FANCM 是一种 DNA 修复蛋白，可识别停滞的复制叉并招募下游修复因子。 FANCM 活性对于利用端粒替代延长 (ALT) 机制的癌细胞的生存也至关重要。 FANCM 通过其对分支 DNA 结构的强亲和力，有效识别基因组或端粒中停滞的复制叉。在这项研究中，我们证明 N 端易位酶结构域驱动这种特定的分支 DNA 识别。转位酶内的 Hel2i 子结构域对于有效的底物接合至关重要，并将 DNA 结合与催化 ATP 依赖性分支迁移结合起来。 Hel2i 的去除或该结构域内关键 DNA 结合残基的突变降低了 FANCM 对连接 DNA 的亲和力并消除了分支迁移活性。重要的是，这些突变的 FANCM 变体未能挽救细胞周期停滞、端粒相关复制应激或耗尽内源 FANCM 的 ALT 阳性癌细胞的致死率。我们的结果表明，Hel2i 结构域是 FANCM 正确接合 DNA 底物的关键，因此通过抑制 ALT 通路的过度激活，在其肿瘤抑制功能中发挥着重要作用。© 作者 2024。由牛津大学出版社出版代表核酸研究。

FANCM is a DNA repair protein that recognizes stalled replication forks, and recruits downstream repair factors. FANCM activity is also essential for the survival of cancer cells that utilize the Alternative Lengthening of Telomeres (ALT) mechanism. FANCM efficiently recognizes stalled replication forks in the genome or at telomeres through its strong affinity for branched DNA structures. In this study, we demonstrate that the N-terminal translocase domain drives this specific branched DNA recognition. The Hel2i subdomain within the translocase is crucial for effective substrate engagement and couples DNA binding to catalytic ATP-dependent branch migration. Removal of Hel2i or mutation of key DNA-binding residues within this domain diminished FANCM's affinity for junction DNA and abolished branch migration activity. Importantly, these mutant FANCM variants failed to rescue the cell cycle arrest, telomere-associated replication stress, or lethality of ALT-positive cancer cells depleted of endogenous FANCM. Our results reveal the Hel2i domain is key for FANCM to properly engage DNA substrates, and therefore plays an essential role in its tumour-suppressive functions by restraining the hyperactivation of the ALT pathway.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.