The protein–protein interaction studies on core NER factors have greatly contributed to the understanding of basal NER reaction (47
). However, only a few studies on the interaction between DDB and core NER factors have been reported (26
), in contrast to numerous reports of DDB-interactive proteins involved in other cellular processes (49
). The first core NER factor shown to interact with DDB is RPA, based on immunoprecipitation using partially purified DDB fraction or nuclear extracts (48
). RPA is a eukaryotic single-stranded DNA-binding protein essential for chromosomal DNA replication as well as NER and shows a preferential binding to damaged DNA compared with native DNA (50
). The interaction between DDB and RPA appears to enhance damaged DNA-binding activity of both proteins (48
). The authors (48
) and we (19
) also observed a ternary complex of DDB, RPA and damaged DNA in an electrophoretic mobility shift assay. In addition, RPA is known to interact with XPA physically and functionally and this interaction again enhances both binding activities to damaged DNA (50
). Together with our previous observation of a higher complex containing DDB, RPA, XPA and damaged DNA (19
), DDB might also play a role in recruiting RPA along with XPA to damaged DNA sites.
Recently, Sugasawa et al.
) have reported that DDB physically interacts with XPC based on immunoprecipitation using cell lysates as well as purified proteins. The interaction between DDB and XPC is implicated in the ubiquitination of XPC by the E3 ligase complex with Cullin4A, Roc1 and DDB, and the XPC polyubiquitination alters its DNA-binding properties. In the literature, XPC is also known to interact with XPA (53
). Furthermore, other group has recently shown that XPC is modified by SUMO-1 and ubiquitin in untransformed cells exposed to UV light and these modifications require not only DDB2 but also XPA (27
). Taken all together, it is plausible that DDB bound to damaged DNA sites may function in the recruitment of XPC-RAD23B, XPA and RPA and further in the modification of XPC at damaged DNA sites.
A recently developed micropore UV irradiation method has contributed to our understanding of sequential assembly of NER factors at damaged DNA sites in the nucleus (43
). We originally found that DDB rapidly accumulates at locally UV-damaged subnuclear regions in the absence of XPC or XPA (20
). Other groups reported similar observations and further showed that the recruitment of XPC to UV-damaged sites, especially CPD, requires functional DDB2 (as a DDB complex) (22–24
). In this study, we showed that the ectopic expression of DDB2 in SV40-transformed XP-F cells enhances the recruitment of XPA to UV-damaged sites (). Using the same local UV irradiation, Volker et al.
) demonstrated that XPC is required for the recruitment of XPA to damaged-DNA sites in primary diploid human fibroblasts containing a normal level of DDB2. DDB may enhance the recruitment of XPA to DNA lesions in vivo
by two possible mechanisms, directly through its interaction with XPA and indirectly through efficient XPC loading onto DNA lesions.
XPA is known to interact with all of other five core NER factors (34
), and their interaction domains as well as DNA-binding domain in XPA have been mapped. The DDB2-interactive domain (residues 185–225) determined in this study partially overlaps with a DNA-binding domain (residues 98–219) reported previously (57
) and is located between an RPA1-binding domain (residues 98–187) (52
) and a C-terminal TFIIH-binding domain (residues 226–273) (34
). We further demonstrated that R207G mutation in the DDB2-interactive domain results in the reduced binding to DDB2 or DDB heterodimer. Importantly, this amino-acid substitution caused the attenuated XPA recruitment to locally UV-damaged subnuclear regions ( and Supplementary Figure 2S
) and impaired enhancement of CPD repair by DDB in vitro
() as well as in vivo
(), clearly indicating that the interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction for CPD.
The R207G mutation was found in the XP129 revertant cell line, which had been isolated from a SV40-transfomed XP12ROSV cell line following the repetitious treatment of methyl methane sulfonate and reported to have a unique DNA repair phenotype of removing 6-4PP normally but not CPD (39
). On the other hand, Kobayashi et al.
) examined the effects of this mutation on NER activity and UV sensitivity using various XP12ROSV transfectants stably expressing wild-type or R207G mutant XPA. Based on the observation that the rate of CPD repair in the transfectant expressing R207G mutant XPA was almost normal, the authors concluded that the R207G mutation might not be responsible for the selective GGR defect for CPD repair in XP129 cells. The apparent discrepancy between the two studies seems explainable by the findings that SV40 transformation reduces GGR activity for CPD significantly (but not completely) due to dysfunction of p53 by the large T antigen (44
) and this repair deficiency can be reversed by ectopic expression of DDB2 (20
). Our data shown in strongly support this possible explanation.
In conclusion, this study has found that DDB2 physically interacts with XPA and this interaction is required for the stimulatory effect of DDB on CPD excision in vitro, the efficient recruitment of XPA to damaged sites in vivo and the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These findings reveal a new link between DDB and the core NER factors, and indicate that XPA interacts with not only five core NER factors but also an accessory factor DDB. The critical role of XPA as a scaffold might explain why XP-A patients or XPA-deficient cells exhibit the most severe phenotypes among seven complementation groups of NER-defective XP.