Periodic Reporting for period 4 - REPLICONSTRAINTS (Dissecting the constraints that define the eukaryotic DNA replication program)
Reporting period: 2021-02-01 to 2022-07-31
REPLICONSTRAINTS
With this project we aim at understanding how the cells in our body manage to duplicate their chromosomes in order to grow and proliferate. This process, known as DNA replication, is specially complex in human cells, because of the shear amount of DNA that has to be copied. DNA replication happens at replication forks, which are composed of different proteins that, together, accomplish to synthesise the new DNA strands. Rather than copying one full chromosome from beginning to the end with one or two replication forks, human cells need to use thousands of forks simultaneously, taking many hours to fully duplicate their genome. Surprisingly, cells are equipped with the potential to replicate even faster, but they have an apparent need to constraint their DNA replication activity. How cells maintain "healthy" levels of DNA replication and why it is normally constrained are questions never addressed before, and which could open avenues to understand how cancer develops and also how to treat it with novel and better drugs.
DNA replication is a natural source of DNA instability, a condition present in many cancers and that increases the rate of changes and mutations in the genome, promoting cancer cells to gain new capabilities and evolve into more aggressive forms. We believe the speed and rates of DNA replication might play an important role in cancer development. Yet, there is another side of this aspect of DNA replication. Understanding how DNA replication rates are controlled, we could envision targeting its essential regulatory pathways to induce a burst of DNA replication in cancer cells and push beyond the boundaries they can sustain. Thus this project its relevant for society, considering it could have a long term impact in how we fight human cancers.
An evidence for the existence of these boundaries is that cells have a limited capacity to sustain DNA in single stranded form or ssDNA. This ssDNA happens naturally during DNA replication, and needs a protein called RPA to be protected. Without RPA, ssDNA is extremely fragile and breaks. The DNA replication machinery of cancer cells has the capacity to overrun this boundary and cause a massive breakage of ssDNA coined "Replication Catastrophe" due to a the exhaustion of RPA molecules.
With these concepts in mind, this project has the following overall objectives:
1. Understand the consequences that higher levels of DNA replication have for cells.
2. Understand how the levels of ssDNA are regulated in the cell.
3. Understand why RPA is so essential to protect ssDNA.
4. Identify new targets and molecules that can be used to eliminate cancer cells by pushing them beyond their DNA replication sustainable boundaries.
With this project we aim at understanding how the cells in our body manage to duplicate their chromosomes in order to grow and proliferate. This process, known as DNA replication, is specially complex in human cells, because of the shear amount of DNA that has to be copied. DNA replication happens at replication forks, which are composed of different proteins that, together, accomplish to synthesise the new DNA strands. Rather than copying one full chromosome from beginning to the end with one or two replication forks, human cells need to use thousands of forks simultaneously, taking many hours to fully duplicate their genome. Surprisingly, cells are equipped with the potential to replicate even faster, but they have an apparent need to constraint their DNA replication activity. How cells maintain "healthy" levels of DNA replication and why it is normally constrained are questions never addressed before, and which could open avenues to understand how cancer develops and also how to treat it with novel and better drugs.
DNA replication is a natural source of DNA instability, a condition present in many cancers and that increases the rate of changes and mutations in the genome, promoting cancer cells to gain new capabilities and evolve into more aggressive forms. We believe the speed and rates of DNA replication might play an important role in cancer development. Yet, there is another side of this aspect of DNA replication. Understanding how DNA replication rates are controlled, we could envision targeting its essential regulatory pathways to induce a burst of DNA replication in cancer cells and push beyond the boundaries they can sustain. Thus this project its relevant for society, considering it could have a long term impact in how we fight human cancers.
An evidence for the existence of these boundaries is that cells have a limited capacity to sustain DNA in single stranded form or ssDNA. This ssDNA happens naturally during DNA replication, and needs a protein called RPA to be protected. Without RPA, ssDNA is extremely fragile and breaks. The DNA replication machinery of cancer cells has the capacity to overrun this boundary and cause a massive breakage of ssDNA coined "Replication Catastrophe" due to a the exhaustion of RPA molecules.
With these concepts in mind, this project has the following overall objectives:
1. Understand the consequences that higher levels of DNA replication have for cells.
2. Understand how the levels of ssDNA are regulated in the cell.
3. Understand why RPA is so essential to protect ssDNA.
4. Identify new targets and molecules that can be used to eliminate cancer cells by pushing them beyond their DNA replication sustainable boundaries.
Objective 1: Elucidate the consequences of non-physiological levels of replication in human cells
We first focused on describing how human limiting factors of replication regulate physiological DNA synthesis, and on validating our hypothesis that over-expression of these factors could create ‘supra DNA replication’. Unfortunately we could not find any solid evidence of limiting factors being correlated to levels of DNA replication. Surprisingly enough, cell lines with very low levels of these factors were able to sustain very high levels of DNA synthesis and origin firing, but we did not undertake an investigation of this. Eventually this main line of work was abandoned.
We took a turn and devised a new strategy to elucidate new factors involved in origin firing using mass spectrometry. We carried out a time-resolved profiling of proteins recruited to origins of replication upon (and after) origin firing, identifying new factors that are involved in the efficacy of this process. We are now currently working on characterising some of these to wrap the work up in a manuscript (Work done by Amitash Sampath).
Also within this objective, we examined the regulators of the S-phase checkpoint as one of the key processes controlling origin firing and the levels of DNA replication in cells. Surprisingly we found that TRESLIN/MTBP, a protein complex so far known for its role in activating origin firing, is also able to monitor S-phase progression and it is the master regulator of the S/G2 cell cycle transition (Work done by Gijs Zonderland). This work was published in 2022 in Molecular Cell.
Objective 2: Identify the molecular players that regulate the exposure of single stranded DNA (ssDNA) at the human replisome
Although we had a very promising analytical pipeline in place to carry out this objective, unfortunately it didn't yield any substantial results that deserved a follow up. Members of the lab working on this objective focused on Objective 1.
Objective 3: Depict the functional role of replication catastrophe by characterizing its regulatory steps
Similar to objective number two, we did not find solid results to follow up.
Objective 4: Characterize novel replication proteins as drivers of RC in the context of cancer therapy
Under this objective we discovered potentially the most important mechanism to suppress ssDNA in the cell. During DNA synthesis, Polymerase alpha activity prevents the uncoupling of leading and lagging strands, which otherwise causes a rapid accumulation of ssDNA and the consequent exhaustion of RPA. This work was published in Cell Reports in 2020, and ignited a pursue to identify novel POLA1 inhibitors as innovative anti cancer agents. Besides, we are working on characterising other consequences that POLA1 inhibition has for the cell, which could be a novel type of replication stress.
OVERVIEW
Overall I am very satisfied with our achievements and the output of our research. Although some of the objectives turned out to be more technically challenging than anticipated, we carried out a purely curiosity driven investigation on those fronts that offered us interesting findings. We have discovered two new major regulatory mechanisms in DNA replication, which are on their own key advances for our field, and set a funding stone for future research stemming from other labs. During this time we have also taken every opportunity to disseminate our work in local and international conferences. Finally, our research opens opportunities for potentially novel therapeutic targets that could be exploited for treating hyperproliferation-related diseases like cancer.
We first focused on describing how human limiting factors of replication regulate physiological DNA synthesis, and on validating our hypothesis that over-expression of these factors could create ‘supra DNA replication’. Unfortunately we could not find any solid evidence of limiting factors being correlated to levels of DNA replication. Surprisingly enough, cell lines with very low levels of these factors were able to sustain very high levels of DNA synthesis and origin firing, but we did not undertake an investigation of this. Eventually this main line of work was abandoned.
We took a turn and devised a new strategy to elucidate new factors involved in origin firing using mass spectrometry. We carried out a time-resolved profiling of proteins recruited to origins of replication upon (and after) origin firing, identifying new factors that are involved in the efficacy of this process. We are now currently working on characterising some of these to wrap the work up in a manuscript (Work done by Amitash Sampath).
Also within this objective, we examined the regulators of the S-phase checkpoint as one of the key processes controlling origin firing and the levels of DNA replication in cells. Surprisingly we found that TRESLIN/MTBP, a protein complex so far known for its role in activating origin firing, is also able to monitor S-phase progression and it is the master regulator of the S/G2 cell cycle transition (Work done by Gijs Zonderland). This work was published in 2022 in Molecular Cell.
Objective 2: Identify the molecular players that regulate the exposure of single stranded DNA (ssDNA) at the human replisome
Although we had a very promising analytical pipeline in place to carry out this objective, unfortunately it didn't yield any substantial results that deserved a follow up. Members of the lab working on this objective focused on Objective 1.
Objective 3: Depict the functional role of replication catastrophe by characterizing its regulatory steps
Similar to objective number two, we did not find solid results to follow up.
Objective 4: Characterize novel replication proteins as drivers of RC in the context of cancer therapy
Under this objective we discovered potentially the most important mechanism to suppress ssDNA in the cell. During DNA synthesis, Polymerase alpha activity prevents the uncoupling of leading and lagging strands, which otherwise causes a rapid accumulation of ssDNA and the consequent exhaustion of RPA. This work was published in Cell Reports in 2020, and ignited a pursue to identify novel POLA1 inhibitors as innovative anti cancer agents. Besides, we are working on characterising other consequences that POLA1 inhibition has for the cell, which could be a novel type of replication stress.
OVERVIEW
Overall I am very satisfied with our achievements and the output of our research. Although some of the objectives turned out to be more technically challenging than anticipated, we carried out a purely curiosity driven investigation on those fronts that offered us interesting findings. We have discovered two new major regulatory mechanisms in DNA replication, which are on their own key advances for our field, and set a funding stone for future research stemming from other labs. During this time we have also taken every opportunity to disseminate our work in local and international conferences. Finally, our research opens opportunities for potentially novel therapeutic targets that could be exploited for treating hyperproliferation-related diseases like cancer.
We have identified two new major regulatory mechanisms in human DNA replication, which have yielded two publications in top international journals. I am very proud of the originality and novelty of this works, which represent clear advancements for our understanding of human cell biology.
- We have revealed that leading and lagging strand synthesis work independently and that inhibition of POLA1 leads to the uncoupling of these activities. This phenomenon, that we coined strand uncoupling, leads to the quick generation of ssDNA, which can exhaust the cell resources of RPA in a matter of minutes. This work has laid the foundation for the identification and development of novel POLA1 inhibitors as anti cancer agents.
- We have discovered what we have come to call "the missing cell-cycle checkpoint", namely the one between S and G2 phases of the cell cycle. TRESLIN-MTBP, a protein complex know for its role in controlling origin firing, orchestrates the monitoring of S-phase progression to determine when DNA replication is finished, and the timely transition to G2.
- We have revealed that leading and lagging strand synthesis work independently and that inhibition of POLA1 leads to the uncoupling of these activities. This phenomenon, that we coined strand uncoupling, leads to the quick generation of ssDNA, which can exhaust the cell resources of RPA in a matter of minutes. This work has laid the foundation for the identification and development of novel POLA1 inhibitors as anti cancer agents.
- We have discovered what we have come to call "the missing cell-cycle checkpoint", namely the one between S and G2 phases of the cell cycle. TRESLIN-MTBP, a protein complex know for its role in controlling origin firing, orchestrates the monitoring of S-phase progression to determine when DNA replication is finished, and the timely transition to G2.