Periodic Reporting for period 1 - DSB Architect (The role of chromosome conformation in DNA double-strand break repair)
Reporting period: 2022-09-01 to 2024-08-31
Eukaryotic cells often face damage to their DNA, including double-strand breaks (DSBs), which need proper repair to maintain the cell’s health. One highly accurate repair process is called homologous recombination. This error-free process uses an undamaged homologous DNA region as a template to repair the break. Given the vast size of human genomes, a random search for these matching regions would be highly inefficient. Indeed, homologous recombination is slower and less efficient when homologous DNA regions are on different chromosomes. However, after DNA replication, thanks the presence of an exact copy of each chromosome, homologous recombination becomes more efficient between the copies, called sister chromatids, due to their specific organization. Cohesin complexes play a crucial role in this organization, thus supporting more effective DNA repair.
Cohesin complexes play two key roles in organizing sister chromatids: they create stable connections between them and form dynamic DNA loops within each chromatid. These loops shape specific domains that influence various processes in the nucleus, including DNA repair. The stable links formed by cohesin are crucial for efficient homologous recombination, as they help locate matching DNA sequences and restructure the repair sites.
However, the exact function of this three-dimensional DNA organization in the repair process has remained unclear until now.
The goal of this project was to understand how the three-dimensional organization of sister chromatids contributed to homologous recombination repair. Specifically, it aimed to 1) explore how the structure of sister chromatids affected and was affected by the repair process, and 2) identify key molecular factors that regulated chromosome conformation for efficient DNA repair.
By combining microscopy- and sequencing-based assays, together with a novel sister chromatid sensitive Hi-C assay developed in the hosting lab, the Fellow has identified a role for sister chromatid organization, regulated by both cohesive and loop-extruding cohesin, in controlling the range and dimensionality of the homology search step during homologous recombination in human cells.
Cohesin complexes play two key roles in organizing sister chromatids: they create stable connections between them and form dynamic DNA loops within each chromatid. These loops shape specific domains that influence various processes in the nucleus, including DNA repair. The stable links formed by cohesin are crucial for efficient homologous recombination, as they help locate matching DNA sequences and restructure the repair sites.
However, the exact function of this three-dimensional DNA organization in the repair process has remained unclear until now.
The goal of this project was to understand how the three-dimensional organization of sister chromatids contributed to homologous recombination repair. Specifically, it aimed to 1) explore how the structure of sister chromatids affected and was affected by the repair process, and 2) identify key molecular factors that regulated chromosome conformation for efficient DNA repair.
By combining microscopy- and sequencing-based assays, together with a novel sister chromatid sensitive Hi-C assay developed in the hosting lab, the Fellow has identified a role for sister chromatid organization, regulated by both cohesive and loop-extruding cohesin, in controlling the range and dimensionality of the homology search step during homologous recombination in human cells.
The project was organized into three work packages (WPs).
WP1 focused on analyzing the native structure of sister chromatids and how it changed upon DNA damage and repair through homologous recombination. Key milestones included developing a system for inducing double-strand breaks (DSBs) with controlled timing at specific genomic sites in replicated synchronized cells. This system was validated using microscopy-based assays and genomic profiling to confirm the proper recruitment of homologous recombination repair factors. The system was then applied to a recently developed technique in the host lab, sister-chromatid-sensitive Hi-C (scsHi-C), to map the native structure of sister chromatids before DNA damage and, for the first time, to reveal structural changes during homologous recombination repair.
WP2 investigated the role of cohesin’s two functions—cohesion and loop extrusion—in shaping sister chromatids during homologous recombination repair. This involved combining the DSB induction system with the targeted and acute depletion of cohesin and its regulators. The updated approach allowed the team to generate genomic profiles of homologous recombination repair factors and produce scsHi-C maps of both undamaged and damaged sister chromatids. The analysis revealed a new role for cohesin’s cohesive and loop-extruding activities in the context of homologous recombination repair in human cells.
WP3 centered on the communication and dissemination of the project. The findings were presented at three international conferences. By the end of the funding period, a manuscript was in preparation, with plans for submission by the end of the calendar year.
WP1 focused on analyzing the native structure of sister chromatids and how it changed upon DNA damage and repair through homologous recombination. Key milestones included developing a system for inducing double-strand breaks (DSBs) with controlled timing at specific genomic sites in replicated synchronized cells. This system was validated using microscopy-based assays and genomic profiling to confirm the proper recruitment of homologous recombination repair factors. The system was then applied to a recently developed technique in the host lab, sister-chromatid-sensitive Hi-C (scsHi-C), to map the native structure of sister chromatids before DNA damage and, for the first time, to reveal structural changes during homologous recombination repair.
WP2 investigated the role of cohesin’s two functions—cohesion and loop extrusion—in shaping sister chromatids during homologous recombination repair. This involved combining the DSB induction system with the targeted and acute depletion of cohesin and its regulators. The updated approach allowed the team to generate genomic profiles of homologous recombination repair factors and produce scsHi-C maps of both undamaged and damaged sister chromatids. The analysis revealed a new role for cohesin’s cohesive and loop-extruding activities in the context of homologous recombination repair in human cells.
WP3 centered on the communication and dissemination of the project. The findings were presented at three international conferences. By the end of the funding period, a manuscript was in preparation, with plans for submission by the end of the calendar year.
This project advanced the fields of chromosome conformation and DNA repair in significant ways.
Until recently, studying the structure of sister chromatids during DNA repair has been challenging due to limitations in conventional chromosome conformation capture methods, which did not detect interactions between identical sequences. To overcome this, the hosting lab of Daniel Gerlich developed a novel technique called sister-chromatid-sensitive Hi-C (scs-HiC). This breakthrough enabled the detailed analysis of both inter- and intra-molecular interactions within replicated human chromosomes, allowing the project to systematically investigate the native structure of sister chromatids and their reorganization following controlled DSB induction. By combining scs-HiC with genomic profiling and microscopy-based techniques, the project provided a comprehensive, multi-dimensional view of how genomic positioning, three-dimensional organization, and dynamic chromatin changes supported HR repair. Furthermore, the study employed targeted depletion of cohesin and its regulators to gain deeper insights into how cohesin’s two functions—cohesion and loop extrusion—govern sister chromatid conformation during HR repair in human cells.
These findings expanded the understanding of a fundamental mechanism that preserves genomic integrity. Beyond its physiological importance in healthy tissues, the project’s insights into homologous recombination repair also shed light on pathological processes. Additionally, understanding how chromosome organization influences homologous recombination repair holds promise for technical applications in genome engineering, where current technologies rely on endogenous DNA repair pathways.
Until recently, studying the structure of sister chromatids during DNA repair has been challenging due to limitations in conventional chromosome conformation capture methods, which did not detect interactions between identical sequences. To overcome this, the hosting lab of Daniel Gerlich developed a novel technique called sister-chromatid-sensitive Hi-C (scs-HiC). This breakthrough enabled the detailed analysis of both inter- and intra-molecular interactions within replicated human chromosomes, allowing the project to systematically investigate the native structure of sister chromatids and their reorganization following controlled DSB induction. By combining scs-HiC with genomic profiling and microscopy-based techniques, the project provided a comprehensive, multi-dimensional view of how genomic positioning, three-dimensional organization, and dynamic chromatin changes supported HR repair. Furthermore, the study employed targeted depletion of cohesin and its regulators to gain deeper insights into how cohesin’s two functions—cohesion and loop extrusion—govern sister chromatid conformation during HR repair in human cells.
These findings expanded the understanding of a fundamental mechanism that preserves genomic integrity. Beyond its physiological importance in healthy tissues, the project’s insights into homologous recombination repair also shed light on pathological processes. Additionally, understanding how chromosome organization influences homologous recombination repair holds promise for technical applications in genome engineering, where current technologies rely on endogenous DNA repair pathways.