Cadmium mutagenicity and human nucleotide excision repair protein XPA: CD,EXAFS and H-1/N-15-NMR spectroscopic studies on the zinc(II)- and cadmium(II)-associated minimal DNA-binding domain (M98-F219)
Gw. Buchko et al., Cadmium mutagenicity and human nucleotide excision repair protein XPA: CD,EXAFS and H-1/N-15-NMR spectroscopic studies on the zinc(II)- and cadmium(II)-associated minimal DNA-binding domain (M98-F219), CARCINOGENE, 21(5), 2000, pp. 1051-1057
Human XPA is a 31 kDa protein involved in nucleotide excision repair (NER),
a ubiquitous, multi-enzyme pathway responsible for processing multiple typ
es of DNA damage in the eukaryotic genome. A zinc-associated, C4-type motif
(C105-X-2-C108-X-17-C126-X-2-C129) located in the minimal DNA-binding regi
on (M98-F219) of XPA. (XPA-MBD) is essential for damaged DNA recognition, C
admium is a known carcinogen and can displace the zinc in many metal-bindin
g proteins, It has been suggested that the carcinogenic properties of cadmi
um may result from structural changes effected in XPA when Cd2+ is substitu
ted for Zn2+ in the metal-binding site. The solution structure of XPA-MBD c
ontaining zinc(Ir) has recently been determined [Buchko ef nl,? (1998) Nucl
eic Acids Res., 26, 2779-2788; Buchko et al,, (1999) Biochemistry, 38, 1511
6-15128], To assess the effects of cadmium(II) substitution on the structur
e of XPA-MBD, XPA-MBD was expressed in minimal medium supplemented with cad
mium acetate to yield a protein that was almost exclusively (>95%) associat
ed with cadmium(II) (CdXPA-MBD). Extended X-ray absorption fine structure s
pectra collected on ZnXPA-MBD and CdXPA-MBD in frozen (77 K) 15% aqueous gl
ycerol solution show that the metal is coordinated to the sulfur atoms of f
our cysteine residues with an average metal-sulfur bond length of 2.34 +/-
0.01 and 2.54 +/- 0.01 Angstrom, respectively. Comparison of the circular d
ichroism, two-dimensional H-1,N-15-HSQC, and three-dimensional N-15-edited
HSQC-NOESY spectra of ZnXPA-MBD and CdXPA-MBD show that there are no struct
ural differences between the two proteins. The absence of major structural
changes upon substituting cadmium(II) for zinc(II) in XPA suggests that cad
mium-induced mutagenesis is probably not due to structural perturbations to
the zinc-binding core of XPA.