The superfamily 2 helicase XPD is a central component of the general transcription factor II H (TFIIH), which is essential for transcription and nucleotide excision DNA repair (NER). Within these two processes, the helicase function of XPD is vital for NER but not for transcription initiation, where XPD acts only as a scaffold for other factors. Using cryo-EM, we deciphered one of the most enigmatic steps in XPD helicase action: the active separation of double-stranded DNA (dsDNA) and its stalling upon approaching a DNA interstrand cross-link, a highly toxic form of DNA damage. The structure shows how dsDNA is separated and reveals a highly unusual involvement of the Arch domain in active dsDNA separation. Combined with mutagenesis and biochemical analyses, we identified distinct functional regions important for helicase activity. Surprisingly, those areas also affect core TFIIH translocase activity, revealing a yet unencountered function of XPD within the TFIIH scaffold. In summary, our data provide a universal basis for NER bubble formation, XPD damage verification and XPG incision.

超家族 2 解旋酶 XPD 是一般转录因子 II H （TFIIH） 的核心组分，对转录和核苷酸切除 DNA 修复 （NER） 至关重要。在这两个过程中，XPD 的解旋酶功能对 NER 至关重要，但对转录起始至关重要，其中 XPD 仅充当其他因子的支架。使用冷冻电镜，我们破译了 XPD 解旋酶作用中最神秘的步骤之一：双链 DNA （dsDNA） 的主动分离及其在接近 DNA 链间交联（一种剧毒的 DNA 损伤形式）时的停滞。该结构显示了 dsDNA 是如何分离的，并揭示了 Arch 结构域在活性 dsDNA 分离中的极不寻常参与。结合诱变和生化分析，我们确定了对解旋酶活性很重要的不同功能区域。令人惊讶的是，这些区域还影响核心 TFIIH 转位酶活性，揭示了 XPD 在 TFIIH 支架内尚未遇到的功能。总之，我们的数据为 NER 气泡形成、XPD 损伤验证和 XPG 切口提供了通用基础。