Mechanism of nickel assault on the zinc finger of DNA repair protein XPA

Abstract

Xeroderma pigmentosum group A complementing protein (XPA) is a member of the protein complex of the nucleotide excision repair (NER) pathway of DNA repair, participating in the assembly of the incision complex. The 4S zinc finger domain of XPA is involved the interactions with other NER proteins. As demonstrated previously, the activity of XPA is compromised by several metal ions implicated in DNA repair inhibition, including Ni(II), Cd(II), and Co(II) (Asmuss, M., Mullenders, L. H. F., Elker, A., and Hartwig, A. (2000) Carcinogenesis 21, 2097-2104). To study the possible molecular mechanisms of XPA inhibition, we investigated Zn(II) and Ni(II) interactions with the synthetic 37 peptide (XPAzf), representing the XPA zinc finger sequence AcDYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHKam. The binding constants were determined using fluorescence and UV-vis spectroscopies, structural insights were provided by CD, and oxidative damage to XPAzf was studied with HPLC. The binding constants for Zn(II) and Ni(II) are (8.5 +/- 1.5) x 10(8) (log value 8.93(7)) and (1.05 +/- 0.07) x 10(6) M(-)(1) (6.02(3)), respectively, in 10 mM phosphate buffer, pH 7.4, and (6 +/- 4) x 10(9) (9.8(2)) and (2.9 +/- 0.5) x 10(6) M(-)(1) (6.46(8)) in 50 mM phosphate buffer, pH 7.4, yielding binding constant ratios Zn(II)/Ni(II) of 800 +/- 100 and 2300 +/- 500, respectively. The Ni(II) ion forms a square planar complex with the sulfurs of XPAzf, opposed to the tetrahedral structure of the native Zn(II) complex. Consequently, the overall zinc finger structure is lost in the Ni(II)-substituted peptide. Zn(II)-saturated XPAzf is remarkably resistant to air oxidation and is only slowly oxidized by 0.01 mM, 0.1 mM, and 1 mM H(2)O(2) in a concentration-dependent fashion. However, the presence of just 10-fold molar excess of Ni(II) is sufficient to accelerate this process for all three H(2)O(2) concentrations tested. Overall, our results indicate that XPAzf can undergo Ni(II) assault in specific conditions.