Specific association between the human DNA repair proteins XPA and ERCC1.

Abstract

Processing ofDNA damage by the nucleotideexcision repair pathway in eukaryotic cells is most likely accomplished by multiprotein complexes. However, the nature of these complexes and the details of the molecular interactions between DNA repair factors are for the most part unknown. Here, we demonstrate both in vivo, using the two-hybrid system, and in vitro, using recombinant proteins, that the human repair factors XPA and ERCC1 specificafly interact. In addition, we report an initial determination of the domains in ERCC1 and XPA that medlate this interaction. These results suggest that XPA may play a role in the iocalation or ld ofan incision complex, composed ofERCC1 and possibly other repair factors, onto a damaged site.Two groups of mutants defective in the early steps of nucleotide-excision repair have been identified in mammalian cells. These are the naturally occurring cell lines obtained from patients with the rare disorders xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy and the laboratory-derived rodent cell lines designated ERCC (excision repair cross-complementing). Eleven genetic complementation groups have been identified in the former series (1, 2) and 11 in the latter (3, 4). Several of the mutants in these two classes fall into the same complementation group; however, the full extent of overlap remains to be determined. Seven of these genes have now been cloned (5-11), setting the stage for investigations into how DNA damage processing is mediated by this system. One aspect of these studies is to determine the nature ofprotein-protein interactions between DNA-repair factors and to define the composition of multifactorial complexes with the eventual goal ofelucidating their function.The yeast two-hybrid system developed by Fields and colleagues (12, 13) is a novel genetic approach that is used for the detection of interacting proteins. In this system, association in yeast between two proteins, one fused to the DNAbinding domain and the second to the activation domain II of GALA, activates promoters containing GALA-binding sites (14). We have used a modification (15, 16) of the original system involving a dual selection scheme for HIS3 and LacZ to screen a human cDNA library for genes whose expression products interact with the human repair factor ERCC1 (5,17). In addition to the isolation of a number of novel genes, this screening also resulted in the identification of the repair protein XPA as a factor that associates with ERCC1.

Domains in the XPA protein important in its role as a processivity factor

“…Within the N-terminal domain of XPA is a region encoded by exon 2 that associates with ERCC1 and is essential for NER activity (Fig. 4) [21,25]. The 12 bp deletion created in this region has been shown to inhibit the ability of XPA to interact with ERCC1 and to increase repair activity in XPA cells or extracts [21].…”

Genetic Characterization and Functional Analysis of the GID1 Gibberellin Receptors inArabidopsis

“…For yeast two-hybrid analysis, pLexA-NLS (containing LexA DB domain; Vojtek et al, 1993) and pACTII (containing transcriptional AD domain; Li et al, 1994) were used as expression vectors. pSLY101 (DB-SLY1), pRG42 (AD-RGA), pRG225 (AD-rga-D17), pGAI102 (AD-GAI), pgai-1 (AD-gai-D17), and AD fusions with GAI truncations (GAI-NT1 [amino acids 1 to 157], GAI-CT1 [amino acids 92 to 532], GAI-CT2 [amino acids 151 to 532], and GAI-CT3 [amino acids 223 to 532]) were made previously .…”

Virus–Human Cell Interactomes

“…A Gateway ® cassette was introduced downstream and in frame with Gal4DB 2. pACT2-gw: pACT2 is commercialized by Clontech (12). It is a prey vector, encoding Gal4AD domain (aa 768-881) under the control of ADH1 promoter (see Note 4) …”