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Dissecting the constraints that define the eukaryotic DNA replication program

Periodic Reporting for period 3 - REPLICONSTRAINTS (Dissecting the constraints that define the eukaryotic DNA replication program)

Reporting period: 2019-08-01 to 2021-01-31

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. Surprisingly, cells are equipped with the potential to replicate DNA faster than they do, but they have an apparent need to constraint their DNA replication activity to "healthy levels". How cells maintain these levels of DNA replication and why are normally constrained are questions that 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 that the majority of cancers have 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. Basically, the inherent potential that human cells have to replicate faster could be exploited by cancer cells to grow more rapidly and, by causing more DNA instability, help the cancer to develop further. 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, which happens naturally during DNA replication. There is a protein called RPA, which is essential to bind and protect ssDNA. When ssDNA levels exceed the protective capacity of the cellular RPA pool, ssDNA at replication sites breaks, leading to the death of the cell.

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 identifying the putative human limiting factors of replication, and on validating our hypothesis that overexpression of these factors could create ‘supra DNA replication’. We are characterising a number of cancer and non cancer cells for the levels of key replication factors, and understand how they correlate with the levels of DNA replication and other parameters in each cell. We are in the process now of creating a library of cell lines with different levels of overexpression of these factors, on which we will carry out an extensive analysis further on. We have also determined that cells with reduced capacity to fire origins have a deficient checkpoint and hyperactive CDKs. These and other results indicate that the checkpoint could serve as a buffer to not only constrain, but also to sustain a determined level of replication.

Objective 2: Identify the molecular players that regulate the exposure of single stranded DNA (ssDNA) at the human replisome

Our main aim is to understand how ssDNA is generated in response to hydroxyurea, and particularly the role of ATR in regulating the balance of ssDNA locally at replication forks. We carried out our first siRNA screen targeting all helicases present in the human genome. Although the screen was performed effectively, unfortunately it didn't yield any substantial results that deserved a follow up. Despite we had a very powerful pipeline in place, we regard this result to the limitations of siRNA technology. We have now put this part on hold and potentially in the future we will try to revisit and design new experimental approaches to tackle this question.

Objective 3: Depict the functional role of replication catastrophe by characterizing its regulatory steps

With the same library targeting all nucleases in the human genome, we plan to address the question of how ssDNA is cleaved after RPA exhaustion. We completed the screening using multiple different experimental inputs and unfortunately we didn't find any substantial positive hit. We regard this to the limitation of siRNA technology and also to the possibility that there is a high degree of redundancy in DNA endo- and exo-nucleases.

Objective 4: Characterize novel replication proteins as drivers of RC in the context of cancer therapy

We are now under review in Molecular Cell to publish this fascinating story. It started with the pilot-scale drug screen I carried out at the end of my postdoc, which led to the identification of a family of compounds with completely unexpected properties. Right before I started my group one these compounds (CD437) was identified by an independent group as a specific inhibitor of Polymerase alpha. We have described that inhibition of Polymerase alpha in vivo does not stop replication forks but causes the uncoupling of leading and lagging strands, which leads to a rapid accumulation of ssDNA at the lagging strand and the consequent exhaustion of RPA. In addition to this result, our data describes the full spectrum of responses of such scenario in mammalian cells and provides initial evidence for the use of Polymerase Alpha inhibitors as anticancer agents.
- We have identified a critical role for replication proteins in regulating CDK activity and cell cycle progression. We expect to characterise these two fundamental aspects in detail and put together publications before the end of the project.

- We have characterised the cellular consequences of PolA inhibition in human cells and discovered a new phenomenon (strand uncoupling) that explains its effects to replication forks. This work will be published soon and we are foreseeing developments with the CRISPR screen mentioned above and also investigating the therapeutic potential of PoLAi with translational studies.
Objectives of the project