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 structurally distinct DNA lesions in the eukaryotic genome. Human XPA, a 31 kDa, zinc-associated protein, is thought to play a major NER role in the recognition of damaged DNA and the recruitment of other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analyses and genetic evidence suggest that zinc is associated with a C4-type motif, C105-X2-C108-X17-C126-X2-C129, located in the minimal DNA binding region of XPA (M98-F219). The zinc-associated motif is essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc associated minimal DNA-binding domain of XPA (ZnXPA-MBD) show directly, for the first time, that the zinc is coordinated to the sulfur atoms of four cysteine residues with an average Zn-S bond length of 2.34+/-0.01 A. XPA-MBD was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily (>95%) cobalt associated (CoXPA-MBD). EXAFS spectra collected on CoXPA-MBD show that the cobalt is also coordinated to the sulfur atoms of four cysteine residues with an average Co-S bond length of 2.33+/-0.02 A.
Hess
N J
NJ
Pacific Northwest National Laboratories, Biogeochemistry Resources, Molecular Biosciences Department, Richland, Washington 99352, USA.
Buchko
G W
GW
Conradson
S D
SD
Espinosa
F J
FJ
Ni
S
S
Thrall
B D
BD
Kennedy
M A
MA
D015502:Absorptiometry, Photon; D001665:Binding Sites / Q000502:physiology; D003035:Cobalt / Q000737:chemistry; D004260:DNA Repair / Q000502:physiology; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005583:Fourier Analysis; D008667:Metalloproteins / Q000737:chemistry; D016601:RNA-Binding Proteins / Q000737:chemistry*; D051760:Xeroderma Pigmentosum Group A Protein; D015032:Zinc / Q000737:chemistry
D004268:DNA-Binding Proteins; D008667:Metalloproteins; D016601:RNA-Binding Proteins; D051760:Xeroderma Pigmentosum Group A Protein; D003035:Cobalt; D015032:Zinc