Mechanisms at the interface of DNA damage repair and transcription

Introduction

Bulky DNA lesions are a major obstacle during gene transcription by RNA polymerase II enzymes (RNAPII). The stalling of RNAPII at DNA lesions triggers a genome-wide transcriptional arrest.

Transcription-Coupled Repair

Transcription-coupled repair (TCR) is a specialized DNA repair pathway that selectively removes DNA lesions from actively transcribed genes to restore transcription. Stalled RNAPII at DNA lesions forms a roadblock for advancing DNA replication forks, resulting in toxic collisions.

Knowledge Gaps

The mechanisms that enable the repair of transcription-blocking DNA lesions, the restoration of transcription after repair, and the resolution of transcription-replication conflicts are poorly understood.

Proposed Approaches

To address these knowledge gaps, I propose to establish a series of innovative approaches aimed at identifying the mechanisms involved in the cellular responses to transcription-blocking DNA damage. We will focus on the functional characterization of known and several promising new TCR factors that we recently identified in combined genome-wide CRISPR and targeted proteomics screens.

Specific Aims

I propose to dissect the role of known and new TCR proteins by:

1. Applying a genome-wide approach for directly measuring TCR activity in combination with proximity-labelling proteomics and genetic-interaction mapping to define how TCR complexes assemble and operate.
2. Identifying the mechanisms in transcription restoration by combining advanced genomics methods to map nascent transcripts, monitor RNAPII occupancy, and correlate these with specific chromatin modifications in a genome-wide manner.
3. Dissecting the mechanisms involved in resolving transcription-replication conflicts by combining functional DNA replication assays with genome-wide approaches to map transcription, R-loops, and DNA replication directionality.

Conclusion

This ERC project will break new ground by offering a detailed understanding of the mechanisms that enable cells to overcome transcriptional roadblocks.