Minute amounts of helicase-deficient truncated RECQL4 are sufficient for DNA replication

RECQL4 is a member of the RecQ family of helicases, playing essential roles in DNA replication and maintaining genome integrity. Mutations in RECQL4 are linked to severe human diseases, including Rothmund-Thomson Syndrome, RAPIDALINO Syndrome, and Baller-Gerold Syndrome. However, we still do not fully understand its functions and genetic interactions. The role of the ATP-dependent helicase activity in RECQL4 remains controversial. To understand RECQL4’s functions further, we conducted a genome-wide forward genetic screen using murine models that closely mimic the RECQL4 mutations found in patients with Rothmund-Thomson syndrome. Our goal was to identify loss-of-function alleles that could rescue the proliferation and viability defects associated with RECQL4 mutation. From our screening we identified the loss of KLHDC3, a substrate-binding subunit of the Cullin-RING ligase (CRL) E3, as the most significant rescue allele. KLHDC3 facilitates the ubiquitin-mediated destruction of proteins with specific C-terminal degron motifs. Its loss normalized cell proliferation and DNA replication rates in cells with mutated RECQL4. Further analysis revealed that the loss of KLHDC3 led to the stabilization of minute levels of a truncated RECQL4 protein. This RECQL4 fragment contained a neo-degron sequence specific for KLHDC3, formed after Cre-mediated recombination of the Recql4 ^fl allele. Although this rescue mechanism does not apply to human RECQL4 mutations, it shows that very low chromatin-bound levels of a truncated RECQL4 protein—comprising only the N-terminal 480 amino acids, including its Sld2-like domain but lacking the ATP-dependent helicase domain and the entire C-terminal portion—are sufficient to support DNA replication in mammalian cells. These results demonstrate that the ATPase activity and helicase domain of RECQL4 are not essential for DNA replication in mammals. Furthermore, our findings suggest that there are unlikely to be monogenic loss-of-function alleles that can rescue RECQL4 mutations. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require the ATP dependent helicase activity.