Interstrand DNA-DNA cross-links (ICLs) are generated by endogenous processes, drugs, and environmental toxins. Understanding the cellular pathways by which various ICLs are repaired is critical to understanding their biological effects. Recent studies showed that replication-dependent repair of an ICL derived from the reaction of an abasic (AP) site with an adenine residue (dA) on the opposing strand of duplex DNA proceeds via a novel mechanism in which the DNA glycosylase NEIL3 unhooks the ICL. Here we examined the ability of the glycosylase domain of murine NEIL3 (MmuNEIL3-GD) to unhook dA-AP ICLs. The enzyme selectively unhooks the dA-AP ICL located at the duplex/single-strand junction of splayed duplexes that model the strand-separated DNA at the leading edge of a replication fork. We show that the ability to unhook the dA-AP ICL is a specialized function of NEIL3 as this activity is not observed in other BER enzymes. Importantly, NEIL3 only unhooks the dA-AP ICL when the AP residue is located on what would be the leading template strand of a model replication fork. The same specificity for the leading template strand was observed with a 5,6-dihydrothymine monoadduct, demonstrating that this preference is a general feature of the glycosylase and independent of the type of DNA damage. Overall, the results show that the glycosylase domain of NEIL3, lacking the C-terminal NPL4 and GRF zinc finger motifs, is competent to unhook the dA-AP ICL in splayed substrates and independently enforces important substrate preferences on the repair process.

中文翻译：

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DNA糖基化酶NEIL3脱钩DNA叉结构上的链间交联。

链间DNA-DNA交联（ICL）是由内源性过程，药物和环境毒素产生的。了解修复各种ICL的细胞途径对于了解其生物学效应至关重要。最近的研究表明，复制的修复依赖于无碱基（AP）位点与双链DNA相对链上的腺嘌呤残基（dA）反应的ICL通过一种新的机制进行，其中DNA糖基化酶NEIL3解除了ICL的连接。在这里，我们检查了鼠NEIL3（MmuNEIL3-GD）的糖基化酶结构域脱钩dA-AP ICL的能力。该酶选择性地脱钩位于展开的双链体的双链体/单链交界处的dA-AP ICL，该双链体/双链体在复制叉的前端模拟了链分离的DNA。我们表明解钩dA-AP ICL的能力是NEIL3的专门功能，因为在其他BER酶中未观察到此活性。重要的是，仅当AP残基位于模型复制叉的主要模板链上时，NEIL3才会解钩dA-AP ICL。用5，6-二氢胸腺嘧啶单加合物观察到对前导模板链的相同特异性，表明该偏好是糖基化酶的一般特征，并且与DNA损伤的类型无关。总体而言，结果表明，缺少C末端NPL4和GRF锌指基序的NEIL3的糖基化酶结构域能够在展开的底物中脱钩dA-AP ICL，并在修复过程中独立执行重要的底物偏好。重要的是，仅当AP残基位于模型复制叉的主要模板链上时，NEIL3才会解钩dA-AP ICL。用5，6-二氢胸腺嘧啶单加合物观察到对前导模板链的相同特异性，表明该偏好是糖基化酶的一般特征，并且与DNA损伤的类型无关。总体而言，结果表明，缺少C末端NPL4和GRF锌指基序的NEIL3的糖基化酶结构域能够在展开的底物中脱钩dA-AP ICL，并在修复过程中独立执行重要的底物偏好。重要的是，仅当AP残基位于模型复制叉的主要模板链上时，NEIL3才会解钩dA-AP ICL。用5，6-二氢胸腺嘧啶单加合物观察到对前导模板链的相同特异性，表明该偏好是糖基化酶的一般特征，并且与DNA损伤的类型无关。总体而言，结果表明，缺少C末端NPL4和GRF锌指基序的NEIL3的糖基化酶结构域能够在展开的底物中脱钩dA-AP ICL，并在修复过程中独立执行重要的底物偏好。6-二氢胸腺嘧啶单加合物，证明这种偏好是糖基化酶的一般特征，与DNA损伤的类型无关。总体而言，结果表明，缺少C末端NPL4和GRF锌指基序的NEIL3的糖基化酶结构域能够在展开的底物中脱钩dA-AP ICL，并在修复过程中独立执行重要的底物偏好。6-二氢胸腺嘧啶单加合物，证明这种偏好是糖基化酶的一般特征，与DNA损伤的类型无关。总体而言，结果表明，缺少C末端NPL4和GRF锌指基序的NEIL3的糖基化酶结构域能够在展开的底物中脱钩dA-AP ICL，并在修复过程中独立执行重要的底物偏好。