HLTF Prevents G4 Accumulation and Promotes G4-induced Fork Slowing to Maintain Genome Stability

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes, but their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected, dual role for the dsDNA translocase HLTF in G4 metabolism. First, we find that HLTF is enriched at G4s in the human genome and suppresses G4 accumulation throughout the cell cycle using its ATPase activity. This function of HLTF affects telomere maintenance by restricting alternative lengthening of telomeres, a process stimulated by G4s. We also show that HLTF and MSH2, a mismatch repair factor that binds G4s, act in independent pathways to suppress G4s and to promote resistance to G4 stabilization. In a second, distinct role, HLTF restrains DNA synthesis upon G4 stabilization by suppressing PrimPol-dependent repriming. Together, the dual functions of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) of HLTF in a U2OS cell line expressing endogenously GFP-tagged HLTF, using a GFP antibody (Abcam, ab290) for IP. This U2OS cell line also constitutively expresses human codon-optimized S. pyogenes Cas9 protein. Cells are either transfected with a non-targeting control single guide RNA (sgRNA) or sgRNA against HLTF. Mouse embryonic stem cells (ESCs) expressing endogenously GFP-tagged MPP8 are used as spike-in control to normalize the ChIP-seq signal.