Sequential Assembly of the Nucleotide Excision Repair Factors In Vivo

Abstract

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.

Regulation of human DNA polymerase delta in the cellular responses to DNA damage

“…We have analyzed the spatiotemporal recruitment of all four Pol d subunits to sites of UV damage by their colocalization with CPDs using fluorescence microscopy. We used the technique of irradiation through UV-opaque polycarbonate filters with 5-lm pores [Volker et al, 2001]. These gave rise to macrofoci that were readily observed and also allowed quantitative analysis of the percentage colocalization with CPD damage [Chea et al, 2012].…”

Dynamic two-stage mechanism of versatile DNA damage recognition by xeroderma pigmentosum group C protein

“…In higher eukaryotes, this sequential reaction is initiated by a versatile DNA damage sensor composed of XPC, Rad23B and centrin-2 [12,13]. XPC is the actual sensor subunit of this initiator complex, whereas Rad23B and centrin-2 exert accessory functions (see section 3).…”

XPG: Its Products and Biological Roles

“…33 GG-NER operates by the sequential assembly of all the proteins involved at sites of DNA damage. [34][35][36] Accordingly, NER proteins diffuse freely trough the cell and are recruited in a defined order to sites of DNA damage and are released again from NER complexes once the damage has been repaired. Current evidence suggests that the XPC/HR23B/centrin-2 protein complex is the initial damage recognition factor in GG-NER (Figure 2) .…”

“…Current evidence suggests that the XPC/HR23B/centrin-2 protein complex is the initial damage recognition factor in GG-NER (Figure 2) . 35,37,38 XPC then recruits TFIIH to sites of UV lesions, and two helicase subunits XPB and XPD of TFIIH initiate the opening of the DNA around the lesion. XPA, RPA and XPG subsequently join the complex to form a stable open structure and recruitment of ERCC1-XPF by XPA initiates the dual incision of the DNA 5' and 3' to the lesion by ERCC1-XPF and XPG, respectively.…”

“…XPG interacts tightly with TFIIH and it is the interaction with TFIIH that recruits XPG to NER complexes at sites of UV damage. 30,35,41 Although investigation of the intracellular behavior of XPG suggests that the proteins has a mobility consistent with it being present in monomeric form, 41 some studies have found a tight and perhaps constitutive association of XPG with TFIIH. 42,50 It is currently believed that this constitutive interaction between XPG and TFIIH is important for the additional roles of the protein outside of NER (see below).…”