Dynamics of DNA repair proteins at nucleosomes – DYPROSOME

The partners at IAB Grenoble and LBMC Lyon, leading specialists in their respective fields, assemble laboratory-reconstructed nucleosome samples with defects inserted into the DNA sequence, mimicking the results of damage typically inflicted on the cell by chemical pathogens, oxidation and cellular aging processes, or interaction with ionizing radiation. These "damaged" nucleosomes are dissolved with BER enzymes descriptors for defect recognition to study their structure in interaction with the damaged DNA. The details of the molecular structure are revealed by cryo-atomic microscopy.

In parallel, the partners at IEMN and UGSF, both based in Lille, perform computer simulations using molecular docking and molecular dynamics methods to reconstruct the structural and dynamic details at the atomic scale of enzyme/DNA interactions. These theoretical methods are capable of describing and tracking molecular movements and interactions with a degree of accuracy superior to experiments, and thus represent a key ingredient for deciphering experimental data.

The DYPROSOME project has led to important advances, particularly in theoretical modelling. We have shown that DNA, when slightly deformed in some compact structures of the nucleus, can temporarily expose normally hidden bases. This openness makes it possible for repair enzymes, such as UDG, to act and then detect and correct abnormalities. These results, confirmed by several successive studies, show that the very structure of DNA, in particular its curvature and its organization around histone proteins, plays an active role in repair mechanisms. This work continues today, in particular to better understand the influence of electrostatic interactions in the context of chromatin, which is the key structure of DNA in the cell nucleus.

On an experimental level, the project encountered significant challenges but also provided promising results. We succeeded in stabilizing a complex between a repair enzyme (OGG1) and damaged DNA within a nucleosome, a key step towards a detailed understanding of these mechanisms. However, the technically demanding production and analysis of samples slowed progress, and some work was halted due to drastic changes in the team. Despite this, the methods developed have laid a solid foundation, which is now being exploited in new collaborations, paving the way for future discoveries on DNA repair.

Although the DYPROSOME project could not be fully completed on the experimental side, it provided important insights into how DNA repair enzymes operate under realistic cellular conditions. Rather than studying DNA in simplified settings, we placed it in its natural environment, where it is tightly packed and organized. We showed that physical forces can slightly deform DNA, briefly exposing hidden defects and making them accessible to repair enzymes. This means that the shape and organization of DNA around proteins (nucleosomes) play an active role in the repair process. We also identified a molecular “anchoring” mechanism that helps the enzyme UDG position itself correctly on DNA.

Experimentally, not all initial goals could be achieved, but the simulations we produced offer solid directions for future validation. They point toward new experimental approaches, such as observing forces acting on DNA at the single-molecule level or tracking, in real time, how repair enzymes locate and interact with DNA. These techniques, once highly challenging, are now becoming increasingly feasible.

The project also highlights the importance of studying more flexible forms of DNA, which appear to be repaired more easily than the rigid structures typically used in research. This could lead to a more accurate understanding of how DNA repair works inside living cells.

Overall, this work shows that DNA repair is not only a matter of chemistry, but also of physical forces that are still largely unexplored. It opens new perspectives for understanding and eventually better controlling these essential processes of life.

We propose a combined computational and experimental study of the molecular mechanisms by which repair proteins identify the earliest stages of DNA damage in chromatin. DNA damage repair processes (DDR) are tightly regulated among several pathways operating for different types of damage. However, the very early stages of DDR are still little explored on a molecular level, since this requires advanced computational and experimental techniques that are only now developing. How do proteins can initially identify damaged DNA regions? Which molecular interactions direct repair to one pathway with respect to another, for a given type of damage? To what extent does the variable degree of compaction of nucleosomes affect the access of repair proteins to damaged DNA? We will focus on DNA lesions localized at nucleosomes, the key compaction units in chromatin structure, and study their interaction with a carefully chosen set of proteins operating at the very early stages of the repair chain.