Buco Paula Armina V, Tandazo-Castillo Wilson, Chalk Alistair M, Pilcher Courtney, Holien Jessica K, Heierhorst Jörg, Tan Tiong Y, Koren Amnon, Smeets Monique F, Walkley Carl R
bioRxiv. 2025 Jul 21:2025.07.21.666025. doi: 10.1101/2025.07.21.666025.
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 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.
RECQL4是解旋酶RecQ家族的成员，在DNA复制和维持基因组完整性方面发挥着重要作用。RECQL4基因的突变与严重的人类疾病相关，包括罗思蒙德 - 汤姆森综合征、RAPIDALINO综合征和巴勒 - 杰罗尔德综合征。然而，我们仍未完全了解其功能和遗传相互作用。RECQL4中ATP依赖的解旋酶活性的作用仍存在争议。为了进一步了解RECQL4的功能，我们使用了小鼠模型进行全基因组正向遗传筛选，该模型紧密模拟了罗思蒙德 - 汤姆森综合征患者中发现的RECQL4突变。我们的目标是识别能够挽救与RECQL4突变相关的增殖和活力缺陷的功能丧失等位基因。通过筛选，我们确定Cullin-RING连接酶（CRL）E3的底物结合亚基KLHDC3的缺失是最显著的挽救等位基因。KLHDC3促进具有特定C末端降解基序的蛋白质的泛素介导的破坏。其缺失使RECQL4突变细胞中的细胞增殖和DNA复制速率正常化。进一步分析表明，KLHDC3的缺失导致截短的RECQL4蛋白的微量水平稳定。这个RECQL4片段包含一个对KLHDC3特异的新降解子序列，是在等位基因的Cre介导的重组后形成的。虽然这种挽救机制不适用于人类RECQL4突变，但它表明仅包含N末端480个氨基酸（包括其Sld2样结构域但缺乏ATP依赖的解旋酶结构域和整个C末端部分）的截短的RECQL4蛋白的极低染色质结合水平足以支持哺乳动物细胞中的DNA复制。这些结果表明RECQL4的ATP酶活性和解旋酶结构域对于哺乳动物中的DNA复制不是必需的。此外，我们的发现表明不太可能存在能够挽救RECQL4突变的单基因功能丧失等位基因。这表明RECQL4是DNA复制和细胞活力的必需且非冗余调节因子，并且这种活性不需要ATP依赖的解旋酶活性。
