HLTF resolves G4s and promotes G4-induced replication fork slowing to maintain genome stability.

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability. [Display omitted] • HLTF is enriched at G4s and suppresses G4 accumulation in all cell cycle phases • HLTF translocase directly resolves G4 structures using its ATPase activity • HLTF restrains DNA synthesis and suppresses PrimPol in response to G4 stabilization • HLTF protects cells from DNA damage and growth defects induced by G4 stabilization Bai et al. identify an unexpected function for the helicase-like transcription factor, HLTF, in suppressing G-quadruplexes (G4s), a DNA secondary structure that can threaten genome stability. HLTF uses its DNA translocase activity to unfold G4s in dsDNA and to restore the DNA duplex, and it restrains DNA synthesis at G4s. [ABSTRACT FROM AUTHOR]