GAP-REPAIR

The goal of this project is to understand how the balance between error-free and mutagenic lesion bypass is regulated in living cells, using Escherichia coli as a model. • Background The genome of all organisms is constantly threatened by numerous agents that cause DNA damage. When an unrepaired lesion hinders the progression of replicative DNA polymerases, repriming downstream generates a single-stranded DNA gap (ssGap). Two strategies are available to bypass the lesion and fill the gap: potentially mutagenic translesion synthesis (TLS) and error-free damage avoidance (DA). However, it is not known today how the balance between these two mechanisms is regulated. In addition, no techniques were available to focus on ssGaps processing. • Novel Approach To overcome these limitations, our group has developed an innovative technique to insert a single lesion into the genome of a living cell, allowing us to generate a ssGap and study its processing and filling. • Research Objectives The main goal of this project is to understand how lesion bypass is regulated in living cells. We will address four key questions: 1. Which factors are responsible for processing ssGaps? 2. How are these factors recruited at ssGaps and which are their interactors/partners? 3. How do these factors affect ssGaps structure, length and resolution, and modulate the kinetics of gap generation, extension and resection? 4. How does ssGap processing modulate the balance between error-free and mutagenic bypass? • Methodology Using our single-lesion approach in Escherichia coli, we will: -Identify proteins recruited to the lesion site -Characterize recruitment modes and identify interactors using computational approaches and single-molecule imaging -Assess the role of these factors in ssGap processing using molecular approaches (Electron Microscopy, NGS, qPCR). -Evaluate their functions in lesion bypass by monitoring TLS and DA levels -Correlate gap size and dynamics with error-free vs. mutagenic bypass outcomes • Potential Impact This research will provide crucial insights into DNA damage response mechanisms, potentially leading to improved understanding of mutagenesis, cancer development, and novel therapeutic strategies for DNA repair-related diseases, while also shedding light on antibiotic resistance mechanisms, which arise from mutagenesis-driven evolution and adaptation to environmental pressures.