Mutational analysis of the structure and function of the xeroderma pigmentosum group A complementing protein. Identification of essential domains for nuclear localization and DNA excision repair.
We showed previously that the xeroderma pigmentosum group A complementing (XPAC) protein involved in the DNA excision repair pathway contains a zinc-finger motif and is localized in the nucleus of normal human cells. For detailed structural and functional analyses of the XPAC protein, we constructed various XPAC cDNAs by site-directed mutagenesis and isolated permanent cell lines expressing mutant proteins. Immunofluorescent analysis of these lines indicated that the nuclear localization signal is located in the region encoded by Exon 1, especially centered at amino acids 30-42. A UV survival study showed that regions from Exons 2 through 6 were essential for DNA repair function, but that Exon 1 was not. Interestingly, deletion of the glutamic acid cluster in the region encoded by Exon 2 resulted in a dramatic loss of DNA repair activity. Furthermore, replacements of each of the 4 cysteines supposed to form a zinc-finger structure in the region encoded by Exon 3 by serine or glycine resulted in similar levels of loss of repair activity. These results suggest that all 4 cysteines forming a zinc-finger structure and also the glutamic acid cluster are important for DNA repair function.
Miyamoto
I
I
Department of Urology, Hyogo College of Medicine, Nishinomiya, Japan.
Miura
N
N
Niwa
H
H
Miyazaki
J
J
Tanaka
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014983:Xeroderma Pigmentosum / Q000378:metabolism; D051760:Xeroderma Pigmentosum Group A Protein; D016335:Zinc Fingers / Q000235:genetics
D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D004247:DNA