DNA Repair and Transcriptional Deficiencies Caused by Mutations in the Drosophila p52 Subunit of TFIIH Generate Developmental Defects and Chromosome Fragility

Abstract

The transcription and DNA repair factor TFIIH is composed of 10 subunits. Mutations in the XPB, XPD, and p8 subunits are genetically linked to human diseases, including cancer. However, no reports of mutations in other TFIIH subunits have been reported in higher eukaryotes. Here, we analyze at genetic, molecular, and biochemical levels the Drosophila melanogaster p52 (DMP52) subunit of TFIIH. We found that DMP52 is encoded by the gene marionette in Drosophila and that a defective DMP52 produces UV light-sensitive flies and specific phenotypes during development: organisms are smaller than their wild-type siblings and present tumors and chromosomal instability. The human homologue of DMP52 partially rescues some of these phenotypes. Some of the defects observed in the fly caused by mutations in DMP52 generate trichothiodystrophy and cancer-like phenotypes. Biochemical analysis of DMP52 point mutations introduced in human p52 at positions homologous to those of defects in DMP52 destabilize the interaction between p52 and XPB, another TFIIH subunit, thus compromising the assembly of the complex. This study significantly extends the role of p52 in regulating XPB ATPase activity and, consequently, both its transcriptional and nucleotide excision repair functions.

The genetic depletion or the triptolide inhibition of TFIIH in p53 deficient cells induce a JNK-dependent cell death in Drosophila

“…Thus, to direct the expression of Dmp52i, we first used the MS1096 driver, which directs the expression of Gal4 in the dorsal domain of the wing pouch; we then crossed these flies with flies carrying dsRNA against Dmp52 (Dmp52i) from the Vienna Resource Center. Consistent with results from our previous work using Dmp52 mutant alleles (Fregoso et al, 2007), we observed a reduction in the size of adult wings in flies expressing Dmp52i compared with flies carrying only the driver (Fig. 3).…”

“…In a previous analysis of mutant alleles in Dmp52, we discovered the presence of chromosomal aberrations in neuroblast metaphasic chromosomes (Fregoso et al, 2007). This defect may be related to the increase in apoptosis observed in the wing discs that have reduced levels of Dmp52.…”

“…In Drosophila, mutant alleles of the Dmp52 gene are lethal, but heteroallelic combinations of these mutants may generate adult organisms that are smaller than wild-type flies with melanotic tumours and chromosomal instabilities (Fregoso et al, 2007). These phenotypes are pleiotropic and may be caused by the accumulation of defects during development; it is therefore difficult to determine the contribution of the genetic interaction of factors such as Dp53.…”

“…Mutations in other subunits of TFIIH associated with syndromes have not yet been reported in humans. Using Drosophila melanogaster as a model to reveal the function of other subunits, it has been demonstrated that Dmp52 mutations generate phenotypes associated with these three syndromes, indicating that Dmp52 plays an important role in regulating the entire activity of TFIIH during several cellular processes Fregoso et al, 2007).…”

Control of the function of the transcription and repair factor TFIIH by the action of the cochaperone Ydj1

“…Along this line, phosphorylation of Ser751 in XPB needs to be carefully regulated to permit the incision during NER (34). XPB retention and ATPase stimulation for appropriate NER and transcription are dependent on the integrity of Ydj1-binding motif present in Tfb2/p52 to avoid strong defects in Drosophila (35). The other two motives present in Tfb2/p52, located in the C-terminal part, contain key residues to establish contact with Tfb5 (36) (Fig.…”