The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.

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.

真核生物中的核苷酸切除修复（NER）由通用转录因子TFIIH的核心形式协调进行，它包含解旋酶XPB、XPD以及五个“结构”亚基，即p62、p44、p34、p52和p8。近期的冷冻电镜结构显示，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过程中调节XPD的解旋酶活性，但p62被认为仅具有结构作用。在此，通过解旋酶和腺苷三磷酸酶测定，我们发现包含p44和p62的复合物使XPD对双链DNA的亲和力比单独的p44提高了3倍。值得注意的是，双链DNA损伤使相对亲和力进一步提高到60倍。直接结合研究表明，这种偏好源于p44/p62对受损单链DNA的高亲和力（20 nM）。不含XPD的p44/p62复合物的单分子成像显示，它们与DNA结合并在其上随机扩散，然而，在存在紫外线诱导的DNA损伤时，这些复合物会停滞。结合对近期冷冻电镜结构的分析，我们认为p44/p62作为一种新型的DNA结合实体，增强了TFIIH中的损伤识别能力。这改变了我们对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。