Abstract
Cells must replicate and segregate their DNA to daughter cells accurately to maintain genome stability and prevent cancer. DNA replication is usually fast and accurate, with intrinsic (proofreading) and extrinsic (mismatch repair) error-correction systems. However, replication forks slow or stop when they encounter DNA lesions, natural pause sites, and difficult-to-replicate sequences, or when cells are treated with DNA polymerase inhibitors or hydroxyurea, which depletes nucleotide pools. These challenges are termed replication stress, to which cells respond by activating DNA damage response signaling pathways that delay cell cycle progression, stimulate repair and replication fork restart, or induce apoptosis. Stressed forks are managed by rescue from adjacent forks, repriming, translesion synthesis, template switching, and fork reversal which produces a single-ended double-strand break (seDSB). Stressed forks also collapse to seDSBs when they encounter single-strand nicks or are cleaved by structure-specific nucleases. Reversed and cleaved forks can be restarted by homologous recombination (HR), but seDSBs pose risks of mis-rejoining by non-homologous end-joining (NHEJ) to other DSBs, causing genome rearrangements. HR requires resection of broken ends to create 3’ single-stranded DNA for RAD51 recombinase loading, and resected ends are refractory to repair by NHEJ. This Mini Review highlights mechanisms that help maintain genome stability by promoting resection of seDSBs and accurate fork restart by HR.
Highlights
Cells maintain relatively stable genomes during cell division to prevent accumulation of potentially oncogenic mutations
It remains unclear how cells choose among various lesion bypass and fork restart pathways, it is likely that the types of blocking lesions, and the extent and duration of replication stress are determining factors
How might cells mitigate risks of classical non-homologous end-joining (cNHEJ) acting on resulting singleended double-strand break (seDSB)? A recently described mechanism operates at forks stalled by collision with opposing transcription R-loops in which MUS81 cleaves the fork, and the resulting ends are rejoined by LIG4XRCC4 with assistance by RAD52-mediated strand annealing and PolD3, a non-catalytic subunit of Pol d (Chappidi et al, 2020)
Summary
Cells maintain relatively stable genomes during cell division to prevent accumulation of potentially oncogenic mutations. Most DNA lesions block replicative polymerases (Pol ε, Pol δ), causing fork stalling and fork collapse, and cells manage this replication stress by activating S phase-specific DDR pathways. Replication stress is caused by conflicts with R-loops formed during transcription, at fragile sites, telomeres, and ribosomal DNA, and by proteins that bind tightly to DNA (Ivessa et al, 2003; Bermejo et al, 2012; Kotsantis et al, 2016; Billard and Poncet, 2019; Gomez-Gonzalez and Aguilera, 2019). These so-called ‘difficult-to-replicate’ sequences are encountered by replisomes in every S phase.
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