THE XPA PROTEIN IS A ZINC METALLOPROTEIN WITH AN ABILITY TO RECOGNIZE VARIOUS KINDS OF DNA-DAMAGE

被引:142
作者
ASAHINA, H
KURAOKA, I
SHIRAKAWA, M
MORITA, EH
MIURA, N
MIYAMOTO, I
OHTSUKA, E
OKADA, Y
TANAKA, K
机构
[1] OSAKA UNIV,INST MOLEC & CELLULAR BIOL,SUITA,OSAKA 565,JAPAN
[2] OSAKA UNIV,INST PROT RES,SUITA,OSAKA 565,JAPAN
[3] HYOGO MED UNIV,DEPT UROL,NISHINOMIYA,HYOGO 663,JAPAN
[4] HOKKAIDO UNIV,FAC PHARMACEUT SCI,KITA KU,SAPPORO,HOKKAIDO 060,JAPAN
[5] SENRI LIFE SCI FDN,TOYONAKA,OSAKA 565,JAPAN
来源
MUTATION RESEARCH-DNA REPAIR | 1994年 / 315卷 / 03期
关键词
XERODERMA PIGMENTOSUM; RECOMBINANT PROTEIN; MICROINJECTION; UNSCHEDULED DNA SYNTHESIS; DNA BINDING; ZINC BINDING PROTEIN;
D O I
10.1016/0921-8777(94)90034-5
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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-108 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 C-4-type zinc finger motif is indispensable for normal protein conformation.
引用
收藏
页码:229 / 237
页数:9
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