Mapping of the regions implicated in nuclear localization of multi-functional DNA repair endonuclease XPF-ERCC1.

The structure-specific endonuclease XPF-ERCC1 is a multi-functional heterodimer that participates in a variety of DNA repair mechanisms for maintaining genome integrity. Both subunits contain C-terminal tandem helix-hairpin-helix (HhH2 ) domains, which are necessary for not only their dimerization but also enzymatic activity as well as protein stability. However, the interdependency of both subunits in their nuclear localization remains poorly understood. In this study, we have analyzed the region(s) that affects the subcellular localization of XPF and ERCC1 using various deletion mutants. We first identified the nuclear localization signal (NLS) in XPF, which was essential for its nuclear localization under the ERCC1-free condition, but dispensable in the presence of ERCC1 (probably as XPF-ERCC1 heterodimer). Interestingly, in the NLS-independent and ERCC1-dependent XPF nuclear localization, the physical interaction between XPF and ERCC1 via C-terminal HhH2 domains was not needed. Instead, the amino acid regions 311-469 of XPF and 216-260 of ERCC1 are required for the nuclear localization. Furthermore, we found that the 311-469 region of XPF interacts with ERCC1 in a co-immunoprecipitation assay. These results suggest that the nuclear localization of XPF-ERCC1 heterodimer is regulated at multiple levels in an interdependent manner.

DCAF7 is required for maintaining the cellular levels of ERCC1-XPF and nucleotide excision repair.

The ERCC1-XPF heterodimer is a structure-specific endonuclease and plays multiple roles in various DNA repair pathways including nucleotide excision repair and also telomere maintenance. The dimer formation, which is mediated by their C-terminal helix-hairpin-helix regions, is essential for their endonuclease activity as well as the stability of each protein. However, the detailed mechanism of how a cellular level of ERCC1-XPF is regulated still remains elusive. Here, we report the identification of DDB1- and CUL4-associated factor 7 (DCAF7, also known as WDR68/HAN11) as a novel interacting protein of ERCC1-XPF by mass spectrometry after tandem purification. Immunoprecipitation experiments confirmed their interaction and suggested dominant association of DCAF7 with XPF but not ERCC1. Interestingly, siRNA-mediated knockdown of DCAF7, but not DDB1, attenuated the cellular level of ERCC1-XPF, which is partly dependent on proteasome. The depletion of TCP1α, one of components of the molecular chaperon TRiC/CCT known to interact with DCAF7 and promote its folding, also reduced ERCC1-XPF level. Finally, we show that the depletion of DCAF7 causes inefficient repair of UV-induced (6-4) photoproducts, which can be rescued by ectopic overexpression of XPF or ERCC1-XPF. Altogether, our results strongly suggest that DCAF7 is a novel regulator of ERCC1-XPF protein level and cellular nucleotide excision repair activity.