Uncovering the Molecular Mechanisms of Collisions Between the DNA Replication and Transcription Machinery (360G-Wellcome-324370_Z_25_Z)

DNA replication and transcription are essential for life, with defects in these processes a major cause of human disease. In eukaryotes, DNA replication is carried out by the replisome, while transcription is performed by RNA polymerases (RNAPs). During S-phase, both machineries operate simultaneously on the same genomic template, leading to transcription-replication conflicts (TRCs) - a significant source of genome instability. However, the molecular mechanisms underlying TRCs remain poorly understood. To develop a mechanistic understanding of TRC formation and outcomes, I will reconstitute these collisions in vitro using purified mammalian proteins and nucleic-acid templates. This system will allow precise control over the identity and concentration of TRC components, collision orientation and the activity of the replication and transcription machinery. Combining biochemical, structural, and single-molecule approaches, I will define the molecular outcomes of reconstituted TRCs - including replisome stalling, its kinetics, and potential resolution mechanisms. An integrated structural approach, combining cryo-EM and cross-linking mass spectrometry, will reveal the architecture of TRCs and sub-complexes formed during collisions. The effects of structure-guided mutations will be tested both in vitro and in human cells. Time-resolved cryo- EM and single-molecule methods will uncover how helicases and nucleases resolve TRCs, providing critical insight for improving therapies targeting replication stress.