The XPA protein is a zinc metalloprotein with an ability to recognize various kinds of DNA damage.
The XPA (xeroderma pigmentosum group A) gene encodes a protein of 273 amino acids with a zinc finger motif. The human XPA cDNA was placed in an Escherichia coli expression vector for the synthesis of the recombinant XPA protein. The molecular weight of the wild-type protein was about 40 kDa in SDS-PAGE. Microinjection of the wild-type protein specifically restored the defect of UV-induced unscheduled DNA synthesis in XP-A cells. Thus, the bacterially expressed XPA protein retains biochemical properties identical to those of natural sources. The wild-type protein binds preferentially to UV-, cis-diamminedichloroplatinum(II) (cisplatin)- or osmium tetroxide (OsO4)-damaged DNA as assayed by retention on nitrocellulose filters. In addition, the data from atomic absorption and UV-CD spectra revealed that the wild-type protein is a zinc metalloprotein with secondary structure. Furthermore, the mutant protein, of which the cysteine-103 residue in the zinc finger motif was replaced with serine, has a vastly different protein conformation resulting in a loss of XP-A correcting and DNA-binding activities. These findings indicate that the XPA protein is a zinc-binding protein with affinity for various DNA damages, and a cysteine residue in the C4-type zinc finger motif is indispensable for normal protein conformation.
Asahina
H
H
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Kuraoka
I
I
Shirakawa
M
M
Morita
E H
EH
Miura
N
N
Miyamoto
I
I
Ohtsuka
E
E
Okada
Y
Y
Tanaka
K
K
D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D004249:DNA Damage; D004279:DNA, Viral / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D008667:Metalloproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum; D051760:Xeroderma Pigmentosum Group A Protein; D015032:Zinc / Q000032:analysis*
D000596:Amino Acids; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D008667:Metalloproteins; D011993:Recombinant Fusion Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA; D015032:Zinc