DNA replication ensures the precise duplication of the genome during the S phase. The replisome, a complex molecular machinery responsible for copying DNA, can be halted by metabolic or external perturbations [1]. External perturbations, including radiation and chemical exposure, can be mutagenic and compromise genome stability. Cancer therapies often target DNA replication to inhibit cell proliferation. Camptothecin (CPT) is a natural alkaloid and anticancer agent that targets and inhibits DNA topoisomerase I (TOP1) [2]. The activity of TOP1 is linked to DNA transcription, replication, and repair. TOP1 acts on the DNA, introducing transient single-stranded breaks (SSBs) to alleviate superhelical tension that accumulates during DNA replication and transcription [3]. TOP1 cleaves the DNA backbone by forming a covalent bond between the DNA and an active site tyrosine residue on TOP1 [4]. This transient covalent linkage between TOP1 and nicked DNA is known as the TOP1 cleavage complex (TOP1cc). The nick allows the 5′ broken DNA strand to rotate around the intact DNA strand, thereby resolving DNA coiling and releasing torsional stress. When torsional stress is released, the free 5′ OH end starts a nucleophilic attack on the covalent linkage between TOP1 and DNA, ensuring re-ligation of the nick and restoration of the intact double-stranded DNA [5], [6].

The inhibitory mechanism of CPT involves binding to and stabilizing TOP1cc, which prevents the re-ligation of TOP1cc-induced DNA nicks. This results in SSBs that require repair to avoid further damage. Unrepaired SSBs can lead to replication run-off and fork collapse when a replication fork encounters a nick, leading to the formation of a double-strand end [7], [8]. At high doses of CPT, two-ended double-strand breaks (DSBs) can also arise if two TOP1ccs form in proximity on opposite strands [9], [10]. Cells employ fork reversal as a protective mechanism to prevent DSB formation in response to CPT. When a replication fork encounters a TOP1cc lesion, it can reverse and form a four-way junction in which the newly synthesized strands anneal. This results in a temporary stall of fork progression, allowing the repair of the lesion. After repair, the reversed forks restart, and DNA replication continues without leaving behind damaged DNA [10], [11], [12], [13]. However, cells cannot always prevent replication run-off, and DSBs can form even at low doses of CPT. The formation of DSBs seems to be the main reason for the cytotoxic effect of CPT [6], [10]. Owing to its toxic side effects, the clinical use of CPT has been phased out in recent years, while new TOP1 inhibitor-based drugs with potentially milder side effects are currently used or under development [14], [15].

The repair of CPT-induced DSBs primarily occurs in the S phase, favoring homologous recombination (HR) [16]. HR begins with binding the MRE11-RAD50-NBS1 (MRN) complex to the DSB. The MRN complex performs end resection to create 3’-overhangs. The single-stranded DNA (ssDNA) overhangs are quickly coated with RPA to prevent secondary structure formation and avoid cleavage by nucleases. Subsequently, the RPA molecules are replaced by RAD51 to form a nucleofilament that invades the homologous template DNA in search of a matching DNA sequence. The RAD51 filament facilitates base pairing between the invading strand and the homologous DNA template, forming a displacement loop (D-loop). A DNA polymerase then extends the 3′ end of the invading strand, using the homologous sequence as a template [17], [18], [19], [20]. RAD51 recombinase is crucial for HR because it forms nucleoprotein filaments on ssDNA and catalyzes strand exchange during DSB repair [21]. While the DSB repair function of RAD51 is well-characterized, other roles of RAD51 in replication stress survival have only recently begun to emerge. For example, RAD51 directly protects stalled replication forks [22], [23]. Similarly, BRCA1 and FANCD2 safeguard stalled replication forks by stabilizing the RAD51 filament. Moreover, RAD51 protects reversed forks caused by CPT, thereby preventing stress-induced DSB formation [24], [25], [26].

Using plasmids as a template and purified proteins, Westhorpe et al. showed that, unlike trapped TOP1ccs located on the leading strand of the fork, TOP1ccs on the lagging strand do not result in fork collapse [27]. Despite years of research, the analysis of replication forks from CPT-treated cells remains enigmatic because of the complexity of the potential DNA damage response (DDR) that the drug can trigger. We hypothesized that cells respond to TOP1 inhibitors depending on the extent and duration of DNA damage, resulting in differential DDR regulation based on the availability of the TOP1 molecules and drug concentration. Using specific molecular markers, in-depth cell cycle investigations, and time course experiments in diverse cellular models, we show that cells treated with TOP1 inhibitors activate DNA repair pathways according to the drug concentration and time of incubation. These parameters have a differential and acute impact on the ongoing DNA replication, suggesting continuous DNA damage and repair cycles. Additionally, we show that DNA damage caused by TOP1 inhibitors can be repaired, albeit only to a limited extent, without compromising cell viability. Finally, we uncover that HR-deficient cancer cells are sensitive to low-dose CPT, allowing us to discuss new therapeutic strategies for cancer subtypes with potentially fewer side effects.