HLTF Promotes Fork Reversal, Limiting Replication Stress Resistance and Preventing Multiple Mechanisms of Unrestrained DNA Synthesis

DNA replication stress can stall replication forks, leading to genome instability. DNA damage tolerance pathways assist fork progression, promoting replication fork reversal, translesion DNA synthesis (TLS), and repriming. In the absence of the fork remodeler HLTF, forks fail to slow following replication stress, but underlying mechanisms and cellular consequences remain elusive. Here, we demonstrate that HLTF-deficient cells fail to undergo fork reversal in vivo and rely on the primase-polymerase PRIMPOL for repriming, unrestrained replication, and S phase progression upon limiting nucleotide levels. By contrast, in an HLTF-HIRAN mutant, unrestrained replication relies on the TLS protein REV1. Importantly, HLTF-deficient cells also exhibit reduced double-strand break (DSB) formation and increased survival upon replication stress. Our findings suggest that HLTF promotes fork remodeling, preventing other mechanisms of replication stress tolerance in cancer cells. This remarkable plasticity of the replication fork may determine the outcome of replication stress in terms of genome integrity, tumorigenesis, and response to chemotherapy. [Display omitted] •HLTF mediates fork reversal in vivo, associated with DSB formation•HLTF prevents unrestrained replication driven by PRIMPOL or the TLS protein REV1•Unrestrained DNA synthesis promotes S phase progression under replication stress•HLTF loss increases cellular resistance to replication stress and ATR inhibition Under replication stress, cells deficient in the fork remodeler HLTF fail to slow DNA replication. Here, Bai et al. report that, when HLTF is disrupted, replication is completed by alternative PRIMPOL- or REV1-dependent mechanisms. Both replication modes are potentially mutagenic and lead to replication stress resistance.