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)
Gw. Buchko et al., 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), MUT R-DNA R, 486(1), 2001, pp. 1-10
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.