Final Report Summary - NDOGS (Nuclear Dynamic, Organization and Genome Stability)
Our genomes are constantly challenged by DNA lesions arising either from environmental stress, such as exposure to irradiation, ultraviolet light or genotoxic agents or from endogenous events, such as reactive oxygen species generated during cellular metabolism or replication fork accidents. The cellular response that follows is a coordinated series of events that allows DNA damage detection, signaling and repair. Among the various forms of DNA damage, double strand breaks (DSBs) are the most genotoxic and their improper repair leads to genomic instability. DSB repair can occur through two different mechanisms: Non Homologous End Joining (NHEJ) and Homologous Recombination (HR). While DNA repair mechanisms have been extensively characterized during the last decades, it is only recently that the chromatin context, in which DNA repair occurs, and the organization of chromatin in the nuclear space have emerged as possible regulators of DNA repair pathway choice and repair outcome. However, the molecular steps regulated remain to be defined.
Focusing on Double strand breaks (DSBs) in response to which cells activate checkpoint and DNA repair pathways, we aimed at characterizing the spatial and temporal behaviour of damaged chromatin and determining how this affects the maintenance of genome integrity using Saccharomyces cerevisiae as an experimental model. Our specific goals were to 1) Define how nuclear organization influences the detection, processing and subsequent repair of DNA lesions; 2) Develop proteomic and microscopy based genome-wide screens to decipher the molecular pathways leading to the repositioning of damaged chromatin in the nucleus; 3) Combine advanced live microscopy and mathematical simulations to define damaged chromatin dynamics and understand the constraints imposed on DNA repair by chromatin packaging and nuclear compartmentalization.
The position of DSB in the nucleus is regulated and influences repair pathways. We previously demonstrated that DSBs relocate to the nuclear periphery where they contact nuclear pores or the nuclear membrane protein Mps3. We dissected the molecular pathways defining the position of DSB in the nucleus by performing genetic and proteomic screens, testing the functional consequence of nuclear position for checkpoint activation and DNA repair by driving the DSB to specific nuclear landmarks and, defining the dynamics of DNA damages in different repair contexts.
To understand further how the different genomic, chromatin, and subnuclear context influence homologous recombination, we developed and characterized an assay that allowed to score spontaneous and DSB induced recombination events between alleles located at different chromosomal positions. We used this assay to measure recombination frequency and outcome in strains in which the physical distances between telomeres and/or the spreading of heterochromatin in subtelomeric regions were modified through overexpression of the Sir3 or Sir3A2Q protein. Using these tools, we observed that loci at different locations in the genome recombine with different efficiency. We further demonstrated that reducing the physical distance between homologous sequences favours recombination, reinforcing the notion that homology search is limiting for recombination efficiency. This unique assay allowed to assess the competition between two recombination pathways, namely gene conversion (GC) or break induced replication (BIR). We showed that BIR is the pathway of choice to repair subtelomeric DSB. At these sites the presence of heterochromatin increased recombination frequencies both through GC and BIR. Our molecular analysis further revealed that heterochromatin fine-tunes DSB resection, limiting the loss of the homologous sequence required to perform strand invasion.
Focusing on Double strand breaks (DSBs) in response to which cells activate checkpoint and DNA repair pathways, we aimed at characterizing the spatial and temporal behaviour of damaged chromatin and determining how this affects the maintenance of genome integrity using Saccharomyces cerevisiae as an experimental model. Our specific goals were to 1) Define how nuclear organization influences the detection, processing and subsequent repair of DNA lesions; 2) Develop proteomic and microscopy based genome-wide screens to decipher the molecular pathways leading to the repositioning of damaged chromatin in the nucleus; 3) Combine advanced live microscopy and mathematical simulations to define damaged chromatin dynamics and understand the constraints imposed on DNA repair by chromatin packaging and nuclear compartmentalization.
The position of DSB in the nucleus is regulated and influences repair pathways. We previously demonstrated that DSBs relocate to the nuclear periphery where they contact nuclear pores or the nuclear membrane protein Mps3. We dissected the molecular pathways defining the position of DSB in the nucleus by performing genetic and proteomic screens, testing the functional consequence of nuclear position for checkpoint activation and DNA repair by driving the DSB to specific nuclear landmarks and, defining the dynamics of DNA damages in different repair contexts.
To understand further how the different genomic, chromatin, and subnuclear context influence homologous recombination, we developed and characterized an assay that allowed to score spontaneous and DSB induced recombination events between alleles located at different chromosomal positions. We used this assay to measure recombination frequency and outcome in strains in which the physical distances between telomeres and/or the spreading of heterochromatin in subtelomeric regions were modified through overexpression of the Sir3 or Sir3A2Q protein. Using these tools, we observed that loci at different locations in the genome recombine with different efficiency. We further demonstrated that reducing the physical distance between homologous sequences favours recombination, reinforcing the notion that homology search is limiting for recombination efficiency. This unique assay allowed to assess the competition between two recombination pathways, namely gene conversion (GC) or break induced replication (BIR). We showed that BIR is the pathway of choice to repair subtelomeric DSB. At these sites the presence of heterochromatin increased recombination frequencies both through GC and BIR. Our molecular analysis further revealed that heterochromatin fine-tunes DSB resection, limiting the loss of the homologous sequence required to perform strand invasion.