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Molecular Characterization of Mutant Alleles of the DNA Repair/ Basal Transcription Factor Haywire/ERCC3 in Drosophila

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Molecular Characterization of Mutant Alleles of the DNA Repair/ Basal Transcription Factor Haywire/ERCC3 in Drosophila Copyright 1999 by the Genetics Society of America Molecular Characterization of Mutant Alleles of the DNA Repair/ Basal Transcription Factor haywire/ERCC3 in Drosophila Leslie C. Mounkes*,²,1 and Margaret T. Fuller² *Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 and ²Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford, California 94309 ABSTRACT The haywire gene of Drosophila encodes a putative helicase essential for transcription and nucleotide excision repair. A haywire allele encoding a dominant acting poison product, lethal alleles, and viable but UV-sensitive alleles isolated as revertants of the dominant acting poison allele were molecularly character- ized. Sequence analysis of lethal haywire alleles revealed the importance of the nucleotide-binding domain, suggesting an essential role for ATPase activity. The viable hay nc2 allele, which encodes a poison product, has a single amino acid change in conserved helicase domain VI. This mutation results in accumulation of a 68-kD polypeptide that is much more abundant than the wild-type haywire protein. HE haywire locus of Drosophila encodes the ¯y ho- ily of DNA/RNA helicases (Gorbalenya et al. 1989; Tmolog of ERCC3, a human gene associated with the Linder et al. 1989). Two of these, the predicted nucleo- DNA repair de®ciency disease xeroderma pigmentosum tide- and magnesium-binding domains, are responsible group B (XP-B) (Mounkes et al. 1992). Mutations in for the essential ATPase activity of the SSL2/RAD25 the Saccharomyces cerevisiae homolog SSL2/RAD25 are de- (Park et al. 1992) and ERCC3 (Ma et al. 1994a) products. fective in overall nucleotide excision and transcription- Helicase activity is associated with the TFIIH transcrip- coupled repair (Sweder and Hanawalt 1994). In addi- tion complex (Schaeffer et al. 1993; Drapkin et al. tion, these proteins appear to play a role in transcrip- 1994), and helicase activity of the ERCC3 protein has tion. The human ERCC3 (Schaeffer et al. 1993) and been demonstrated in vitro (Ma et al. 1994b). Helicase S. cerevisiae SSL2/RAD25 (Feaver et al. 1993) gene prod- activity of ERCC3 requires the remaining conserved heli- ucts are found to be associated with the basal transcrip- case domains, which have been shown by mutational tion factor TFIIH. Puri®ed TFIIH complements the ex- analysis to function in excision repair of UV-sensitive cision repair defect of lysates from cells lacking ERCC3 cell cultures (Ma et al. 1994a). function (Drapkin et al. 1994). Consistent with a func- The Drosophila ERCC3 homolog haywire was originally tion in transcription, SSL2/RAD25 is an essential gene identi®ed through an unusual mutant allele, hay nc2, that (Park et al. 1992), and a temperature-sensitive allele acted as a dominant enhancer of mutations in the testis- of SSL2/RAD25 inhibits transcription at the restrictive speci®c b-tubulin B2t. Males heterozygous for either temperature (Qui et al. 1993). Different alleles of SSL2/ hay nc2 or B2t null and wild type are fertile at 258. However, RAD25 show defects that can separate the roles of the ¯ies transheterozygous for one copy of hay nc2 and one protein in excision repair and transcription (Guzder copy of B2t null are male sterile at 258. The genetic interac- et al. 1994). As for SSL2/RAD25 of yeast, extreme alleles tion between hay nc2 and B2t null suggested the haync2 allele of haywire are lethal (Regan and Fuller 1990), whereas encoded a poison product because ¯ies transheterozy- viable mutant alleles are UV sensitive (Mounkes et al. gous for B2t mutations and a de®ciency of haywire were 1992). Only a few cases of XP-B are known to date, male fertile (Regan and Fuller 1988). Revertant alleles suggesting that ERCC3 is also essential in humans, and of hay nc2 abolish the genetic interaction between hay nc2 that only patients carrying special weak mutant alleles of and B2t null, but still result in novel abnormal phenotypes ERCC3 survive to birth (Weeda et al. 1990; Vermeulen et with respect to male fertility (Regan and Fuller 1990), al. 1994). A viable point mutation in ERCC3 now de®nes suggesting that a poison product encoded by the hay nc2 a new trichothidystrophy complementation group which, allele is responsible for the failure to complement tu- however, does not cause XP-B (Weeda et al. 1997). bulin mutations. The ¯y, yeast, and human ERCC3 homologs show hall- In this article, we report the molecular characteriza- mark conserved domains characteristic of the superfam- tion of mutations that identify regions of haywire that are important either for viability or DNA repair. In addi- tion, we show data indicating that the hay nc2 allele causes Corresponding author: Margaret T. Fuller, Department of Develop- production of a 68-kD polypeptide that accumulates mental Biology, Stanford University School of Medicine, Stanford, CA 94305-5427. E-mail: [email protected] to much higher levels than wild-type haywire protein. 1 Present address: California Paci®c Medical Center Research Institute, Mutant haywire alleles that revert the genetic interaction Stern Building, 2330 Clay St., San Francisco, CA 94115. between hay nc2 and B2t null abrogate production of the Genetics 152: 291±297 ( May 1999) 292 L. C. Mounkes and M. T. Fuller stable, truncated protein, suggesting that the same de- haywire antibody production and puri®cation: A 2.5-kb fect causes accumulation of the 68-kD polypeptide and HindIII-NotI fragment from the haywire cDNA plasmid was treated with Klenow (Mounkes et al. 1992), inserted into the poison product activity. pQE30 (Qiagen) digested with SmaI, and phosphatase treated to make a construct encoding full-length haywire protein tagged with six histidine residues at the N terminus of haywire. MATERIALS AND METHODS Induction of fusion protein expression with 2 mm IPTG Fly strains and culture: Fly stocks and crosses were main- (Sigma, St. Louis) and harvesting of fusion protein were per- tained using standard ¯y media at 258. Balancer chromosomes, formed as described (Mounkes and Fuller 1998) with the visible markers, Df(1)w, B2t null, and Df(3L)E(z)7e11 (67E1-4;67F1- following differences. haywire fusion protein was not soluble in 8 m urea and was solubilized in 6 m guanidine/TN1. Super- 3) are described in Lindsley and Zimm (1992). The following 21 haywire alleles were used: hay nc2, a recessive male sterile muta- natants with soluble protein were applied to a Ni ±NTA col- tion that fails to complement mutations in B2t (Regan and umn (Qiagen) equilibrated in 6 m guanidine/TN1. The col- Fuller 1988), and six alleles recovered as revertants of the umn was washed with 6 m guanidine/TN1, and fusion protein failure of hay nc2 to complement B2t null (Regan and Fuller was eluted in TN1/8 m urea and 60 mm imidazole. Antiserum 1990). Fertility of mutant combinations of alleles was deter- to puri®ed protein was raised in rabbits (Josman Laboratories, mined by testis dissections, as described (Mounkes et al. 1992). Napa, CA). Antibodies were af®nity puri®ed using the pure, Sequencing of haywire alleles: The coding region of haywire original antigen coupled to Af®-gel 10 (Bio-Rad, Richmond, was divided into halves by two sets of PCR primers, each ampli- CA) essentially as described (Mounkes and Fuller 1998). fying z1.5-kb fragments from haywire. Primers used to amplify Western analysis: Protein samples were made by grinding the 59 half of haywire were 59-GGAAGCTTGCCATACGTGCT 20 adult ¯ies in an Eppendorf tube in 0.15 ml of solution GTGTTAC-39, and 59-CCAAGCTTCCAGCACCATGATGC A (0.1 m Tris, pH 9.0, 0.1 m EDTA, 1% SDS, 0.5% DEPC) CCC-39. Primers used to amplify the 39 half of haywire were plus 1 mm PMSF. Samples were spun in the microfuge for 30 59-GGGCTGTGGAATTCTTGTGACCACATAC-39 and 59-GGA sec to pellet large body parts. Supernatants were transferred AGCTTGGGTTACTGCAGTGGTAAAG-39. For the viable al- to a new tube, 10 ml each were brought to 13 sample buffer leles hay nc2 and hay nc2rv8, templates for the PCR reactions were (Laemmli 1970), and proteins were separated by 10% SDS- genomic DNA made from ¯ies heterozygous for the viable PAGE. Gels were blotted to nitrocellulose (Schleicher & haywire allele and a de®ciency for haywire, Df(3L)E(z)7e11. For Schuell, Keene, NH) as described (Towbin et al. 1979). haywire lethal haywire alleles, template DNA for the PCR reactions was was visualized by enhanced chemiluminescence (ECL) stain- made from ¯ies heterozygous for the lethal revertant allele ing as described by the manufacturer (Amersham). Af®nity- and the parent chromosome, hay nc2. PCR products were puri- puri®ed primary antibody was used at 1:1500. Horseradish ®ed and sequenced as described (Sanger et al. 1977; Biggin peroxidase-conjugated donkey anti-rabbit secondary antibody et al. 1983; Kretz et al. 1989). One strand of the revertant (Amersham) was used at 1:4000. alleles was sequenced, except in areas where there were Germline transformations: PCR mutagenesis of a 380-nucle- changes. Changes were veri®ed by sequencing the comple- otide BglII-SacI fragment of the 39 end of the haywire cDNA mentary strand. was performed to introduce the same C-to-T transition found Northern analysis: Total RNA was isolated by RNAzol extrac- in the hay nc2 allele. The mutagenic oligonucleotide had the tion according to Cinna Scienti®c. RNA (30 mg) was run on sequence 59-CCAGATCTCTTCGCATGGCGGCTCTTGTCGT 0.1 m sodium phosphate gels, blotted onto a Hybond mem- CAGGAGG-39. The mutagenized BglII-SacI fragment was sub- brane (Amersham, Arlington Heights, IL), and hybridized as cloned into a 4.5-kb BamHI fragment of the 39 half of a geno- described (Sambrook et al.
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