Final Report Summary - DAMAGE BYPASS (Mechanistic analysis of DNA damage bypass in the context of chromatin and genome replication)
DNA, the carrier of our genetic information, is particularly vulnerable to decay during its replication, and the capacity of cells to deal with replication stress is a major factor protecting us from genome instability and cancer. In oder to complete the replication of a damaged genome, cells employ pathways collectively called DNA damage bypass, which either employ specialised DNA polymerases that copy the damaged DNA in a mutagenic reaction or involve a recombination-like process that avoids the use of the damaged template altogether. Hence, damage bypass contributes to genome maintenance, but can itself be a source of genomic instability and mutagenesis. It is therefore not surprising that the process is highly regulated and needs to be limited to the appropriate situations by stringent control mechanisms.
The aim of this ERC-funded project has been to investigate how DNA damage bypass is regulated in time and space in its physiological setting. To this end, we have investigated the relationship between damage bypass and global damage signalling. We found that in contrast to situations where the replisome itself is compromised, lesions in the replication template elicit a checkpoint response mainly via postreplicative daughter-strand gaps that are also the substrates of damage bypass. These results have for the first time offered a mechanistic explanation for the dichotomy between replisome- versus template-induced checkpoint signalling. We have also established a novel experimental tool to induce defined replication-stalling DNA lesions into a specific region of the yeast genome, thus allowing us to monitor the passage of the replication machinery over the affected region in real time. This approach has given unprecedented insight into the orchestration of DNA damage bypass at molecular resolution, revealing an apparent separation of replicative polymerases, differential replication of leading and lagging strand, and further processing of the damaged DNA after passage of the replisome. In the course of this investigation, we have developed a new next-generation sequencing method for the genome-wide detection of single-stranded DNA breaks and various other lesions. As a complementary strategy, we have established methods to follow damage bypass in live cells by means of fluorescence microscopy, using both common replication factors as well as custom-made sensors of activated DNA damage bypass pathways as reporters. In this manner, we were able to show that the bulk of damage bypass activity occurs in spatial separation from ongoing replication.
In a second part of the project, we have systematically investigated the contributions of chromatin components and dynamics on damage bypass. In the course of these experiments, we identified novel factors that impinge on the efficiency of the reaction, such as the posttranslational modification of histone H2B by the small protein ubiquitin. In addition, we were able to assign a new role to the chromatin remodelling INO80 complex during a late step of DNA damage bypass.
Finally, we investigated how modifications to a central replication factor, PCNA, impinge on its interactions and functions in DNA replication and damage bypass. By designing and constructing a series of tailor-made ubiquitin protein ligases, we were able to manipulate the linkage of the polyubiquitin chain on PCNA that normally triggers damage bypass, thus for the first time assigning distinct functional consequences to different linkages on one and the same substrate.
Overall, this project has given important new insight into the mechanism of DNA damage tolerance by defining its activities in relationship to the replication machinery in the environment of native chromatin.
The aim of this ERC-funded project has been to investigate how DNA damage bypass is regulated in time and space in its physiological setting. To this end, we have investigated the relationship between damage bypass and global damage signalling. We found that in contrast to situations where the replisome itself is compromised, lesions in the replication template elicit a checkpoint response mainly via postreplicative daughter-strand gaps that are also the substrates of damage bypass. These results have for the first time offered a mechanistic explanation for the dichotomy between replisome- versus template-induced checkpoint signalling. We have also established a novel experimental tool to induce defined replication-stalling DNA lesions into a specific region of the yeast genome, thus allowing us to monitor the passage of the replication machinery over the affected region in real time. This approach has given unprecedented insight into the orchestration of DNA damage bypass at molecular resolution, revealing an apparent separation of replicative polymerases, differential replication of leading and lagging strand, and further processing of the damaged DNA after passage of the replisome. In the course of this investigation, we have developed a new next-generation sequencing method for the genome-wide detection of single-stranded DNA breaks and various other lesions. As a complementary strategy, we have established methods to follow damage bypass in live cells by means of fluorescence microscopy, using both common replication factors as well as custom-made sensors of activated DNA damage bypass pathways as reporters. In this manner, we were able to show that the bulk of damage bypass activity occurs in spatial separation from ongoing replication.
In a second part of the project, we have systematically investigated the contributions of chromatin components and dynamics on damage bypass. In the course of these experiments, we identified novel factors that impinge on the efficiency of the reaction, such as the posttranslational modification of histone H2B by the small protein ubiquitin. In addition, we were able to assign a new role to the chromatin remodelling INO80 complex during a late step of DNA damage bypass.
Finally, we investigated how modifications to a central replication factor, PCNA, impinge on its interactions and functions in DNA replication and damage bypass. By designing and constructing a series of tailor-made ubiquitin protein ligases, we were able to manipulate the linkage of the polyubiquitin chain on PCNA that normally triggers damage bypass, thus for the first time assigning distinct functional consequences to different linkages on one and the same substrate.
Overall, this project has given important new insight into the mechanism of DNA damage tolerance by defining its activities in relationship to the replication machinery in the environment of native chromatin.