Journal Article

Cadmium mutagenicity and human nucleotide excision repair protein XPA: CD, EXAFS and <sup>1</sup>H/<sup>15</sup>N-NMR spectroscopic studies on the zinc(II)- and cadmium(II)-associated minimal DNA-binding domain (M98–F219)

Garry W. Buchko, Nancy J. Hess and Michael A. Kennedy

in Carcinogenesis

Volume 21, issue 5, pages 1051-1057
Published in print May 2000 | ISSN: 0143-3334
Published online May 2000 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/21.5.1051
Cadmium mutagenicity and human nucleotide excision repair protein XPA: CD, EXAFS and 1H/15N-NMR spectroscopic studies on the zinc(II)- and cadmium(II)-associated minimal DNA-binding domain (M98–F219)

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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-X2-C108-X17-C126-X2-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 Cd2+ is substituted for Zn2+ 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 Å, respectively. Comparison of the circular dichroism, two-dimensional 1H,15N-HSQC, and three-dimensional 15N-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.

Keywords: CD, circular dichroism; CdXPA-MBD, cadmium associated XPA-MBD; EXAFS, extended X-ray absorption fine structure; HSQC, heteronuclear single quantum coherence; NER, nucleotide excision repair; NMR, nuclear magnetic resonance; UV, ultraviolet; XPA-MBD, minimal DNA-binding domain of human NER protein XPA (M98–F219); ZnXPA-MBD, zinc associated XPA-MBD.

Journal Article.  5790 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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