link to G4 unwinding however is not fully understood, yet. In genetic disorders in which helicases are deficient, mutations are observed around regions with a strong potential to form G4 structures. This data suggests that helicases do not act at G4 structures globally, but only at a certain subset. How this specificity is achieved is not known. Using bakers yeast (S. cerevisiae) as a model organism we aim to identify proteins that are important for G4 formation and unfolding. The characterization of these proteins is helping us to understand the question when and why these specific DNA structures form. We aim to learn how the cell uses G4 structures to up/down regulate transcription or to protect telomeres. Furthermore, by deleting or mutating these proteins, we want to investigate the question how changes in the G4 structure biology has an impact on other biological processes and genome stability. The overall aim of the proposal was to understand how G4 structures are regulated and how and these structures can lead to DNA damage (mutations and deletions). Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti We have identified over 60 novel G4-binding proteins in yeast. By combining biochemical, molecular and global approaches we further characterized the function of these proteins on G4 structures and genome stability. To this date we have characterized Slx9, Mms1, Mgs1, Zuo1 and DHX36. The obtained results are all published in international peer-review journals. Here, I summarize a few of these findings: Slx9 binds very robustly to G4 structures in vitro. In normal wildtype cells however it does not bind to G4 structures. We could reveal that Slx9 binds only in the presence of DNA damage and in absence of specific helicases to G4 structures where it supports DNA repair and genome stability. Further, we characterized Mgs1/binding to G4 structures in vitro and in vivo. Here it was interesting that it binds well to G4 structures in vitro and in vivo but that its function at G4s is not essential for genome stability. We came to the conclusion that the major function of Mgs1 at G4s is not during DNA replication or DNA repair. These two examples demonstrate that it is essential not only to identify proteins binding to G4s but that it is important to characterize their function and relevance at G4 in living cells.For Mms1, Zuo1 and DHX36 we could reveal the binding parameters as well as the function and relevance on G4 structures. We demonstrated that Mms1 binds throughout the cell cycle to G4 motifs on the lagging strand. Mms1-binding to G4 structures is required to support DNA replication and to prevent the accumulation of mutations and deletions at G4 sites. In detailed molecular analysis we identified that the major function of Mms1 at G4s is to support binding of the major G4-unwinding helicase Pif1. Without Mms1 less Pif1 binds to G4 structures, which leads to replication stalls, mutations, deletions and overall enhanced genome instability.In one of our recent publications we identified and characterized Zuo1 as a novel G4-binding and stabilizing protein. Zuo1 binds upon UV damage to G4 structures. Zuo1-binding to G4s is essential for cell to cope with lesions after UV irradiation. We could reveal that G4 structures themselves are key to recruit proteins of the nucleotide excision repair machinery, which is essential for an efficient and functional DNA repair after UV irradiation. This work was to our knowledge one of the first demonstrations that G4 structures formation positively supports genome stability events by modulating the binding and function of DNA repair proteins.In human cells we demonstrated that DHX36 binds to G4 RNA structures and by this modulates their mRNA abundance. This accumulation of G4 structures within a given mRNA renders it translational incompetent and leads to stress granule formation.At the start of this project G4-forming regions were mapped mainly computationally in yeast. We aimed to develop a novel method to map and quantify G4 structures in living cells. Different G4-specific antibodies are published to this end. In a first publication we validated those antibodies and could demonstrate that the 1H6 antibody binds in addition to G4s also poly(dT) regions and consequently is not valid for our research question. In recent years changes in G4 structure formation were shown to differ among cells and different stimuli (e.g. UV). We aimed to develop a method for the fast and accurate quantification of G4 levels in living cells. We created a protocol to quantify G4 levels by flow cytometry (BG-Flow). This methods allows scientists to monitor G4 levels in different cell types and even in mixed cell populations. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) We have contributed to an overall understanding of the functions and relevance of G4 binding protein in living cells. Our work provided significant novel findings on how and why G4 contribute to genome stability. Based on this finding we postulate that specific G4 structures form in a controlled and programmed manner with the help of proteins. These programmed G4s support biological processes and by this genome stability in living cells. However, upon specific stimuli (DNA damage) or in the absence of proteins (e.g. helicases) G4 formation is not programmed and can lead to mutations, deletions and/or transcriptional changes that all can drive genome instability. Role of G4 for Genome Integrity
