Drosophila Damaged DNA-Binding Protein 1 Is an Essential Factor for Development

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ABSTRACT

The damaged DNA-binding protein (DDB) complex, thought to recognize (6-4) photoproducts and other lesions in DNA, has been implicated to have a role in global genomic nucleotide excision repair (NER) and E2F-1-mediated transcription. The complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2), the latter also being known as XPE. We reported previously that in Drosophila expression of the DDB1 (D-DDB1) gene is controlled by the DRE/DREF system, and external injury to DNA is not essential for D-DDB1 function. In the present study of the function of D-DDB1 in a multicellular system, we prepared transgenic flies, which were knocked down for the D-DDB1 gene due to RNA interference (RNAi), and performed immunocytochemistry to ascertain the distribution of D-DDB1 in the eye imaginal disc. It was found to be abundant in the anterior of the morphogenetic furrow (MF). Whole-body overexpression of dsRNA of D-DDB1 in Drosophila using a GAL4-UAS targeted expression system induced melanotic tumors and caused complete lethality. When limited to the eye imaginal disc, a severe rough eye phenotype resulted. Correspondingly, all of the D-DDB1 gene knocked-out flies also died. D-DDB1 therefore appears to be an essential development-associated factor in a multicellular organism.

THE damaged DNA-binding protein (DDB) complex, which is a heterodimeric protein composed of 127-kD (DDB1) and 48-kD (DDB2) subunits, has been shown to recognize many types of DNA lesions (FELDBERG 1980; CAREW and FELDBERG 1985; HIRSCHFELD et al. 1990; KEENEY et al. 1993; REARDON et al. 1993; PAYNE and CHU 1994). DDB1 can interact with SPT3-TAFII31-GCN5L acetylase (STAGA) complex (MARTINEZ et al. 2001) and p300 histone acetyltransferase (RAPIC-OTRIN et al. 2002), while DDB2 can interact with CREB-binding protein (DATTA et al. 2001). DDB might function as a repair protein to alter chromatin structure and recruit nucleotide excision repair (NER) factors to DNA damage sites. DDB2 is also known as xeroderma pigmentosum complementation group E (XPE) and cells with mutations in this gene are mildly defective in NER of DNA damage (SHIYANOV et al. 1999a; LIU et al. 2000). However, DDB was found not to be required in NER reconsti- BRFtution studies in vitro (ABOUSSEKHRA and WOOD 1995; MU et al. 1995; KAZANTSEV et al. 1996) despite the fact that damaged DNA-binding activity of DDB is absent in cells of a subset of XPE patients (CHU and CHANG 1988; KATAOKA and FUJIWARA 1991; KEENEY et al. 1992, 1993). In vivo studies have shown that XPE cells (DDB2 mutants) are selectively defective in global genomic repair (GGR; HWANG et al. 1999). No mutations of mammalian DDB1 have been described although ZOLEZZI et al. (2002) discussed that although DDB1 is not necessarily a lethal factor in a unicellular system, the very potent defects that result as a consequence of the Schizosaccharomyces pombe DDB1 deletion would suggest that mutations in the mammalian DDB1 gene are likely to be lethal.

Because functions of DDB other than directly in DNA repair have recently been suggested (NICHOLS et al. 2000; TAKATA et al. 2002; ZOLEZZI et al. 2002), the present study was performed to investigate the properties of DDB1 in the whole body of Drosophila melanogaster. DDB2 binds to E2F-1, which is a cell cycle regulatory transcription factor (HAYES et al. 1998). DDB binds to cullin 4A, which is believed to be a ubiquitin-protein isopeptide ligase (type E3; SHIYANOV et al. 1999b). The apolipoprotein B (apoB) gene regulatory factor-2 (BRF-2)/human hepatitis B virus X-associated protein-1 (XAP-1)/DDB1 may belong to a new family of transcription factors and control apoB gene transcription (KRISHNAMOORTHY et al. 1997). DDB1 also binds to viral transcriptional transactivators, including the hepatitis B virus X protein (HBVx; BUTEL et al. 1995). DDB1 has roles in chromosome segregation and the aberrant nuclear structures observed in the ddb1Δ strain in S. pombe (ZOLEZZI et al. …