2.3. TFIIH Recruitment and functions in GG-NER
GG-NER is responsible for the repair of bulky adducts across the genome, and is particularly important for the suppression of mutations and potential carcinogenesis associated with UV lesions and other bulky adducts. In GG-NER, damage such as a CPD lesion is first recognized by the UV-DDB protein and then transferred to XPC, through UV-DDB-mediated XPC polyubiquitylation (Sugasawa et al., 2005). XPC forms a heterodimer with RAD23 and binds to thermodynamically destabilized DNA (Min & Pavletich, 2007), instead of a specific type of lesion. This low lesion specificity allows GG-NER to repair a broad range of lesions, such as UV photolesions, cisplatin-induced intrastrand crosslinks, benzopyrene adducts, and other helix-distorting lesions. However, the low specificity may enable XPC to bind to other types of damage not commonly repaired by NER, including DNA mismatch bubbles (Chen et al., 2015; Y. S. Krasikova et al., 2013). Thus, the importance of TFIIH’s damage verification function is inexplicably tied to the role of XPC to avoid incisions at sites without bulky adducts.
TFIIH is directly recruited by XPC in GG-NER. Biochemical data indicates that the p62 and XPB subunits of TFIIH physically interact with XPC (Uchida et al., 2002; Yokoi et al., 2000). This finding is further supported by the structural data for the yeast XPC-TFIIH-DNA complex, which shows that the N-terminus of XPC contacts with the pleckstrin homology (PH) domain of p62, while the XPC C-terminal domain interacts with the C-terminal helix of XPB (van Eeuwen et al., 2021).
After recruitment by XPC, TFIIH plays two major roles in GG-NER: DNA unwinding and damage verification, both of which are mainly dependent on the XPD subunit (Figure 2 ). XPD is the major helicase to unwind the two strands in the NER pathway. While XPB was initially suggested as another helicase with an opposite polarity to XPD (Fuss & Tainer, 2011), other studies show that XPB’s ATPase, but not the helicase function, is required for DNA repair (Coin et al., 2007). This raises an interesting model that XPB functions as an ATPase for the initial unwinding and anchoring TFIIH to the DNA strand, resulting in a helix opening action and engagement of XPD to the DNA. In agreement with this model, Cryo-EM analysis of the yeast XPC-TFIIH-DNA complex has provided structural insights into the coordinated action between XPB and XPC in initiating DNA unwinding (van Eeuwen et al., 2021). The data shows that XPB binds to the 5’ side relative to the damage, whereas XPC holds the 3’ side. XPB uses its ATP-dependent DNA translocase activity to generate torsion stress and unwind DNA. XPC holds the other side as an anchor to avoid DNA free rotation. Hence, XPB and XPC function in a cooperative manner to initiate DNA unwinding. The partially opened DNA state promoted by XPB and XPC is then delivered to XPD for further bubble formation and damage verification. These structural data thus suggest that XPB and XPD act sequentially to promote formation of the NER bubble structure. Defects in either of XPB or XPD can result in failure of strand separation around the damage and incomplete repair. In line with this notion, yeast genetics evidence has shown that truncation of the C-terminal portion of XPB or point mutations deactivating XPD’s helicase activity leads to extremely high UV sensitivity and low or even undetectable GG-NER (Duan et al., 2020; van Eeuwen et al., 2021).
XPD also performs a sliding function to verify if a genuine NER damage is present. This damage verification function is carried out by sliding a single-stranded DNA (ssDNA) through the central tunnel of XPD protein formed by an iron-sulfur cluster and an arch domain (Kuper et al., 2014). This sliding function will stop or be impeded if the DNA has a bulky lesion and can aid in the translocation of the TFIIH complex to the location of the damage. The stalling of XPD by a bulky lesion thus serves as a critical damage verification mechanism before NER initiates strand excision. Mutations of several amino acids near XPD’s central tunnel abolishes the damage verification function, but does not impact the DNA helicase activity (Mathieu et al., 2013), suggesting that DNA unwinding and damage verification are conducted by different functional domains in XPD. Once the DNA has been opened and the damage verified, it allows for further proteins of the preincision complex to be recruited at the lesion site to excise the damaged nucleotides. A critical protein for the preincision complex assembly is XPA. XPA binds to the 5’ end of the damage and facilitates the recruitments of replication protein A (RPA) and repair endonuclease XPF-ERCC1 (Sugitani et al., 2016). RPA is a single-stranded DNA binding protein that binds approximately 30 nucleotides on the undamaged strand. It functions together with XPA as the central scaffold to ensure proper positioning of the two repair endonucleases, XPF and XPG, at the site of damage in the DNA (Schärer, 2013).