Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein

Abstract

Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed ␣-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.

Biochemical and Structural Domain Analysis of Xeroderma Pigmentosum Complementation Group C Protein

“…Notably, our results from sequence analysis were in agreement with the general predictions deposited in the Pfam-A database (61,62), which also suggest that the region 520-870 is fundamentally distinct from the rest of XPC. The prediction of structure in the region 520-870 is also fully consistent with the mapping of XPC interactions with DNA (6), RAD23B (6), and CETN2 (11,27) to functional domains within XPC-C (Fig. 1C).…”

“…Damage recognition by XPC is stimulated through its protein-protein interactions with the ubiquitin-like protein human homologue of Rad23 B (RAD23B) (10) and the EF-hand calcium binding protein centrin-2 (CETN2) (11). The heterotrimeric XPC-RAD23B-CETN2 complex participates in the first fundamental step of GG-NER, DNA damage recognition, and sets the stage for the following two steps, DNA damage excision and gap-filling synthesis and ligation.…”

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

“…(B) Scheme of the homologous domains in the human XPC sequence. Also shown are the region of sequence similarity with OB-folds [42], the domains involved in interactions with Rad23B [44], XPA [41], centrin-2 [30] and TFIIH [44], as well as the newly identified minimal DNA damage sensor interface [54]. …”

“…XPC protein itself possesses DNA-binding activity, whereas the ubiquitin-binding Rad23B and the calcium-binding centrin-2 protect the initiator complex from degradation and stimulate its activity in DNA repair [28,30]. In double-mutant mouse cells lacking Rad23B as well as the functionally redundant Rad23A ortholog, XPC protein is completely degraded by proteasomal activity [31].…”

“…The N-terminal region of human XPC is responsible for an association with XPA [41]. On the other hand, the carboxy-terminal tail of XPC protein harbors domains that interact with centrin-2 (residues 847-863) and TFIIH (residues 816-940) [30,44].…”