Periodic Reporting for period 1 - Irrev Fork Arrest (Deciphering the mechanism of irreversible replication fork arrest)
Período documentado: 2020-04-01 hasta 2022-03-31
The replication or copy of genomic DNA is a complex and regulated process, requiring the coordinated action of multiple specialised replication proteins. In eukaryotic cells, like those of human and yeast cells, the genome is made of double-stranded DNA (dsDNA) forming the famous DNA double helix. To replicate it, DNA must be first unwound, exposing the two single DNA strands, and then two new copies of DNA can be synthesised. The replication proteins can elegantly coordinate the unwinding of DNA with the synthesis of the new daughter DNA strands. In eukaryotes, DNA replication initiates simultaneously at multiple sites along the genome known as origins of replication (Figure 1A). In each origin of replication, dsDNA is unwound leading to the formation of a DNA bubble with edges that resemble a fork, known as replication forks (Figure 1B). Replication forks and their replisomes move in opposing directions as they unwind parental DNA and synthesise the new DNA copies (Figure 1C).
DNA replication is an essential process for live, but it is also a very challenging and dangerous process. DNA is usually wrapped and protected from external threats; however, to allow DNA replication, it needs to be exposed and unwound, posing risk to it being damaged. Moreover, despite DNA replication process is extremely efficient and accurate, errors can occur leading to mutations or to the synthesis of incomplete copies of DNA. To repair DNA damage, correct mutations, and ensure that the genome is replicated entirely, cells possess multiple safeguard mechanisms. The safeguard mechanisms can act any time that a cell replicates its DNA, but they are especially important to ensure cell survival in the presence of what is known as “replication stress”. Replication stress is a term used to describe a situation where DNA replication is affected and cannot proceed normally. Although not exclusively, cells famous for having a lot of replication stress are the cancer cells, putatively due to the presence of damaged DNA in these cells, that is likely more difficult to replicate. Moreover, replication stress in cancer cells could be due to exhaustion of the replication components caused by the increased replication and division of these cells. More knowledge is required to understand replication stress and its consequences to, for example, address if replication stress is cause or consequence of cancer cell transformation.
In the presence of replication stress, cells need to protect replication forks to prevent further DNA damage, and at the same time, they need to ensure that replication forks will be able to restart and complete genome replication. The DNA damage checkpoint pathway (the checkpoint) is one of the key safeguard mechanisms to survive to replication stress. It has been shown that, in the absence of an intact checkpoint, stressed replication forks fail to restart replication after episodes of replication stress, which leads to incomplete genome replication, massive accumulation of DNA damage and cell death. Despite the importance of DNA replication and of the checkpoint pathway to ensure that cells can survive to replication stress, there are still many questions remaining to fully understand these processes. Better understanding can be key to understand the transformations that precede the formation of anomalous cells such as cancer cells, and to help identify future treatment avenues to prevent or revert such transformations.
The main objectives of this project, summarised in Figure 1D, aimed to study:
1. Special characteristics of stressed replication forks.
2. Requirements of replication forks to restart after episodes of replication stress.
3. How the checkpoint protects forks to allow DNA replication restart after replication stress.
From our results, we can conclude that in the presence of replication stress, several aspects of replication forks are changed, and their ability to restart after stress episodes can be affected. Moreover, we have identified putative mechanisms by which the checkpoint could protect forks from replication stress to allow restart, ensuring genome replication and cell survival.
DNA replication is an essential process for live, but it is also a very challenging and dangerous process. DNA is usually wrapped and protected from external threats; however, to allow DNA replication, it needs to be exposed and unwound, posing risk to it being damaged. Moreover, despite DNA replication process is extremely efficient and accurate, errors can occur leading to mutations or to the synthesis of incomplete copies of DNA. To repair DNA damage, correct mutations, and ensure that the genome is replicated entirely, cells possess multiple safeguard mechanisms. The safeguard mechanisms can act any time that a cell replicates its DNA, but they are especially important to ensure cell survival in the presence of what is known as “replication stress”. Replication stress is a term used to describe a situation where DNA replication is affected and cannot proceed normally. Although not exclusively, cells famous for having a lot of replication stress are the cancer cells, putatively due to the presence of damaged DNA in these cells, that is likely more difficult to replicate. Moreover, replication stress in cancer cells could be due to exhaustion of the replication components caused by the increased replication and division of these cells. More knowledge is required to understand replication stress and its consequences to, for example, address if replication stress is cause or consequence of cancer cell transformation.
In the presence of replication stress, cells need to protect replication forks to prevent further DNA damage, and at the same time, they need to ensure that replication forks will be able to restart and complete genome replication. The DNA damage checkpoint pathway (the checkpoint) is one of the key safeguard mechanisms to survive to replication stress. It has been shown that, in the absence of an intact checkpoint, stressed replication forks fail to restart replication after episodes of replication stress, which leads to incomplete genome replication, massive accumulation of DNA damage and cell death. Despite the importance of DNA replication and of the checkpoint pathway to ensure that cells can survive to replication stress, there are still many questions remaining to fully understand these processes. Better understanding can be key to understand the transformations that precede the formation of anomalous cells such as cancer cells, and to help identify future treatment avenues to prevent or revert such transformations.
The main objectives of this project, summarised in Figure 1D, aimed to study:
1. Special characteristics of stressed replication forks.
2. Requirements of replication forks to restart after episodes of replication stress.
3. How the checkpoint protects forks to allow DNA replication restart after replication stress.
From our results, we can conclude that in the presence of replication stress, several aspects of replication forks are changed, and their ability to restart after stress episodes can be affected. Moreover, we have identified putative mechanisms by which the checkpoint could protect forks from replication stress to allow restart, ensuring genome replication and cell survival.
To develop this project, we have used a biochemical approach to study DNA replication reconstituted using purified proteins and template DNA. First, we purified the proteins needed for DNA replication, and then we optimised the conditions to perform DNA replication in vitro. We then moved on to introduce perturbations to the system to induce replication stress. We wanted to set up a new system where we could induce enough replication stress to impair DNA replication progression but, importantly, we needed to set up a system where we could also “release” replication forks form the stress and study replication restart. Using this reversible stress system, we identified several interesting changes to stressed forks that could potentially have implications for fork restart.
Next, we aimed to characterise the ability of stressed forks to restart after the stress. We stressed forks in the presence of different proteins and identified conditions and specific proteins that can modify the ability of forks to restart after stress. We are currently working to further characterise how these proteins interact with each other and with stressed replication forks to promote or impair replication restart.
Finally, we aim to understand how the checkpoint protects stressed forks to maintain genome integrity and cell viability. We have combined prior knowledge of the checkpoint with our recent findings of stressed forks to speculate about putative mechanisms. We have identified two important functions of the checkpoint that could have key roles in fork protection to replication stress. Our characterisation of stressed forks could be key to understand the role of the checkpoint, which would validate our initial strategy. In the future, the system that we have set up could serve as a platform for us and others to study the role of other pathways such as DNA repair and recombination in relation with stressed replication forks. Moreover, our workflow can now be used to compare low dNTP stress with other sources of replication stress.
Next, we aimed to characterise the ability of stressed forks to restart after the stress. We stressed forks in the presence of different proteins and identified conditions and specific proteins that can modify the ability of forks to restart after stress. We are currently working to further characterise how these proteins interact with each other and with stressed replication forks to promote or impair replication restart.
Finally, we aim to understand how the checkpoint protects stressed forks to maintain genome integrity and cell viability. We have combined prior knowledge of the checkpoint with our recent findings of stressed forks to speculate about putative mechanisms. We have identified two important functions of the checkpoint that could have key roles in fork protection to replication stress. Our characterisation of stressed forks could be key to understand the role of the checkpoint, which would validate our initial strategy. In the future, the system that we have set up could serve as a platform for us and others to study the role of other pathways such as DNA repair and recombination in relation with stressed replication forks. Moreover, our workflow can now be used to compare low dNTP stress with other sources of replication stress.
Interestingly, we have identified some new characteristics of stressed replication forks that can be useful to understand how stress affects forks and how the checkpoint protects them. This could help understand how cells maintain genome integrity and ensure cell survival in the face of replication stress, which could be especially important to study diseases such as cancer. We consider that the ability of the new system that we have established to identify a putative link with the checkpoint serves to validate our strategy and offers a new system to study different aspects of replication fork stability. We envision that the system that we have set up can serve as a platform to study the role of other proteins in the future, for example to study processes of DNA repair and recombination linked to stressed replication forks. Moreover, we believe it would be very interesting to use our system and use it to compare low dNTP stress with other sources of replication stress.