Interplay of the Tfb1 Pleckstrin homolog domain with Rad2 and Rad4 in transcription coupled and global genomic nucleotide excision repair

Transcription-coupled repair (TCR) and global genomic repair (GGR) are two subpathways of nucleotide excision repair (NER). The TFIIH subunit Tfb1 contains a Pleckstrin homology domain (PHD), which was shown to interact with one PHD-binding segment (PB) of Rad4 and two PBs (PB1 and PB2) of Rad2 in vitro. Whether and how the different Rad2 and Rad4 PBs interact with the same Tfb1 PHD, and whether and how they affect TCR and GGR within the cell remain mysterious. We found that Rad4 PB constitutively interacts with Tfb1 PHD, and the two proteins may function together within one stable module for damage recognition in TCR and GGR. Rad2 PB1 interacts with Tfb1 PHD at a basal level when NER is inactive, and their interaction augments in a late step of NER. The augmented interaction contributes to both TCR and GGR. Rather than interacting with Tfb1 PHD, Rad2 PB2 constrains the basal interaction between Rad2 PB1 and Tfb1 PHD, thereby permitting their augmented interaction after NER is activated in the cell. Remarkably, the constraint also contributes to TCR and GGR. Our results elucidate the intricate interplay among Tfb1, Rad4, and Rad2 during NER, and unveil previously unexpected regulation mechanisms for NER.

转录偶联修复（Transcription-coupled repair, TCR）与全局基因组修复（Global Genomic Repair, GGR）是核苷酸切除修复（Nucleotide Excision Repair, NER）的两条亚通路。通用转录因子IIH（Transcription Factor IIH, TFIIH）的亚基Tfb1包含一个普列克斯特林同源结构域（Pleckstrin homology domain, PHD），体外实验已证实其可与Rad4的一段PHD结合片段（PHD-binding segment, PB）以及Rad2的两段PB（PB1与PB2）发生相互作用。但不同来源的Rad2与Rad4的PB如何与同一Tfb1 PHD结合，以及它们如何在细胞内调控TCR与GGR，其具体机制仍不明晰。本研究发现，Rad4 PB可与Tfb1 PHD发生组成型相互作用，二者可作为一个稳定的功能模块协同参与TCR与GGR中的损伤识别过程。当NER未激活时，Rad2 PB1仅以基础水平与Tfb1 PHD结合，而在NER的晚期步骤中二者的相互作用会增强。这种增强的相互作用对TCR与GGR均具有调控作用。Rad2 PB2并不与Tfb1 PHD结合，而是通过抑制Rad2 PB1与Tfb1 PHD之间的基础相互作用，使得细胞内NER激活后二者的相互作用得以增强。值得注意的是，这种抑制作用同样对TCR与GGR具有调控意义。本研究结果阐明了NER过程中Tfb1、Rad4与Rad2之间复杂的相互调控关系，并揭示了此前未被发现的NER调控机制。