DNA interstrand cross-links (ICLs) are a form of DNA damage that requires the interplay of a number of repair proteins including those of the Fanconi anemia (FA) and the homologous recombination (HR) pathways. Pathogenic variants in the essential gene BRCA2/FANCD1, when monoallelic, predispose to breast and ovarian cancer, and when biallelic, result in a severe subtype of Fanconi anemia. BRCA2 function in the FA pathway is attributed to its role as a mediator of the RAD51 recombinase in HR repair of programmed DNA double-strand breaks (DSB). BRCA2 and RAD51 functions are also required to protect stalled replication forks from nucleolytic degradation during response to hydroxyurea (HU). While RAD51 has been shown to be necessary in the early steps of ICL repair to prevent aberrant nuclease resection, the role of BRCA2 in this process has not been described. Here, based on the analysis of BRCA2 DNA-binding domain (DBD) mutants (c.8488-1G>A and c.8524C>T) discovered in FA patients presenting with atypical FA-like phenotypes, we establish that BRCA2 is necessary for the protection of DNA at ICLs. Cells carrying BRCA2 DBD mutations are sensitive to ICL-inducing agents but resistant to HU treatment consistent with relatively high HR repair in these cells. BRCA2 function at an ICL protects against DNA2–WRN nuclease–helicase complex and not the MRE11 nuclease that is implicated in the resection of HU-induced stalled replication forks. Our results also indicate that unlike the processing at HU-induced stalled forks, the function of the SNF2 translocases (SMARCAL1, ZRANB3, or HLTF), implicated in fork reversal, are not an integral component of the ICL repair, pointing to a different mechanism of fork protection at different DNA lesions.

中文翻译：

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BRCA2 在响应羟基脲和 DNA 链间交联的复制叉保护中发挥独特作用。


DNA 链间交联 (ICL) 是 DNA 损伤的一种形式，需要许多修复蛋白相互作用，包括范可尼贫血 (FA) 和同源重组 (HR) 途径的修复蛋白。必需基因BRCA2/FANCD1 的致病性变异，当为单等位基因时，易患乳腺癌和卵巢癌，当为双等位基因时，会导致严重的范可尼贫血亚型。 BRCA2 在 FA 途径中的功能归因于其在程序性 DNA 双链断裂 (DSB) 的 HR 修复中作为 RAD51 重组酶介导的作用。 BRCA2 和 RAD51 功能也需要保护停滞的复制叉在羟基脲 (HU) 响应过程中免受溶核降解。虽然 RAD51 已被证明在 ICL 修复的早期步骤中对于防止异常核酸酶切除是必要的，但 BRCA2 在此过程中的作用尚未描述。在此，基于对在表现出非典型 FA 样表型的 FA 患者中发现的BRCA2 DNA 结合域 (DBD) 突变体（c.8488-1G>A 和 c.8524C>T）的分析，我们确定 BRCA2 对于ICL 上 DNA 的保护。携带BRCA2 DBD 突变的细胞对 ICL 诱导剂敏感，但对 HU 治疗有抵抗力，这与这些细胞中相对较高的 HR 修复一致。 ICL 上的 BRCA2 功能可以防止 DNA2-WRN 核酸酶-解旋酶复合物的侵害，但不能防止与 HU 诱导的停滞复制叉切除有关的 MRE11 核酸酶的侵害。 我们的结果还表明，与 HU 诱导的停滞叉的处理不同，与叉反转有关的 SNF2 易位酶（SMARCAL1、ZRANB3 或 HLTF）的功能不是 ICL 修复的组成部分，这表明了不同的机制不同 DNA 损伤处的叉保护。