Uncovering the mechanism of stress-resistant DNA replication in cancer cells using a modern single-molecule technique

C.E. Credits: P.A.C.E. CE Florida CE
Speakers
  • Postdoctoral Fellow, Chemical and Systems Biology, Stanford University School of Medicine
    Biography
      When I obtained my Pharmacist (Pharm. D) degree several years ago, I asked myself what I really wanted to do and what career path would allow me to reach my goals. The answer was clear and obvious in my mind: I wanted to do research and work on understanding cancer development and finding new ways to fight cancer. To this end, I joined the faculty of pharmacy of Montpellier, France, where I obtained a Master's degree in research and development of pharmaceutical drugs. At that time I worked preclinical drug development at Sanofi pharmaceuticals in France. I was in charge of establishing new in vitro/in silico models for study of drugs pharmacokinetics. After that, the next step was to obtain a PhD degree. I joined Dr. Domenico MAIORANO's group in Montpellier, France, and I worked mainly on establishing a functional interaction between the DNA damage tolerance (DDT) and the DNA damage response (DDR) in cells. My work uncovered a novel mechanism responsible for the silencing of the DNA damage checkpoint in early embryos, and explained how translesion DNA polymerases are recruited to DNA damage foci. After a successful PhD concluded by obtaining the Best Young Scientist Award in Cancer research, I joined Professor Karlene Cimprich's lab at Stanford to acquire new experience, new way of working and thinking, and to learn new techniques. Most importantly, this lab is one of top labs in the field of replication stress and replication fork reversal. During my postdoc, I learned new innovative techniques; high-content microscopy (QIBC), single-molecule methods (DNA spreading and DNA combing), high throughput CRIPR-Cas9 genome-wide screening, and bioinformatics. I worked on understanding how cancer cells tolerate replication stress. I am particularly interested in studying several aspects of replication stress in cancer stem cells. This sub-population of cancer cells characterized by its amazingly high resistance to stress inducing agents but the basis for this is unknown. The next step in my career will be going back to biotech company to do a more transnational work in order to discover new innovative therapies to cure/regress cancer. Immune therapy is one of the very promising ways to do that.

    Abstract:

    Accurate DNA replication is essential to transmit the genetic information from one generation to another. However, replication is frequently challenged by barriers that originate from exogenous and endogenous sources. These obstacles perturb normal DNA replication by slowing or stalling replication fork progression, a process known as replication stress. High intrinsic replication stress is a hallmark of cancer. Intriguingly, cancer cells can proliferate and replicate their DNA under replication stress leading to therapy resistance, but how they do so is not understood.

    One of the mechanisms that helps cells to deal with replication stress and restart DNA replication is fork reversal, a pathway that can facilitate replication stress relief. This mechanism involves replication fork remodelers, among them the DNA translocase HLTF. Inactivation of several DNA translocases leads to defective fork restart, slowed fork progression, hypersensitivity to genotoxic agents, and increased double-strand break formation. However, we showed that HLTF-deficient cells display several phenotypes unique among known remodelers, including unrestrained replication fork progression and resistance to replication stress. Using a novel single-cell imaging technique, we have shown that these cells move faster through S-phase under replication stress. To further investigate this phenotype, we took advantage of a single molecule technique called “DNA spreading”. This amazing technique allows us to directly visualize replication fork progression in vivo at a single molecule level through labeling the replicating DNA using different nucleotide analogues. By combining this technique to genetic manipulations of our cells (CRISPR knock out), we have shown that all these phenotypes were dependent on the primase/polymerase PrimPol, mediating discontinuous DNA replication in HLTF-deficient cells. We hypothesize that, in the absence of HLTF, PrimPol mediates active fork repriming downstream of the block leaving gaps behind the fork.

    Altogether, our data suggest that the molecular composition of the replication fork machinery is changed in HLTF-deficient cells, allowing the recruitment of specialized polymerases (i.e. PrimPol) that help the cells to bypass replication obstacles, progress unrestrained under replication stress and proliferate. As HLTF is silenced in many cancers, this phenotype could explain how cancer cells sustain the stress-resistant replication that drives their proliferation and resistance to therapy. Therefore, understanding how HLTF-deficient cells deal with replication stress at the molecular and cellular levels is paramount to improve cancer therapy and DNA spreading will largely contribute to shed the light on our understanding of replication under stress conditions.

    Learning Objectives:

    1. Understand how replication stress tolerance can lead to cancer therapy resistance.

    2. Explain an elegant single-molecule approach to study replication stress in vivo in cells.


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