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)

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.