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DNA Replication: From Physiology to Replication Stress in Human Cancer

Periodic Reporting for period 1 - REPLISTRESS (DNA Replication: From Physiology to Replication Stress in Human Cancer)

Reporting period: 2018-10-01 to 2020-03-31

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 aim to define precisely where the DNA replication origins are located in the genome and the specific DNA sequences that are needed for DNA replication to begin at a specific site. We also want understand how DNA replication is coordinated with gene transcription. Transcription is needed to generate RNA and eventually for protein synthesis. The DNA replication machinery and the transcription machinery use the same DNA template. We intend to understand how collisions between the DNA replication and transcription machineries are avoided. These studies are relevant for understanding cancer development and progression. In cancer, there are new DNA replication origins that fire, which origins do not fire in normal cells. Moreover, in cancer cells, there are collisions between the DNA replication and transcription machineries that lead to DNA damage. Therefore, this research project may provide insights that help the development of new cancer therapies.
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.

We have already mapped origin firing sites in human cells, genome-wide, at a resolution of 1 kb, which is the maximum resolution we can achieve by our EdUseq method. We have identified one motif that is present close to the origin firing sites and that is absolutely essential for origin firing and we are in the process of identifying additional motifs.

Aim 2. Determine the mechanisms by which transcription in G1 suppresses firing of intragenic origins.

We are pursuing two mechanisms. Either the transcription machinery displaces loaded MCMs or it modifies the chromatin in a way that prevents origin firing. We are currently working to distinguish between these two possibilities.

Aim 3. Determine why forks from intergenic origins do not collapse when replicating highly transcribed genes.

We studied origin firing and nascent transcription in normal mouse cells and have observed that most transcribed genes are replicated from origins that are upstream of the gene. This ensures that DNA replication and transcription are codirectional. This could explain why forks from intergenic origins do not collapse, when replicating transcribed genes.
We anticipate that the REPLISTRESS Project, when completed, will provide a much better understanding of the process of DNA replication in human cells and how it is deregulated in cancer.
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