Physical and functional interaction between DDB and XPA in nucleotide excision repair

Abstract

Damaged DNA-binding protein (DDB), consisting of DDB1 and DDB2 subunits recognizes a wide spectrum of DNA lesions. DDB is dispensable for in vitro nucleotide excision repair (NER) reaction, but stimulates this reaction especially for cyclobutane pyrimidine dimer (CPD). Here we show that DDB directly interacts with XPA, one of core NER factors, mainly through DDB2 subunit and the amino-acid residues between 185 and 226 in XPA are important for the interaction. Interestingly, the point mutation causing the substitution from Arg-207 to Gly, which was previously identified in a XP-A revertant cell-line XP129, diminished the interaction with DDB in vitro and in vivo. In a defined system containing R207G mutant XPA and other core NER factors, DDB failed to stimulate the excision of CPD, although the mutant XPA was competent for the basal NER reaction. Moreover, in vivo experiments revealed that the mutant XPA is recruited to damaged DNA sites with much less efficiency compared with wild-type XPA and fails to support the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These results suggest that the physical interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction.

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

“…Recent reports have identified that the N terminus of DDB2 is also PARylated in vivo in response to UV damage, resulting in stabilization of UV-DDB on damaged chromatin (50,51). How posttranslational modifications such as phosphorylation (52), SUMOylation (53), ubiquitylation (21), and PARylation (50, 51), as well as interacting partners such as XPC and XPA (21,54,55), may regulate the dimerization and lifetimes of the kinetic intermediates identified in this work need to be further investigated.…”

HIV-1 Vpr Induces the K48-Linked Polyubiquitination and Proteasomal Degradation of Target Cellular Proteins To Activate ATR and Promote G 2 Arrest

“…Surprisingly, however, relatively few cellular proteins have been shown to date to be regulated by these complexes. CRL4A (DDB2) promotes the ubiquitination of histones 2A, 3, and 4 (28,53); of xeroderma pigmentosum group C protein (XPC) (48); and probably of xeroderma pigmentosum group A protein (XPA) (52) to facilitate UV damage repair. CRL4A (CSA) and CRL4A (DET1-COP1) induce the proteolysis of Cockayne syndrome type B gene product (CSB) (18) and c-JUN (56), respectively.…”

Regulation of nucleotide excision repair through ubiquitination

“…The DDB complex can bind damaged DNA independently of XPC or any other core components of NER [113], suggesting that it is the first recognition protein to reach the UV-damage site and then help to recruit XPC-HR23 to the lesion, followed by the recruitment of XPA for lesion verification, triggering the NER process [114,115]. Support for the above notion includes observations that the tertiary complex of DDB1, DDB2, and XPA is assembled at the damaged DNA site in the early stage of NER [116]. Although (6-4) PPs can also be recognized by XPC, the relocation of XPC to CPDs requires the presence of functional DDB2 [114].…”