Extended X-ray absorption fine structure evidence for a single metal binding domain in Xenopus laevis nucleotide excision repair protein XPA

Nucleotide excision repair (NER) is an important cellular mechanism, conser ved from bacteria to humans, responsible for eliminating multiple types of structurally distinct DNA lesions from the genome. The protein XPA appears to play a central role in NER, recognizing and/or verifying damaged DNA and recruiting other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analysis and genetic evidence suggest that zinc, which is essential for DNA binding, is associated with a C4-type motif, C-X-2-C-X-17 -C-X-2-C. Sequence analysis suggests that a second, H2C2-type zinc-binding motif may be present near the C-terminal. Seventy percent of the amino acid sequence of Xenopus laevis XPA (xXPA) is identical to human XPA and both p utative zinc-binding motifs are conserved in all known XPA proteins. Electr ospray ionization-mass spectroscopy data show that xXPA contains only one z inc atom per molecule. EXAFS spectra collected on full-length xXPA in froze n (77 K) 15% glycerol aqueous solution unequivocally show that the zinc ato m is coordinated to four sulfur atoms with an average Zn-S bond length of 2 .33 +/- 0.02 Angstrom Together, the EXAFS and mass spectroscopy data indica te that xXPA contains just one C4-type zinc-binding motif. (C) 1999 Academi c Press.