On March 6, 2025 our colleague Dávid Lukáč, a researcher at the Laboratory of Genome Integrity, defended his dissertation thesis on "DNA Replication and Cancer".
Supervisor: Pavel Moudrý
Opponents: Apolinar Maya-Mendoza (Danish Cancer Institut, Copenhagen, Denmark) and Juan Bautista de Sanctis
Congratulations!
DNA Replication and Cancer
Dávid Lukáč
Abstarct:
DNA replication is an essential process for duplicating the genome during cell division with precise regulation to prevent genomic instability. DNA replication proceeds bi-directionally, with the leading strand synthesized continuously by DNA polymerase, and the lagging strand synthesized discontinuously by DNA polymerase in the form of short fragments known as Okazaki fragments.The lagging strand synthesis has been identified as a targetable vulnerability in cancer cells, as evidenced by several studies. It has been hypothesized that Okazaki fragments processing might be a limiting factor in the PARP-inhibitor induced replication fork acceleration. To test this hypothesis, emetine, a compound reported as a specific lagging strand synthesis inhibitor, was employed. However, it was observed that emetine does not exclusively target lagging strand synthesis, rather it inhibits DNA replication on both leading and lagging strand. Emetine does not cause strand uncoupling, as indicated by the absence of ssDNA formation and lack of RPA32 accumulation. Furthermore, emetine did not induce DNA damage response or checkpoint activation, suggesting that DNA replication inhibition by emetine is not caused by replication stress. It was demonstrated that emetine´s inhibition of proteosynthesis is the mechanism by which it suppresses DNA replication, rather than specific inhibition of lagging strand synthesis. Poly-(ADP)-ribosylation is a post-translational modification in which chains of poly (ADP-ribose; PAR) are added to proteins by the enzyme (poly (ADP-ribose) polymerase; PARP). In the context of DNA damage, PARylation plays an important role in the cell's response to DNA damage.
Research from other groups has indicated that increased PARylation is specifically present during the S-phase at unprocessed Okazaki fragments during DNA replication. Using specific POLA1 inhibitors, such as ADA and CD437, it was observed that S-phase specific PARylation is not reduced but rather increased in response to replication stress caused by strand uncoupling. In line with these findings, mild (low-dose, long-duration) POLA1 inhibition resulted in strand uncoupling together with decrease of PARylation without triggering DNA damage response, suggesting that lagging strand synthesis could be specifically inhibited without inducing replication stress. Additionally, the depletion of POLA1 did not decrease PARylation; however, a subset of cells exhibited significantly higher PARylation and yH2AX phosphorylation. Excluding these high-signal cells confirmed a reduction in the S-phase specific PARylation after POLA1 depletion. Finally, it was proposed that for specifically investigating lagging strand synthesis inhibition, mild POLA1 inhibition is the best alternative, as emetine inhibits DNA replication primarily by preceding proteosynthesis inhibition rather than targeting lagging strand synthesis specifically.
The full thesis available here.
Photo: Denisa Pavelková
