The polymerase δ (Polδ) is a critical enzyme responsible for synthesizing nascent DNA strands in the eukaryotic genome [1]. This enzyme replicates the lagging strand DNA with extraordinary high accuracy [2], [3], [4]. Polδ comprises four subunits: the catalytic Pold1 and the non-catalytic Pold2, Pold3, and Pold4 [5], [6]. The non-catalytic subunit, Pold2, maintains the integrity of the Polδ holo-enzyme and is vital in early murine embryogenesis [7], [8]. Although the other non-catalytic subunits, Pold3 and Pold4, are not essential, their roles in the release of stalled replication forks have been documented [9], [10], [11], [12]. Pold4 undergoes degradation during the S-phase and in response to DNA damage [13], [14], [15], resulting in the enhanced proofreading activity of Polδ [11]. The depletion of Pold4 in human lung cancer cells leads to genome instability, suggesting a potential role for Pold4 in regulating Polδ's response to genome damage threats [16], [17]. Nevertheless, the exact role of Pold4 remains to be fully elucidated.

Camptothecin (CPT) is an inhibitor of topoisomerase 1 (Top1), which mediates DNA nicking, rotation, and resealing, processes associated with topological relaxation during DNA replication [18], [19]. CPT inhibits Top1 following DNA nicking, inducing single-strand breaks (SSBs) covalently attaching Top1 at its 3’ end [18], [19]. When replication occurs across SSBs, it induces DNA double-strand breaks (DSBs) through replication run-off [20], [21], [22], leading to replication fork collapse. Eukaryotic cells have mechanisms for both repairing DSBs and suppressing their formation [23], [24]. Firstly, DSBs formed during replication fork passage across SSBs are exclusively mended via homologous recombination (HR)-mediated DSB repair [25], wherein BRCA1 plays a pivotal role in initiating this reaction [26]. The involvement of non-homologous end joining (NHEJ) in this lesion could result in abnormal chromosome fusion and is inhibited by RNF8 [25]. Secondly, fork reversal suppresses DSBs by reversing the replication fork, converting DSBs back into SSBs via forming four-way DNA junctions known as chicken foot intermediates. In this, both Poly[ADP-ribose] polymerase I (PARP1) and the proofreading exonuclease activity of Polε play essential roles [27], [28]. This fork reversal can be detected as fork slowing using a DNA fiber assay [29]. The synthetic lethality seen in cells deficient in both BRCA1 and PARP1 underscores this relationship [30], [31]. The replisome complex, which includes Polε, would be the first to encounter SSBs on the template strand, inducing fork reversal when leading strand replication stalls at SSBs. However, the mechanism behind fork reversal during lagging-strand replication stalling at SSBs remains unexplored.

In this study, we demonstrate that the fourth subunit of Polδ, Pold4, has a pivotal role in cellular tolerance to CPT by promoting safe fork slowing upon CPT treatment. Furthermore, we reveal that Pold4, in concert with Polε exonuclease and PARP1, contributes to cellular tolerance to CPT, suggesting a significant role for Pold4 in the fork reversal mediated by the PARP1-Polε exonuclease axis. This research highlights a previously unrecognized mechanism of fork reversal initiated by the Pold4 subunit of Polδ.