Fernandez Anthony, Zhou Tingting, Esworthy Steven, Shen Changxian, Liu Helen, Hess Jessica D, Yuan Hang, Liu Nian, Shi Guojun, Zhou Mian, Kosiyatrakul Settapong, Gaur Vikas, Sommers Joshua, Edelman Winfried, Li Guo-Min, Brosh Robert, Chai Weihang, Lee Marietta Y W T, Zhang Dong, Schildkraut Carl, Zheng Li, Shen Binghui
Departments of Cancer Genetics and Epigenetics Beckman Research Institute, City of Hope, Duarte, CA 91007, USA.
These authors contributed equally to this work.
bioRxiv. 2025 Apr 6:2025.04.04.647332. doi: 10.1101/2025.04.04.647332.
G-quadruplexes (G4s) are widely existing stable DNA secondary structures in mammalian cells. A long-standing hypothesis is that timely resolution of G4s is needed for efficient and faithful DNA replication. , G4s may be unwound by helicases or alternatively resolved via DNA2 nuclease mediated G4 cleavage. However, little is known about the biological significance and regulatory mechanism of the DNA2-mediated G4 removal pathway. Here, we report that DNA2 deficiency or its chemical inhibition leads to a significant accumulation of G4s and stalled replication forks at telomeres, which is demonstrated by a high-resolution technology: Single molecular analysis of replicating DNA (SMARD). We further identify that the DNA repair complex MutSα (MSH2-MSH6) binds G4s and stimulates G4 resolution via DNA2-mediated G4 excision. MSH2 deficiency, like DNA2 deficiency or inhibition, causes G4 accumulation and defective telomere replication. Meanwhile, G4-stabilizing environmental compounds block G4 unwinding by helicases but not G4 cleavage by DNA2. Consequently, G4 stabilizers impair telomere replication and cause telomere instabilities, especially in cells deficient in DNA2 or MSH2.
G-四链体（G4s）是哺乳动物细胞中广泛存在的稳定DNA二级结构。一个长期存在的假说是，及时解开G4s对于高效且准确的DNA复制是必需的。G4s可能会被解旋酶解开，或者通过DNA2核酸酶介导的G4切割来解开。然而，对于DNA2介导的G4去除途径的生物学意义和调控机制知之甚少。在此，我们报告DNA2缺陷或其化学抑制会导致G4s显著积累以及端粒处的复制叉停滞，这通过一种高分辨率技术得以证明：复制DNA的单分子分析（SMARD）。我们进一步确定DNA修复复合物MutSα（MSH2-MSH6）结合G4s并通过DNA2介导的G4切除刺激G4的解开。MSH2缺陷与DNA2缺陷或抑制一样，会导致G4积累和端粒复制缺陷。同时，稳定G4的环境化合物会阻止解旋酶解开G4，但不会阻止DNA2切割G4。因此，G4稳定剂会损害端粒复制并导致端粒不稳定，尤其是在DNA2或MSH2缺陷的细胞中。
