Visualisering av DNA-Reparation - En Molekyl i Taget

Forskningsprojekt, 2021 – 2024

The DNA of our cells is constantly exposed to damage and DNA double strand breaks (DSBs) are among the most dangerous types. Homologous recombination (HR) is one of the two main repair pathways for DSBs. The first steps of this pathway involve keeping the two strands of the DSB together so that efficient and correct repair can occur. The MRN complex and its cofactor CtIP are heavily involved in this process, but the exact mechanisms are not fully understood.Single DNA molecule techniques have revolutionized the understanding of DNA-protein interactions, but there are few such studies on the first steps of HR. This is due to that DSB repair occurs on DNA ends and existing methods require that the DNA is anchored in its ends. We overcome this by confining the DNA in nanofluidic channels where long DNA molecules are stretched, while keeping the two DNA ends free. Our novel nanofluidic device also allows positioning of two ends in close proximity for repair to occur, allowing studies that have so far been impossible on the single DNA molecule level. We will focus on investigating how MRN and CtIP bind and bridge DNA ends and how they cooperate in structuring the ends for correct repair to occur. By including structural and disease-causing mutants of the different proteins we will reveal details that will aid in understanding these processes and why and how they malfunction in disease, including developmental disorders and cancer.