Background

A basic principle of the cell cycle is to ensure that mitosis follows the completion of DNA replication. During S phase, various replication stresses, such as DNA lesions, conflicts with transcription, and tightly bound protein complexes, interfere with DNA replication and cause a genomic instability related to various genetic diseases (Muñoz and Méndez, 2017). To prevent or minimize the deleterious consequences of replication stress, cells have evolved DNA damage response mechanisms, including DNA repair pathways (Yoshida and Fujita, 2021). Stalled replication forks activate checkpoint pathways that halt the cell cycle progression (Lemmens and Lindqvist, 2019; Mocanu and Chan, 2021). However, it has been revealed that upon mild replication stress that modestly slows down the replication fork, cells can enter the mitosis with some under-replicated DNA (Lukas et al., 2011; Bertolin et al., 2020; Lezaja and Altmeyer, 2021; Mocanu and Chan, 2021). Mild replication stress causes incomplete replication without activating G2/M checkpoint, especially at difficult-to-replicate regions such as chromosomal fragile sites, centromeres, and telomeres (Sarlós et al., 2017; Özer and Hickson, 2018; Lokanga et al., 2021). Such regions with under-replicated DNA activate mitotic DNA synthesis (MiDAS) in early mitosis to complete DNA replication, whereas some regions remain incomplete and will be resolved in the subsequent cell cycle (Minocherhomji et al., 2015; Özer and Hickson, 2018; Bertolin et al., 2020; Lezaja and Altmeyer, 2021).

MiDAS is a kind of homology-directed repair DNA synthesis that involves repair of stalled replication intermediates and POLD3 (an accessory subunit of polymerase δ)-dependent conservative DNA synthesis, analogous to break-induced replication (Kockler et al., 2021; Epum and Haber, 2022). Basic protocols for detection of MiDAS were originally developed by Hickson and colleagues (Minocherhomji et al., 2015; Bhowmick et al., 2016; Garribba et al., 2018). Briefly, cells are treated with low dose (0.4 μM) aphidicolin and RO-3306. Aphidicolin, a DNA polymerase inhibitor, slows down DNA replication and induces under-replication, while RO-3306, an inhibitor of cyclin-dependent kinase 1, arrests cells at G2 phase. Cells are then released into mitosis in the presence of a nucleoside analog ethynyl deoxyuridine (EdU) to label newly synthesized DNA. Following click reaction, MiDAS is visualized as EdU foci on prophase chromatin by fluorescence microscopy.

Here, we describe a detailed method for detection of MiDAS at under-replicated regions evoked by repetitive lacO sequences in human U2OS 40-2-6 cells (Figure 1). U2OS 40-2-6 cell, a U2OS-derived cell line carrying lacO array and estrogen receptor (ER^T2)–fused LacI gene, was generated to investigate DNA damage response to replication stress induced by tight DNA–protein complexes on the human chromosome (Ishimoto et al., 2021). Treatment with 4-hydroxytamoxifen (4-OHT) induces rapid formation of lacO–LacI complexes that interfere with DNA replication. Notably, we also found that even in the absence of LacI, repetitive lacO sequences are intrinsically difficult to replicate, remaining under-replicated until mitosis and consequently triggering MiDAS. In the method presented here, nascent DNA is labeled by another nucleoside analog bromodeoxyuridine (BrdU), and lacO array is visualized by immunostaining of LacI proteins bound to the array. In our ER–LacI induction system, replication stress is induced at the specific chromosomal locus using the sequence-specific DNA-binding proteins. MiDAS at the specific locus can therefore be easily detected using common immunostaining methods, without fluorescent in situ hybridization (FISH) used in the protocol developed by Hickson and colleagues. This protocol may be optimized for other repetitive fragile sites where MiDAS functions, such as telomeres, centromeres, and ribosomal DNA arrays.

Figure 1. Flowchart of the assay for detection of mitotic DNA synthesis at the lacO array.