Cadmium mutagenicity and human nucleotide excision repair protein XPA: CD, EXAFS and (1)H/(15)N-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 types 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 region (M98-F219) of XPA (XPA-MBD) is essential for damaged DNA recognition. Cadmium is a known carcinogen and can displace the zinc in many metal-binding proteins. It has been suggested that the carcinogenic properties of cadmium may result from structural changes effected in XPA when Cd(2+) is substituted for Zn(2+) in the metal-binding site. The solution structure of XPA-MBD containing zinc(II) has recently been determined [Buchko et al., (1998) Nucleic Acids Res., 26, 2779-2788; Buchko et al., (1999) Biochemistry, 38, 15116-15128]. To assess the effects of cadmium(II) substitution on the structure of XPA-MBD, XPA-MBD was expressed in minimal medium supplemented with cadmium acetate to yield a protein that was almost exclusively (>95%) associated with cadmium(II) (CdXPA-MBD). Extended X-ray absorption fine structure spectra collected on ZnXPA-MBD and CdXPA-MBD in frozen (77 K) 15% aqueous glycerol solution show that the metal is coordinated to the sulfur atoms of four cysteine residues with an average metal-sulfur bond length of 2.34 +/- 0.01 and 2.54 +/- 0.01 A, respectively. Comparison of the circular dichroism, two-dimensional (1)H,(15)N-HSQC, and three-dimensional (15)N-edited HSQC-NOESY spectra of ZnXPA-MBD and CdXPA-MBD show that there are no structural differences between the two proteins. The absence of major structural changes upon substituting cadmium(II) for zinc(II) in XPA suggests that cadmium-induced mutagenesis is probably not due to structural perturbations to the zinc-binding core of XPA.
Buchko
G W
GW
Pacific Northwest National Laboratories, Environmental Molecular Sciences Laboratory and Biogeochemistry Resources, Richland, WA 99352, USA.
Hess
N J
NJ
Kennedy
M A
MA
D002104:Cadmium / Q000378:metabolism* / Q000633:toxicity*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004577:Electron Probe Microanalysis; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009153:Mutagens / Q000633:toxicity*; D009587:Nitrogen Isotopes; D011485:Protein Binding; D011522:Protons; D014983:Xeroderma Pigmentosum / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein; D015032:Zinc / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009153:Mutagens; D009587:Nitrogen Isotopes; D011522:Protons; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D002104:Cadmium; D004247:DNA; D015032:Zinc