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

XPA is a central protein component of nucleotide excision repair (NER), a u biquitous, multi-component cellular pathway responsible for the removal and repair of many structurally distinct DNA lesions from the eukaryotic genom e. The solution structure of the minimal DNA-binding domain of XPA (XPA-MBD : M98-F219) has recently been determined and chemical shift mapping experim ents with N-15-labeled XPA-MBD show that XPA binds DNA along a basic surfac e 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 N-15/ H-1 HSQC spectrum of XPA-EM closely maps onto the N-15/H-1 HSQC spectrum of XPA-MBD, suggesting the DNA-binding domain is intact in the larger XPA fra gment. Such a conclusion is corroborated by chemical shift mapping experime nts of XPA-EM with a single strand DNA oligomer, dCCAATAACC (d9), that show the same set of N-15/H-1 HSQC cross peaks are effected by the addition of DNA. However, relative to DNA-free XPA-MBD, the N-15/H-1 HSQC cross peaks o f 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 r egion of XPA-EM may associate transiently with the basic DNA-binding surfac e. While the DNA-binding domain in XPA-EM is structured and functional, N-1 5-edited NOESY spectra of XPA-EM indicate that the acidic ERRC1-binding reg ion is unstructured. If the structural features observed for XPA-EM persist in XPA, transient intramolecular association of the ERCC1-binding domain w ith the DNA-binding region may play a role in the sequential assembly of th e NER components. (C) 2001 Elsevier Science B.V. All rights reserved.