DNA interstrand crosslink (ICL) repair requires a complex network of DNA damage response pathways. Removal of the ICL lesions is vital, as they are physical barriers to essential DNA processes that require the separation of duplex DNA, such as replication and transcription. The Fanconi anemia (FA) pathway is the principal mechanism for ICL repair in metazoans and is coupled to DNA replication. In Saccharomyces cerevisiae, a vestigial FA pathway is present, but ICLs are predominantly repaired by a pathway involving the Pso2 nuclease, which is hypothesized to use its exonuclease activity to digest through the lesion to provide access for translesion polymerases. However, Pso2 lacks translesion nuclease activity in vitro, and mechanistic details of this pathway are lacking, especially relative to FA. We recently identified the Hrq1 helicase, a homolog of the disease-linked enzyme RecQ-like helicase 4 (RECQL4), as a component of Pso2-mediated ICL repair. Here, using genetic, biochemical, and biophysical approaches, including single-molecule FRET (smFRET)– and gel-based nuclease assays, we show that Hrq1 stimulates the Pso2 nuclease through a mechanism that requires Hrq1 catalytic activity. Importantly, Hrq1 also stimulated Pso2 translesion nuclease activity through a site-specific ICL in vitro. We noted that stimulation of Pso2 nuclease activity is specific to eukaryotic RecQ4 subfamily helicases, and genetic and biochemical data suggest that Hrq1 likely interacts with Pso2 through their N-terminal domains. These results advance our understanding of FA-independent ICL repair and establish a role for the RecQ4 helicases in the repair of these detrimental DNA lesions.

中文翻译：

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酵母 Hrq1 解旋酶刺激 Pso2 转损伤核酸酶活性，从而促进 DNA 链间交联修复。

DNA 链间交联 (ICL) 修复需要复杂的 DNA 损伤反应途径网络。去除 ICL 损伤至关重要，因为它们是需要分离双链 DNA 的基本 DNA 过程（例如复制和转录）的物理障碍。范可尼贫血 (FA) 途径是后生动物 ICL 修复的主要机制，并与 DNA 复制相关。在酿酒酵母中，存在残留的 FA 途径，但 ICL 主要通过涉及 Pso2 核酸酶的途径进行修复，推测 Pso2 核酸酶利用其核酸外切酶活性消化病变部位，为跨病变聚合酶提供通道。然而，Pso2 在体外缺乏跨损伤核酸酶活性，并且缺乏该途径的机制细节，尤其是与 FA 相关的机制细节。我们最近鉴定了 Hrq1 解旋酶，疾病相关酶 RecQ 样解旋酶 4 (RECQL4) 的同源物，作为 Pso2 介导的 ICL 修复的组成部分。在这里，我们使用遗传、生物化学和生物物理方法，包括单分子 FRET (smFRET) 和基于凝胶的核酸酶测定，表明 Hrq1 通过需要 Hrq1 催化活性的机制刺激 Pso2 核酸酶。重要的是，Hrq1 还通过体外位点特异性 ICL 刺激 Pso2 跨损伤核酸酶活性。我们注意到，Pso2 核酸酶活性的刺激是真核 RecQ4 亚家族解旋酶特有的，遗传和生化数据表明 Hrq1 可能通过其 N 末端结构域与 Pso2 相互作用。这些结果增进了我们对不依赖 FA 的 ICL 修复的理解，并确定了 RecQ4 解旋酶在修复这些有害 DNA 损伤中的作用。