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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - G4DSB (G-quadruplex DNA Structures and Genome Stability)

Teaser

Summary

\"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. Of course, 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.
In the last years 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 we 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 in order 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 a regulatory tool that fine tunes biological processes is correct proteins are needed that 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 which have been described to regulate G4 structures have been implicated in human health, but the direct link to G4 unwinding 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. These 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 as a biological tool to up/down regulate transcription or to protect telomeres. Furthermore, by deleting or mutating these proteins, we want to shed light on the question how changes in the G4 structure formation biology has an impact on biological processes and genome stability. The overall aim of the proposal is to understand how G4 structures are regulated and how and these structures can lead to DNA damage (mutations and deletions).
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Work performed

As part of this research proposal we have identified Mms1 as a novel G4 interacting protein in yeast. In depth molecular, biochemical and genetic analyses showed that Mms1 binds specifically to G4 motifs on the lagging strand throughout the cell cycle. In the absence of Mms1 DNA replication stalls, which results into mutations and deletions at G4 motifs. A similar effect was observed if the helicase Pif1 is mutated. Pif1 is a DNA helicase known to unwind G4 structures efficiently in vitro. Using different in vivo techniques in yeast, we revealed that Mms1 supports Pif1 binding to G4 structures on the lagging strand. Overall, our data revealed that Mms1 is an important new factor that supports Pif1 function at G4 structures. Without Pif1 or Mms1 G4 structures cannot be resolved in time during DNA replication, which leads to stalls of the replication machinery and consequently accumulation of mutations/deletions.
To this date, G4-forming regions were mapped only computationally in yeast. To better understand where in the genome G4 structures form in vivo, we aimed to map G4 structures using a specific antibodies. Different G4-specific antibodies are published and we validated those antibodies using different techniques. In a subsequent publication, we revealed that the antibody 1H6 binds in addition to G4s poly(dT) regions and consequently is not valid for our research question.

Final results

We expect to identify and characterize additional novel G4 binding proteins until the end of the funding period in vitro and in vivo. We will link the function of these proteins - and the state of G4 structures - to different genome stability events. A genome-wide map of G4 structures in yeast, using different alternative methods proposed in aim2, will furthermore enhance our understanding of G4 function, regulation and impact on genome stability.