cDNA Cloning of the Chicken DDB1 Gene Encoding the p127 Subunit of Damaged DNA-binding Protein

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  • Fu DongTao
    Laboratory of Molecular Human Genetics Faculty of Pharmaceutical Sciences Kanazawa University
  • Wakasugi Mitsuo
    Laboratory of Molecular Human Genetics Faculty of Pharmaceutical Sciences Kanazawa University
  • Ishigaki Yasuhito
    Laboratory of Molecular Human Genetics Faculty of Pharmaceutical Sciences Kanazawa University
  • Nikaido Osamu
    Laboratory of Molecular Human Genetics Faculty of Pharmaceutical Sciences Kanazawa University
  • Matsunaga Tsukasa
    Laboratory of Molecular Human Genetics Faculty of Pharmaceutical Sciences Kanazawa University

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Abstract

DDB (damaged DNA-binding protein) is a heterodimer, comprised of p48 (DDB2) and p127 (DDB1) subunits, which has a high affinity for a variety of DNA lesions including UV-photoproducts. The mutations in DDB2 gene have been found in a subset of xeroderma pigmentosum complementation group E patients. However, no natural mutation has been identified so far in the cDNA of human DDB1 and the precise roles of DDB1 are still unknown. We have cloned the DDB1 cDNA from the chicken B lymphocyte line DT40 and revealed an open reading frame of 3420 bp encoding a polypeptide of 1140 amino acids, which is identical in size to the orthologs of human, monkey, mouse, rat and Drosophila melanogaster in databases. The amino acid sequence deduced from the chicken DDB1 cDNA shows a high homology to the mammalian DDB1 orthologs (96-97% identity). Northern blot analysis using 5' portion of the chicken DDB1 cDNA as a probe detected a single transcript of ~ 4.3 kb in chicken DT40 cells as well as in human HeLa cells and mouse embryonic fibroblasts. Furthermore, the chicken DDB1 (tagged with enhanced GFP) transiently expressed in human cells mainly localized in the cytoplasm, and coexpression of human DDB2 dramatically changed the localization from the cytoplasm to nucleus. These results suggest that DDB1 is evolutionarily conserved in the primary structure and function, and may play a fundamental role in higher eukaryotes.<br>

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