Nucleic Acids Research, 1992, Vol. 20, No. 21 5541-5548
© 1992
MOLECULAR BIOLOGY |
Cloning and characterization of the Drosophila homolog of the xeroderma pigmentosum complementation-group B correcting gene, ERCC3
Department of Cell Biology and Genetics, Medical Genetics Centre, Erasmus University PO Box 1738, 3000DR Rotterdam 1Department of Radiation Genetics and Chemical Mutagenesis, Medical Genetics Centre, State University Leiden PO Box 9503, 2300 RA Leiden 2J.A.Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection Wassenaarseweg 62, 2333 AL Leiden, The Netherlands
* To whom correspondence should be addressed
Received September 11, 1992. Revised October 8, 1992. Accepted October 8, 1992.
Previously the human nucleotide excision repair gene ERCC3 was shown to be responsible for a rare combination of the autosomal recessive DNA repair disorders xeroderma pigmentosum (complementation group B) and Cockayne's syndrome (complementation group C). The human and mouse ERCC3 proteins contain several sequence motifs suggesting that it is a nucleic acid or chromatin binding helicase. To study the significance of these domains and the overall evolutionary conservation of the gene, the homolog from Drosophila melanogaster was isolated by low stringency hybridizations using two flanking probes of the human ERCC3 cDNA. The flanking probe strategy selects for long stretches of nucleotide sequence homology, and avoids isolation of small regions with fortuitous homology. In situ hybridization localized the gene onto chromosome III 67E3/4, a region devoid of known D.melanogaster mutagen sensitive mutants. Northern blot analysis showed that the gene is continuously expressed in all stages of fly development. A slight increase (2–3 times) of ERCC3Dm transcript was observed in the later stages. Two almost full length cDNAs were isolated, which have different 5' untranslated regions (UTR). The SD4 cDNA harbours only one long open reading frame (ORF) coding for ERCC3Dm. Another clone (SD2), however, has the potential to encode two proteins: a 170 amino acids polypeptide starting at the optimal first ATG has no detectable homology with any other proteins currently in the data bases, and another ORF beginning at the suboptimal second startcodon which is identical to that of SD4. Comparison of the encoded ERCC3Dm protein with the homologous proteins of mouse and man shows a strong amino acid conservation (71% identity), especially in the postulated DNA binding region and seven ‘helicase’ domains. The ERCC3Dm sequence is fully consistent with the presumed functions and the high conservation of these regions strengthens their functional significance. Microinjection and DNA transfection of ERCC3Dm into human xeroderma pigmentosum (c.g. B) fibroblasts and group 3 rodent mutants did not yield detectable correction. One of the possibilities to explain these negative findings is that the D.melanogaster protein may be unable to function in a mammalian repair context.
+Present addresses: Hôpital St Louis, Laboratoire des Rétrovirus et Rétrotransposons des Vertèbres, Batiment INSERM, 1610 Rue de la Grange aux Belles, 75010 Paris, France
Department of Anthropogenetics, Academical Medical Centre, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
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