The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.
The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.
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DOI：
10.1093/nar/gkaa973
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发表时间：
2020-12-16
影响因子：
14.9
通讯作者：
Kad NM
中科院分区：
文献类型：
--
作者：
Barnett JT;Kuper J;Koelmel W;Kisker C;Kad NM
真核生物中的核苷酸切除修复（NER）由通用转录因子TFIIH的核心形式协调，TFIIH包含解旋酶XPB、XPD和五个“结构”亚基p62、p44、p34、p52和p8。最近的cryo-EM结构表明，p62与p44广泛接触，并部分占据XPD的DNA结合位点。虽然已知p44在NER期间调节XPD的解旋酶活性，但p62被认为是纯粹的结构性的。在这里，使用解旋酶和腺苷三磷酸酶测定，我们表明，含有p44和p62的复合物增强XPD对dsDNA的亲和力3倍于单独的p44。值得注意的是，相对亲和力通过dsDNA损伤进一步增加至60倍。直接结合研究表明，这种偏好源于p44/p62对受损ssDNA的高亲和力（20 nM）。没有XPD的p44/p62复合物的单分子成像显示它们与DNA结合并在DNA上随机扩散，然而，在UV诱导的DNA损伤的存在下，这些复合物停滞。结合最近的冷冻EM结构的分析，我们认为，p44/p62作为一种新的DNA结合实体，增强损伤识别TFIIH。这修正了我们对TFIIH的理解，并促使对核心亚基在DNA修复和/或转录过程中的积极作用进行研究。
Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five ‘structural’ subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD’s DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD’s affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62’s high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.
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