Periodic Reporting for period 4 - REPLISTRESS (DNA Replication: From Physiology to Replication Stress in Human Cancer)
Période du rapport: 2023-04-01 au 2024-09-30
Proliferation of cells requires that the genome becomes duplicated, before each cell division. This ensures that the daughter cells have the same genomic information as the mother cell. Duplication of the genome is performed by a process called DNA replication. The genome contains specific sites, where DNA replication begins. These sites are called origins of DNA replication. In this Project we defined precisely where the DNA replication origins are located in the human and mouse genomes and the specific DNA sequences that are needed for DNA replication to begin at these sites. A second objective of the Project was to understand how DNA replication is coordinated with gene transcription. Transcription is the process by which RNAs are copied from genomic DNA; RNAs provide the information needed to synthesize proteins. The DNA replication machinery and the transcription machinery use the same DNA template. Thus, if the two processes are not coordinated, the transcription and replication machineries may collide, leading to DNA damage. In this Project we discovered how these collisions are avoided. Interestingly, the sensor for impending collisions is the protein PARP1. Inhibitors of PARP1 are already used to treat hereditary breast and ovarian cancers. Therefore, this Project elucidated the mechanism by which PARP1 inhibitors exert their therapeutic effect. Another unanticipated discovery made in the course of this Project is that aging of the liver, and possibly of many other organs, is caused by DNA damage that accumulates over time in cells and which damage remains undetected until the cells start replicating their DNA. At this moment, if the DNA damage exceeds a certain threshold the cells fail to complete replication of their genomic DNA and become senescent. Thus, this Project, beyond its goal to understand DNA replication in normal and cancer cells, provided also unexpected insights on the aging process.
The proposed project has 3 Aims.
Aim 1. Determine whether origin firing in human cells is stochastic or deterministic; map the origin firing sites at high resolution; and identify functionally important origin motifs.
Using mouse embryonic stem cells (ESCs) we demonstrated that origin firing in early replicating genomic domains is deterministic, whereas in late replicating genomic domains it may be either stochastic or deterministic. Further, we mapped DNA replication origins at high resolution in human cells in tissue culture and, importantly, also in normal hepatocytes of mouse regenerating livers in vivo. Finally, we identified a sequence motif within DNA replication origins that is bound by the OCT4 pluripotency factor and which motif is required for firing of specific origins in mouse embryonic stem cells. An unexpected discovery made while studying origin firing in vivo in mouse regenerating livers was that old mice exhibited an origin firing defect and that this defect was due to DNA damage that had accumulated in the hepatocytes during aging, but had not been repaired.
Aim 2. Determine the mechanisms by which transcription in G1 suppresses firing of intragenic origins.
We explored two mechanisms: origin licensing occurs only in early G1 and then transcription removes any licensed origins within transcribed genes or transcription modifies chromatin in a way that prevents firing of licensed origins. Our results support the second mechanism, as we found that origin licensing requires Cdk4 activity, which is a late event in G1. These observations change how we think about origin licensing and firing, as they show that origin licensing requires Cdk activity, whereas previously it was thought (based on experiments in yeast) that Cdk activity inhibited origin licensing.
Aim 3. Determine why forks from intergenic origins do not collapse when replicating highly transcribed genes.
We determined the mechanism by which impending transcription-replication conflicts (TRCs) are sensed by cells. We found that TRCs are sensed by PARP1, which becomes activated and associates with Timeless, a protein that is part of the replication machinery. The interaction between activated PARP1 and Timeless pauses the DNA replication machinery, thereby averting collisions with the transcription machinery and the induction of DNA double-strand breaks and genomic instability. These findings explain how PARP inhibitors exert their therapeutic effect in the context of cancers that have defects in repair of DNA double-strand breaks, notably hereditary breast and ovarian cancers.
In the context of this Project we have published eleven papers. Two of these have been published in the most prestigious scientific journals: Nature and Cell, the remaining have been published in highly respected journals, including Nature Communications and Cell Reports. In addition, we have presented our findings in more than 15 international scientific meetings over the course of the past six years.
Aim 1. Determine whether origin firing in human cells is stochastic or deterministic; map the origin firing sites at high resolution; and identify functionally important origin motifs.
Using mouse embryonic stem cells (ESCs) we demonstrated that origin firing in early replicating genomic domains is deterministic, whereas in late replicating genomic domains it may be either stochastic or deterministic. Further, we mapped DNA replication origins at high resolution in human cells in tissue culture and, importantly, also in normal hepatocytes of mouse regenerating livers in vivo. Finally, we identified a sequence motif within DNA replication origins that is bound by the OCT4 pluripotency factor and which motif is required for firing of specific origins in mouse embryonic stem cells. An unexpected discovery made while studying origin firing in vivo in mouse regenerating livers was that old mice exhibited an origin firing defect and that this defect was due to DNA damage that had accumulated in the hepatocytes during aging, but had not been repaired.
Aim 2. Determine the mechanisms by which transcription in G1 suppresses firing of intragenic origins.
We explored two mechanisms: origin licensing occurs only in early G1 and then transcription removes any licensed origins within transcribed genes or transcription modifies chromatin in a way that prevents firing of licensed origins. Our results support the second mechanism, as we found that origin licensing requires Cdk4 activity, which is a late event in G1. These observations change how we think about origin licensing and firing, as they show that origin licensing requires Cdk activity, whereas previously it was thought (based on experiments in yeast) that Cdk activity inhibited origin licensing.
Aim 3. Determine why forks from intergenic origins do not collapse when replicating highly transcribed genes.
We determined the mechanism by which impending transcription-replication conflicts (TRCs) are sensed by cells. We found that TRCs are sensed by PARP1, which becomes activated and associates with Timeless, a protein that is part of the replication machinery. The interaction between activated PARP1 and Timeless pauses the DNA replication machinery, thereby averting collisions with the transcription machinery and the induction of DNA double-strand breaks and genomic instability. These findings explain how PARP inhibitors exert their therapeutic effect in the context of cancers that have defects in repair of DNA double-strand breaks, notably hereditary breast and ovarian cancers.
In the context of this Project we have published eleven papers. Two of these have been published in the most prestigious scientific journals: Nature and Cell, the remaining have been published in highly respected journals, including Nature Communications and Cell Reports. In addition, we have presented our findings in more than 15 international scientific meetings over the course of the past six years.
Three achievements during the past six years qualify, in our opinion, as progress beyond the state of the art.
First, this Project was the first to map DNA replication origins in a living mammal. Prior to this Project, mammalian DNA replication origins had been mapped only in tissue culture cells and the results from study to study were not consistent. Our origin mapping method proved to be very robust, such that the results from mouse to mouse were very consistent.
Second, we found that old animals had an origin firing defect that could be fully rescued by inhibiting the DNA replication checkpoint. The rescue of the defect implied the existence of DNA damage that had accumulated during aging, but had not been repaired. When the replication machinery encountered this "hidden" (cryptic) DNA damage, the DNA replication checkpoint was activated and suppressed further origin firing. These findings provide a new understanding of the aging process. Importantly, if it were possible to repair the cryptic DNA damage before the cells replicated their DNA, then the aged cells would not exhibit an origin firing defect.
Third, we discovered the mechanism by which impending collisions between the transcription and DNA replication machineries are sensed. Specifically, we found that PARP1 serves as a sensor for impending collisions. PARP1 inhibitors are used as anti-cancer therapeutics. Therefore, we also discovered the mechanism by which PARP1 inhibitors exert their therapeutic effect.
First, this Project was the first to map DNA replication origins in a living mammal. Prior to this Project, mammalian DNA replication origins had been mapped only in tissue culture cells and the results from study to study were not consistent. Our origin mapping method proved to be very robust, such that the results from mouse to mouse were very consistent.
Second, we found that old animals had an origin firing defect that could be fully rescued by inhibiting the DNA replication checkpoint. The rescue of the defect implied the existence of DNA damage that had accumulated during aging, but had not been repaired. When the replication machinery encountered this "hidden" (cryptic) DNA damage, the DNA replication checkpoint was activated and suppressed further origin firing. These findings provide a new understanding of the aging process. Importantly, if it were possible to repair the cryptic DNA damage before the cells replicated their DNA, then the aged cells would not exhibit an origin firing defect.
Third, we discovered the mechanism by which impending collisions between the transcription and DNA replication machineries are sensed. Specifically, we found that PARP1 serves as a sensor for impending collisions. PARP1 inhibitors are used as anti-cancer therapeutics. Therefore, we also discovered the mechanism by which PARP1 inhibitors exert their therapeutic effect.