Human nucleotide excision repair protein XPA: NMR spectroscopic studies of an XPA fragment containing the ERCC1-binding region and the minimal DNA-binding domain (M59-F219)

Abstract

XPA is a central protein component of nucleotide excision repair (NER), a ubiquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genome. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD: M98-F219) has recently been determined and chemical shift mapping experiments with ${}^{15}\text{N}$-labeled XPA-MBD show that XPA binds DNA along a basic surface located in the C-terminal loop-rich subdomain. Here, XPA–DNA interactions are further characterized using an XPA fragment containing the minimal DNA-binding domain plus the ERCC1-binding region (XPA-EM: M59-F219). The ${}^{15}\text{N}\text{/}{}^1\text{H}$ HSQC spectrum of XPA-EM closely maps onto the ${}^{15}\text{N}\text{/}{}^1\text{H}$ HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fragment. Such a conclusion is corroborated by chemical shift mapping experiments of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of ${}^{15}\text{N/}{}^1\text{H}$ HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the ${}^{15}\text{N/}{}^1\text{H}$ HSQC cross peaks of many of the basic residues in the loop-rich subdomain of DNA-free XPA-EM are less intense, or gone altogether, suggesting the acidic ERRC1-binding region of XPA-EM may associate transiently with the basic DNA-binding surface. While the DNA-binding domain in XPA-EM is structured and functional, ${}^{15}\text{N}$-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding region is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain with the DNA-binding region may play a role in the sequential assembly of the NER components.