BEYOND THE BREAK: Exploring mechanisms of DNA damage and repair

Our bodies consist of trillions of cells that continuously divide to sustain growth and tissue maintenance. This process requires accurate duplication (replication) and distribution (mitosis) of our DNA. However, errors during these processes can lead to genetic instability, threatening cell survival.

One of the most serious types of DNA damage is the double-strand break (DSB). These breaks can be caused by internal stress (e.g., replication errors) or external factors like radiation and chemotherapy. If not properly repaired, DSBs can result in chromosomal instability, cell death or cancer. Cells have developed repair mechanisms, primarily non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is quick but error-prone, while HR is more accurate but limited to specific cell cycle phases (S and G2). Other alternatives, such as microhomology-mediated end joining (MMEJ) also exist.

This thesis explores how human cells detect, signal and repair DSBs. A key question is whether the chromatin environment surrounding a break influences its toxicity. Using CRISPR/Cas9 to induce breaks in various genomic regions, the study finds that chromatin context has little effect on break toxicity. Instead, toxicity is determined by how efficiently CRISPR/Cas9 can induce breaks.

We identiy MND1 as an important protein for HR repair in the G2 phase. It works with HOP2 to support RAD51 in homology search. In MND1’s absence, other proteins like RAD51AP1 and RAD54L become essential, suggesting more complex HR pathways.

We introduce live-cell imaging techniques using fluorescently labelled MND1 to better understand HR. This visualizes HR filaments forming over time, with proteins like cohesin and RAD54L influencing their size and dynamics. We also present a conceptual framework for the homology search process, challenging existing models and emphasizing its complexity.

The genome’s stability is maintained by cell cycle checkpoints that halt cell division when DNA damage or chromosomal errors are detected. A key regulator is the tumor suppressor protein p53, which is often mutated in cancers. We explore what happens when p53 is absent. Cells then rely more on MMEJ, particularly during mitosis, to repair remaining DNA damage. This shift may help cancer cells survive despite high DNA damage levels.

Additionally, we investigate how oncogenic signaling, a hallmark of cancer, can be exploited therapeutically. Inhibition of the phosphatase PP2A, combined with WEE1 inhibition, triggers replication stress, premature mitosis, and cell death, especially in tumor cells. This combination effectively halts growth in patient-derived tumors.

In conclusion, this thesis reveals that DNA repair in human cells is more complex than previously thought, with multiple overlapping pathways. Understanding how these mechanisms function and are regulated offers important insights into cancer biology and potential new treatment strategies targeting DNA repair vulnerabilities.