Mapping Origins of DNA Replication
DNA replication begins at specific positions on chromosomes that are called DNA replication origins. There are about five to ten thousand origins in normal human cells. Analyses from our laboratory suggest that cancer cells fire about one to two thousand more DNA replication origins that normal cells and that the firing of these cancer-specific origins is the underlying cause of oncogene-induced DNA replication stress [1].
Analysis of DNA replication origins by our laboratory and many other laboratories is still ongoing. In collaboration with the group of Professor Deborah Stroka at the Department for BioMedical Research, we have been able to map the position of DNA replication origins on the genome in vivo, specifically in the regenerating livers of mice [2]. These studies uncovered a defect in origin firing in aged mice, which may explain why ageing is associated with a weak regenerative potential. We now aim to map DNA replication origins at a ten to hundred-fold higher resolution than what we have currently achieved with the goal of identifying DNA sequence elements that define the position of origins on the genome.
Understanding the Synergistic Lethality of PARP1 and ATR Inhibitors
Protecting the replisome, the machinery that replicates the DNA of cells, is important for accurate DNA replication. In cancer cells, collisions of the replisome with the transcription machinery are an important driver of oncogene-induced DNA replication stress (OiRS) [1]. We showed recently that PARP1 protects DNA replication forks from collisions with transcription complexes [3]. When PARP1 is inhibited, the replisome may collapse after colliding with a transcription complex; such collapsed replisomes are detected by ATR, which facilitates replication restart. We have determined that inhibiting both PARP1 and ATR compromises survival of cancer cells in a synergistic manner, whereas normal cells tolerate much better the combined inhibition of PARP1 and ATR. In this project, we aim to understand the mechanistic basis for the synergy obtained when both PARP1 and ATR are inhibited in cancer cells.
Liver Regeneration in Aged Mice
Together with the laboratory of Professor Deborah Stroka we proposed that normal hepatocytes accumulate DNA damage during ageing and that some of this damage is not repaired, but remains cryptic, only to be discovered when the hepatocytes start replicating their DNA during liver regeneration [2]. The nature of this cryptic DNA damage is still unclear. We aim to use state-of-the-art methods to identify DNA damage lesions in hepatocytes from aged mice. If successful, these studies will reveal what type of DNA lesions can remain undetected in cells in vivo. We hypothesize that it will be the accumulation of this type of DNA lesions that drives ageing in humans.
[1] Macheret, Halazonetis. (2018). Intragenic origins due to short G1 phases underlie oncogene−induced DNA replication stress. Nature 555, 112−116.
[2] Rossetti, Dommann, Karamichali, Dionellis, Asensio Aldave, Yarahmadov, Rodriguez-Carballo, Keogh, Candinas, Stroka, Halazonetis. (2024). In vivo DNA replication dynamics unveil aging-dependent replication stress. Cell 187, 6220-6234.
[3] Petropoulos, Karamichali, Rossetti, Freudenmann, Iacovino, Dionellis, Sotiriou, Halazonetis. (2024). Transcription-replication conflicts underlie sensitivity to PARP inhibitors. Nature 628, 433-441.
