Thesis assessment by Hana Hanzlikova
The submitted PhD thesis of Barbora Boleslavska entitled "Molecular mechanisms of genome integrity maintenance under conditions of replication stress" focuses on the characterization of molecular events in response to replication stress. Replication stress is recognized as a critical threat to genome stability and as an important driver of cancer formation. The key source of replication stress is the conflicts between transcription and replication and the formation of R-loops, which Barbora revealed fundamental insights into. The work describes several very important discoveries and consists of a total of three papers, one of which has Barbora as the first author and was published in 2022 in one of the leading journals in the field, Nuclei Acids Research.
In addition, this thesis includes two articles Barbora co-authored, one published in Molecular Cell in 2020, the other in International Journal of Molecular Sciences in 2021.
The thesis contains an introduction into the general topic of replication stress, collisions between DNA replication and transcription complexes and R-loops formation and provides up-to-date overview. Overall, the author covers all relevant issues and the literature is mostly properly cited. However, in my opinion, the thesis is unfortunately not very well structured and is difficult for readers without background knowledge to read and understand. I would appreciate a better balance in the introductory chapter, which would promote a better flow of the text. Some parts are too detailed and others too brief and deserve a more detailed description. Sections from introduction containing data from the author's papers should be better placed within the results and discussion chapter.
The main goals of the study are to contribute to a better understanding of the occurrence of RNA-loops arising during replication-transcription collisions and their removal so that they do not disturb the genome stability. The selected scientific questions are original and important and show substantial scientific value.
In the results and discussion chapter, Barbora tried to link published works, which is once again a bit unfortunate and difficult for the reader to navigate. I would recommend results to start focusing on the description and optimization of novel protocols for the detection and specific identification of R-loop-associated proteins, which is a great achievement of the author and has significantly contributed to two co-authored publications. Moreover, thanks to this sophisticated method that Barbora established, she identified the DDX17 helicase and its new role in the resolution of R-loop-mediated transcription-replication conflicts, which is her main work. The discussion shows that the author has an overview of the topic and is able to formulate a hypothesis for further
2
testing based on the knowledge gained. The research described in this work will be of interest to a wide range of scientists working in the fields of DNA replication stress, genome stability and cancer.
In this direction, the work brings a whole range of new findings, which in a substantial and non-trivial way develop the submitted work to high quality. This is further supported by publications in prestigious journals and demonstrates that the author has mastered advanced methods of molecular and cell biology and is able to design an experimental procedure.
In summary, this thesis describes an extensive set of studies on the molecular mechanisms of R-loop-mediated transcription-replication conflicts resolution. Although the thesis has minor flaws and is not written in a complete flow and logical structure, is it an experimental work of a high standard and I am pleased to recommend the submitted dissertation for the defense and award of the PhD.
Questions:
1. What is the difference between R-loops that accumulate after CPT or HU induction?
Do you know and/or wonder if DDX17 is also enriched at R-loops after HU treatment?
2. I miss a recent citation on the DDX18 helicase and its role in maintenaning genome stability (Lin WL et al., Cell Rep. 2022 Jul 19;40(3):1110890). Does DDX17 also accumulate at sites of DNA damage? Does DDX17-depletion-induced R-loop accumulation affect DNA repair? Are DDX17-depleted cells more sensitive to DNA damage (eg. IR/HU/CPT)?
3. Please explain in more detail why a PARP inhibitor promotes replication restart. The PARP inhibitor also induces more DNA-single strand breaks which might lead to formation of R-loops. Did you find PARP1 enriched at R-loops in your experiments?
What is the role for PARP1 at R-loops?
4. Which another potential hit from your MS analysis would you like to follow if possible and why?
Prague, 25th February 2023 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Hana Hanzlikova, PhD
