HUMAN NUCLEOTIDE EXCISION-REPAIR PROTEIN XPA - EXTENDED X-RAY-ABSORPTION FINE-STRUCTURE EVIDENCE FOR A METAL-BINDING DOMAIN

The ubiquitous, multi-enzyme, nucleotide excision repair (NER) pathway is responsible for correcting a wide range of chemically and structur ally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kD a, zinc-associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, inc luding RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type mo tif, C105-X-2-C108-X-17-C126-X-2-C129, located in the minimal DNA bind ing region of XPA (M98-F219). The zinc-associated motif is essential f or damaged DNA recognition. Extended X-ray absorption fine structure ( EXAFS) spectra collected on the zinc associated minimal DNA-binding do main of XPA (ZnXPA-MBD) show directly, for the first time, that the zi nc is coordinated to the sulfur atoms of four cysteine residues with a n average Zn-S bond length of 2.34 +/- 0.01 Angstrom. XPA-MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily (>95%) cobalt associated (C oXPA-MBD). EXAFS spectra collected on CoXPA-MBD show that the cobalt i s also coordinated to the sulfur atoms of four cysteine residues with an average Co-S bond length of 2.33 +/- 0.02 Angstrom.