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*,†,1 and Margaret T. Fuller† *Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado 80309 and †Departments 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 characterized. 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.
T
HE haywire locus of Drosophila encodes the fly homolog of ERCC3, a human gene associated with the DNA repair deficiency disease xeroderma pigmentosum group B (XP-B) (Mounkes et al. 1992). Mutations in the Saccharomyces cerevisiae homolog SSL2/RAD25 are defective in overall nucleotide excision and transcriptioncoupled repair (Sweder and Hanawalt 1994). In addition, these proteins appear to play a role in transcription. The human ERCC3 (Schaeffer et al. 1993) and S. cerevisiae SSL2/RAD25 (Feaver et al. 1993) gene products are found to be associated with the basal transcription factor TFIIH. Purified TFIIH complements the excision repair defect of lysates from cells lacking ERCC3 function (Drapkin et al. 1994). Consistent with a function in transcription, SSL2/RAD25 is an essential gene (Park et al. 1992), and a temperature-sensitive allele of SSL2/RAD25 inhibits transcription at the restrictive temperature (Qui et al. 1993). Different alleles of SSL2/ RAD25 show defects that can separate the roles of the protein in excision repair and transcription (Guzder et al. 1994). As for SSL2/RAD25 of yeast, extreme alleles of haywire are lethal (Regan and Fuller 1990), whereas viable mutant alleles are UV sensitive (Mounkes et al. 1992). Only a few cases of XP-B are known to date, suggesting that ERCC3 is also essential in humans, and that only patients carrying special weak mutant alleles of ERCC3 survive to birth (Weeda et al. 1990; Vermeulen et al. 1994). A viable point mutation in ERCC3 now defines a new trichothidystrophy complementation group which, however, does not cause XP-B (Weeda et al. 1997). The fly, yeast, and human ERCC3 homologs show hallmark conserved domains characteristic of the superfam-
Corresponding author: Margaret T. Fuller, Department of Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5427. E-mail:
[email protected] 1 Present address: California Pacific Medical Center Research Institute, Stern Building, 2330 Clay St., San Francisco, CA 94115. Genetics 152: 291–297 ( May 1999)
ily of DNA/RNA helicases (Gorbalenya et al. 1989; Linder et al. 1989). Two of these, the predicted nucleotide- and magnesium-binding domains, are responsible for the essential ATPase activity of the SSL2/RAD25 (Park et al. 1992) and ERCC3 (Ma et al. 1994a) products. Helicase activity is associated with the TFIIH transcription complex (Schaeffer et al. 1993; Drapkin et al. 1994), and helicase activity of the ERCC3 protein has been demonstrated in vitro (Ma et al. 1994b). Helicase activity of ERCC3 requires the remaining conserved helicase domains, which have been shown by mutational analysis to function in excision repair of UV-sensitive cell cultures (Ma et al. 1994a). The Drosophila ERCC3 homolog haywire was originally identified through an unusual mutant allele, hay nc2, that acted as a dominant enhancer of mutations in the testisspecific b-tubulin B2t. Males heterozygous for either hay nc2 or B2t null and wild type are fertile at 258. However, flies transheterozygous for one copy of hay nc2 and one copy of B2t null are male sterile at 258. The genetic interaction between hay nc2 and B2t null suggested the haync2 allele encoded a poison product because flies transheterozygous for B2t mutations and a deficiency of haywire were male fertile (Regan and Fuller 1988). Revertant alleles of hay nc2 abolish the genetic interaction between hay nc2 and B2t null, but still result in novel abnormal phenotypes with respect to male fertility (Regan and Fuller 1990), suggesting that a poison product encoded by the hay nc2 allele is responsible for the failure to complement tubulin mutations. In this article, we report the molecular characterization of mutations that identify regions of haywire that are important either for viability or DNA repair. In addition, we show data indicating that the hay nc2 allele causes production of a 68-kD polypeptide that accumulates to much higher levels than wild-type haywire protein. Mutant haywire alleles that revert the genetic interaction between hay nc2 and B2t null abrogate production of the
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stable, truncated protein, suggesting that the same defect causes accumulation of the 68-kD polypeptide and the poison product activity. MATERIALS AND METHODS Fly strains and culture: Fly stocks and crosses were maintained using standard fly media at 258. Balancer chromosomes, visible markers, Df(1)w, B2t null, and Df(3L)E(z)7e11 (67E1-4;67F13) are described in Lindsley and Zimm (1992). The following haywire alleles were used: hay nc2, a recessive male sterile mutation that fails to complement mutations in B2t (Regan and Fuller 1988), and six alleles recovered as revertants of the failure of hay nc2 to complement B2t null (Regan and Fuller 1990). Fertility of mutant combinations of alleles was determined by testis dissections, as described (Mounkes et al. 1992). Sequencing of haywire alleles: The coding region of haywire was divided into halves by two sets of PCR primers, each amplifying z1.5-kb fragments from haywire. Primers used to amplify the 59 half of haywire were 59-GGAAGCTTGCCATACGTGCT GTGTTAC-39, and 59-CCAAGCTTCCAGCACCATGATGC CCC-39. Primers used to amplify the 39 half of haywire were 59-GGGCTGTGGAATTCTTGTGACCACATAC-39 and 59-GGA AGCTTGGGTTACTGCAGTGGTAAAG-39. For the viable alleles hay nc2 and hay nc2rv8, templates for the PCR reactions were genomic DNA made from flies heterozygous for the viable haywire allele and a deficiency for haywire, Df(3L)E(z)7e11. For lethal haywire alleles, template DNA for the PCR reactions was made from flies heterozygous for the lethal revertant allele and the parent chromosome, hay nc2. PCR products were purified and sequenced as described (Sanger et al. 1977; Biggin et al. 1983; Kretz et al. 1989). One strand of the revertant alleles was sequenced, except in areas where there were changes. Changes were verified by sequencing the complementary strand. Northern analysis: Total RNA was isolated by RNAzol extraction according to Cinna Scientific. RNA (30 mg) was run on 0.1 m sodium phosphate gels, blotted onto a Hybond membrane (Amersham, Arlington Heights, IL), and hybridized as described (Sambrook et al. 1989). Blots were probed with a haywire riboprobe made from a HindIII-linearized cDNA construct (Mounkes et al. 1992) and a random-primer-labeled 1.5-kb EcoRI-SalI fragment from the plasmid containing rp49 (O’Connell and Rosbash 1984).
haywire antibody production and purification: A 2.5-kb HindIII-NotI fragment from the haywire cDNA plasmid was treated with Klenow (Mounkes et al. 1992), inserted into 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. Induction of fusion protein expression with 2 mm IPTG (Sigma, St. Louis) and harvesting of fusion protein were performed as described (Mounkes and Fuller 1998) with the following differences. haywire fusion protein was not soluble in 8 m urea and was solubilized in 6 m guanidine/TN1. Supernatants with soluble protein were applied to a Ni21–NTA column (Qiagen) equilibrated in 6 m guanidine/TN1. The column was washed with 6 m guanidine/TN1, and fusion protein was eluted in TN1/8 m urea and 60 mm imidazole. Antiserum to purified protein was raised in rabbits ( Josman Laboratories, Napa, CA). Antibodies were affinity purified using the pure, original antigen coupled to Affi-gel 10 (Bio-Rad, Richmond, CA) essentially as described (Mounkes and Fuller 1998). Western analysis: Protein samples were made by grinding 20 adult flies in an Eppendorf tube in 0.15 ml of solution A (0.1 m Tris, pH 9.0, 0.1 m EDTA, 1% SDS, 0.5% DEPC) plus 1 mm PMSF. Samples were spun in the microfuge for 30 sec to pellet large body parts. Supernatants were transferred to a new tube, 10 ml each were brought to 13 sample buffer (Laemmli 1970), and proteins were separated by 10% SDSPAGE. Gels were blotted to nitrocellulose (Schleicher & Schuell, Keene, NH) as described (Towbin et al. 1979). haywire was visualized by enhanced chemiluminescence (ECL) staining as described by the manufacturer (Amersham). Affinitypurified primary antibody was used at 1:1500. Horseradish peroxidase-conjugated donkey anti-rabbit secondary antibody (Amersham) was used at 1:4000. Germline transformations: PCR mutagenesis of a 380-nucleotide BglII-SacI fragment of the 39 end of the haywire cDNA was performed to introduce the same C-to-T transition found in the hay nc2 allele. The mutagenic oligonucleotide had the sequence 59-CCAGATCTCTTCGCATGGCGGCTCTTGTCGT CAGGAGG-39. The mutagenized BglII-SacI fragment was subcloned into a 4.5-kb BamHI fragment of the 39 half of a genomic haywire clone (Mounkes et al. 1992). The resulting BamHI fragment was then used to replace the corresponding wild-type fragment by cloning into a 5.2-kb BamHI fragment obtained by partial digestion of the haywire genomic clone. The entire 7.8kb haywire genomic fragment with the C-to-T change was then
TABLE 1 Alterations in lethal and viable haywire alleles
Allele
Typea
Nucleic acid change
Amino acid change
Residue
b
L L L L V, uv L V, uv
CAG → TAG TGC → TAC CGA → TGA TGG → TGA GAG → AAG Rearranged CGT → TGT
Gln → STOP Cys → Tyr Arg → STOP Trp → STOP Glu → Lys Deletes GAGKS Arg → Cys
657 377 380 441 278 265–269 652
haync2rv1 hay nc2rv2 b hay nc2rv3 b hay nc2rv7 b hay nc2rv8 c hay nc2rv12 b hay nc2 b
a
haywire alleles are either lethal (L) or viable (V) and UV sensitive (uv). Single nucleotide changes in hay nc2rv1,2,3,7,8 and haync2 are consistent with the mode of induction of these alleles by EMS mutagenesis (Regan and Fuller 1990), a potent inducer of point mutations (Ashburner 1989) that favors GC-to-AT transitions (Williams and Shaw 1987). c The hay nc2rv12 mutation was induced by diepoxybutane (L. C. Mounkes and K. Schuske, unpublished results), a known inducer of deficiencies (e.g., Olsen and Green 1982). b
haywire Alleles
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Figure 2.—The hay nc2 mutation results in accumulation of a 68-kD polypeptide (nc2) not seen in the wild type. Western analysis of whole-fly homogenates from haywire alleles. WT in first lanes (A and B) indicates homogenates from the wildtype background strain, red e, on which the hay nc2 mutation and subsequent revertant alleles were made. Positions of molecular weight markers are indicated on the left in kilodaltons. (A) Mutant haywire alleles over a wild-type balancer. (B) Comparison of mutant haywire alleles in combination with a deficiency (Df ) or wild-type balancer chromosome (1).
Figure 1.—Alterations in lethal and viable haywire alleles. Amino acid changes are indicated above the appropriate residues. X indicates sequences deleted in the e11 wild-type strain; $ indicates a mutation to a stop codon. The conserved helicase domains are defined by dashed lines underneath the sequences. The asterisk indicates the conserved lysine within the P loop of the nucleotide-binding domain. WT, wild type; rv2, hay nc2rv2; rv3, hay nc2rv3; rv8, hay nc2rv8; rv12, hay nc2rv12; rv7, hay nc2rv7; rv1, hay nc2rv1. Alleles in boldface type are homozygous viable. All other mutant haywire alleles are lethal.
excised with HindIII and cloned into the pCaSpeR transformation vector (Pirotta 1988) previously linearized with HindIII and treated with calf intestinal phosphatase (CIP). All cloning junctions within haywire and the mutated site were sequenced to verify sequence integrity at the cloning junctions and introduction of the mutation. Restriction enzymes, CIP, and Taq DNA polymerase were purchased from Boehringer Mannheim (Indianapolis). DNA (200 mg/ml) twice purified by cesium chloride banding was injected by standard methods (Rubin and Spradling 1982) into Df(1)w embryos with p[ry1, D2–3] (Robertson et al. 1988) at 200 mg/ml as a source of transposase. One transformant linked to the X chromosome was isolated.
RESULTS
Molecular lesion in the hay nc2 allele responsible for the dominant genetic interaction with B2t: The original hay nc2 mutation caused replacement of arginine 652 with a cysteine (Table 1) in conserved helicase domain VI
(Figure 1). This missense mutation was present in all the revertant alleles of hay nc2, as expected. As hay nc2 homozygotes are viable, arginine 652 is not absolutely required for haywire function in basal transcription, but it may be important in DNA repair since the hay nc2 allele is UV sensitive (Mounkes et al. 1992). The hay nc2 mutation resulted in accumulation of a polypeptide that migrated on SDS polyacrylamide gels as 68 kD, cross-reacted with anti-haywire antisera, and was present at much higher levels in the mutant than in the wild type (Figure 2). The 68-kD apparent molecular weight polypeptide was 26 kD less than expected for the full-length protein, suggesting the possibility that the hay nc2 allele results in accumulation of a shortened form of the haywire protein. Although truncation at the site of the hay nc2 mutation (Arg652-to-Cys) would produce a predicted 68-kD N-terminal peptide, the region of haywire protein present in the 68-kD polypeptide is not known. It is also formally possible that the 68-kD polypeptide does not correspond to a fragment of the haywire protein, but instead is an unrelated polypeptide that is overproduced specifically in the hay nc2 mutant background and that cross-reacts with the anti-haywire antisera. The 68-kD polypeptide did not accumulate in either the wild-type background strain (red e, Figure 2) or in flies carrying revertant alleles of hay nc2 (Figure 2), indicating that accumulation of the 68-kD product results from the hay nc2 allele. The affinity-purified, antihaywire antibodies recognized many bands smaller than the expected wild-type size in Western blots. Although some of these smaller peptides could be proteins with cross-reacting epitopes, some could also be degradation products of haywire. To demonstrate that the Arg652-to-Cys mutation was
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TABLE 2 Transgenic animals made by introduction of the Arg652-to-Cys change of hay nc2 into a genomic rescue fragment show a genetic interaction with the B2t null allele
1b 2c 3c 4
Figure 3.—The hay nc2 missense mutation is responsible for production of the 68-kD polypeptide (nc2 trunc). Western analysis of hay nc2 transgenic animal made by site-directed mutagenesis of the wild-type gene. Molecular weight markers (kilodaltons) are indicated on the right. WT, wild-type haywire protein; nc2 trunc, truncated product accumulating in hay nc2 flies. The lanes are as follows: 1, wild-type background strain (red e); nc2/1, hay nc2 over a wild-type balancer (TM3); P[A3,w1], transgenic animal with mutated copy of haywire carrying the hay nc2 C→T transition introduced into an otherwise wild-type haywire genomic fragment; w; ScO/CyO, genetic background in which the transgene was propagated; P[A, ry1], wild-type copy of haywire introduced into flies. Note: Both P[A3, w1] and P[A, ry1] have two wild-type endogenous copies of haywire.
responsible for the production of the 68-kD polypeptide in hay nc2 animals, we constructed a transgenic animal with the hay nc2 C-to-T transition introduced into an otherwise wild-type haywire genomic fragment. The mutated haywire gene was introduced into a background wild type for haywire, and the resulting transformants were tested for production of the truncated hay nc2 product. Several transformant lines established from a single Go fly all made and accumulated the 68-kD polypeptide seen in the original hay nc2 flies (Figure 3). Transformants carrying the Arg652-to-Cys change also showed the genetic interaction with B2t null characteristic of the original hay nc2 mutation. One copy of the altered gene carried on the insert plus one wild-type endogenous copy of haywire resulted in male sterility in flies heterozygous for the B2t null allele (Table 2, line 5). (The flies shown in Table 2, line 5, have one of the two endogenous copies of haywire deleted.) One copy of the altered transgene also caused reduced male fertility in B2t null/1 heterozygotes, even in the presence of two wildtype endogenous copies of haywire (Table 2, line 7),
Fertilea
Sterilea
0
35
35 27
0 0
32
0
1
51
1 1
42
0
1 B2t null
23
8
33
0
1 B2t null haync2 1 B2t null/1 haync2/1 1 B2t null Df(hay) 1
5d
c
P[A3, w1]/.;
1 B2t null Df(hay) 1
6
P[A3, w1]/.;
1 Df(hay)
7
P[A3, w1]/.;
1 1
8
P[A3, w1]/.;
1 1
Lines 1–3, Sibling progeny from the cross hay nc2/TM3 3 B2t null/TM3. Line 4, Df(hay) indicates Df(3L)E(z)7e11, a deletion that removes haywire. Test class flies were derived from crossing Df(1)w; B2t null/TM3 and Df(1)w ; E(z)7e11/TM3 flies. A deletion of hay complements B2t null, while the original hay nc2 allele fails to complement B2t null. Lines 5–7, sibling progeny from the cross P[A3, w1]/P[A3,w1]; Df(3L)E(z)7e11/TM3 virgin females 3 Df(1)w; riB2t nulle/TM6B males. The hay nc2 transgene caused failure to complement B2t null in the presence of one endogenous wild-type copy of haywire partially alleviated male sterility (line 7), suggesting that the poison product encoded by the hay nc2 allele on the transgene acts as an antimorph and competes with wild-type haywire protein. Line 8, The transgene insert did not cause male sterility on its own. a Fertility was scored by visual inspection of dissected testes. b The original genetic interaction between hay nc2 and B2t null. c Control class siblings. d P[A3, w1] indicates an insertion on the X chromosome of the mutant copy of haywire, introducing the same Arg652-toCys change found in the haync2 allele.
although these flies were not as sterile as hay nc2 1/1 B2t null. Thus, the genetic interaction appeared to be sensitive to the ratio between mutant and wild-type haywire (Table 2, compare lines 5 and 7). Similar behavior was observed in the failure of a missense mutation in a-tubulin to complement B2t null (Hays et al. 1989), and it is consistent with hay nc2 encoding an antimorphic poison that can compete with the wild-type protein. The Arg652-to-Cys altered transgene caused both accumulation of the 68-kD polypeptide and failure to complement B2t mutations, demonstrating that the single mutation was responsible for both phenotypes associated with hay nc2.
haywire Alleles
Figure 4.—Flies carrying the hay nc2 allele accumulate normal levels of haywire transcript. Northern analysis of flies hemior heterozygous for mutant haywire alleles. Molecular weight markers (in kilobases) are indicated to the left of the blot. A 2.5-kb haywire transcript (hay) was observed in all haywire alleles. The blot was reprobed for rp49 (20) as a loading control. Lanes: 1, red e ; 2, E(z)7e11/TM3; 3, hay nc2/E(z)7e11; 4, hay nc2rv8/ E(z)7e11; 5, hay nc2/TM3; 6, hay nc2rv8/TM3; 7, hay nc2rv1/TM3; 8, hay nc2rv2/TM3; 9, hay nc2rv3/TM3; 10, hay nc2rv7/TM3; 11, hay nc2rv12/ TM3.
The high levels of the 68-kD possible fragment of haywire accumulating in the hay nc2 mutant did not appear to result from increased transcription or stability of the haywire mRNA encoded by hay nc2. Northern blot analysis of all the alleles sequenced revealed relatively normal levels of haywire transcript in all cases (Figure 4), with the possible exception of the hay nc2rv3 and hay nc2rv7 alleles. hay nc2rv3/TM3 and hay nc2rv7/TM3 flies appeared to have reduced levels of haywire mRNA compared to other alleles tested over the same balancer chromosome. As both of these alleles introduce stop codons early in the haywire protein-coding region (below), they may cause destabilization of the mRNA encoding the mutant allele (Talesa et al. 1995). In addition, accumulation of the 68-kD polypeptide in hay nc2 did not appear to be caused by translation of an abnormally processed message, as no alterations in haywire message size were detected in flies carrying the hay nc2 allele. Lethal alleles isolated as revertants of hay nc2: Several mutations that revert the failure of hay nc2 to complement mutations in B2t introduce stop codons into the haywire coding region, supporting the hypothesis that hay nc2 fails to complement B2t because of the production of a poison product (Table 1). hay nc2rv1 introduces a stop codon in helicase domain VI, hay nc2rv3 results in a stop codon in the conserved nucleotide-binding domain, and hay nc2rv7 has a stop codon six amino acids before the predicted magnesium-binding domain (helicase domain II, Figure 1). These nonsense mutations are all lethal, supporting an essential role in flies for the haywire gene product, probably in basal transcription.
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A missense mutation (hay nc2rv2) and small rearrangement (hay nc2rv12) indicate that the nucleotide-binding domain of haywire is essential for viability. The hay nc2rv2 mutation replaced cysteine 377 with a tyrosine residue (Table 1). This cysteine, which lies eight amino acids downstream of the P loop (Walker et al. 1982) in the nucleotidebinding domain (Figure 1), is conserved among the Drosophila, human, mouse, and yeast homologs. The hay nc2rv12 lesion was a small chromosomal rearrangement located at the ‘GAGKS’ P loop of the nucleotide-binding site (brackets in Figure 1). All these revertants of the genetic interaction of hay nc2 also reverted the defect that leads to accumulation of the 68-kD polypeptide (Figure 2). A viable revertant of hay nc2: The hay nc2rv8 viable mutation changed glutamic acid 278 to lysine, introducing a charge change and possibly affecting protein structure or stability. hay nc2rv8 homozygotes are viable, indicating that glutamic acid 278 is not essential for basal transcription. Glu278 could be important for the repair of UV damage, since this allele also showed UV sensitivity (Mounkes et al. 1992). Alternatively, the UV sensitivity could be caused by the still-present hay nc2 lesion, while the hay nc2rv8 reversion merely alleviated the poison nature of the hay nc2 product. Again, the hay nc2rv8 missense mutation also reverted the defect leading to accumulation of the 68-kD polypeptide characteristic of hay nc2. Altered amino acids encoded by wild-type haywire alleles: The sequences of three wild-type alleles from different genetic backgrounds revealed regions of haywire/ ERCC3 that are not essential (Figure 1). A wild-type cDNA from a dp cn bn strain and two different wild-type genomic sequences (red e and e11) differed at several positions within the amino acid coding region. Alanine residues at positions 238 and 244 in the dp cn bn and red e backgrounds were changed to threonine and proline, respectively, in wild-type haywire from the e11 chromosome. Amino acid 663 was a methionine in the e11 strain but a leucine in both the red e and dp cn bn strains. In addition, the e11 wild-type allele of haywire had a fouramino-acid deficiency at positions 253–256 compared with haywire on both the red e and dp cn bn chromosomes. The four consecutive amino acids missing in the e11 background are valine/valine/alanine/alanine. Most of the wild-type polymorphisms occur in regions that are poorly conserved between the Drosophila, yeast, and mammalian homologs, and may identify residues of haywire not essential for function. DISCUSSION
The single nucleotide mutation in the hay nc2 allele that causes an arginine-to-cysteine change is responsible for both the production of a shortened polypeptide associated with the hay nc2 allele and the genetic interaction observed between hay nc2 and B2t mutations. Several molecular mechanisms of reverting the genetic interac-
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tion between hay nc2 and B2t null are suggested by sequence analysis of the haywire alleles obtained as revertants of hay nc2. Destruction of the P loop of the nucleotide-binding domain reverted the genetic interaction between hay nc2 and B2t null (hay nc2rv12, Table 1) possibly by destroying the ATP-binding or helicase function of the hay nc2 primary product. Two other revertant alleles, hay nc2rv2 and hay nc2rv8, caused missense mutations that could alter protein conformation or stability. hay nc2rv2, which caused a change from a cysteine to tyrosine, altered a potential disulfide bond partner. hay nc2rv8, which caused a change from a glutamic acid to lysine, caused a change from a negatively charged residue to a positively charged residue. Finally, nonsense mutations hay nc2rv1, hay nc2rv3, and haync2rv7 reverted the genetic interaction between hay nc2 and Bt2 null. The finding that at least four out of six revertants of hay nc2 (the three stop codons and the P-loop mutation) are mutations that should knock out haywire function supports the idea that the original hay nc2 allele encodes a poison product. The shortened polypeptide that accumulates in hay nc2 flies is likely to be either responsible for or a direct result of the poison nature of the hay nc2 allele, as the 68-kD polypeptide no longer accumulated in six out of six mutants that reverted the dominant enhancer effect of hay nc2 (Figure 2). The 68-kD polypeptide that accumulates in hay nc2 flies is not likely to be caused by a simple truncation of the haywire protein near the site of the nc2 mutation. The stop codon mutation in hay nc2rv1 (Table 1) suggests that a simple truncation event near amino acid 652 results in neither production of a poison product nor accumulation of the 68-kD polypeptide (Figure 2). Furthermore, the hay nc2rv1 allele is lethal, perhaps because of truncation of the protein within the last conserved helicase domain, whereas the haync2 allele is viable (Table 1). The hay nc2 allele encodes a product that must contain at least some wild-type function, as hay nc2/Df(hay) flies were fully viable. As the mutation responsible for causing accumulation of the truncated product is not a stop codon, full-length haywire protein could be initially expressed from the hay nc2 allele. It is possible that this full-length but mutant haywire protein could provide sufficient haywire function for viability when initially expressed. However, defects in functioning or processing of the defective protein because of the Arg652-to-Cys mutation could alter or disrupt normally occurring processing of the full-length haywire protein, resulting in accumulation of a shortened form of the protein in hay nc2. The observation that the 68-kD shortened polypeptide accumulated to much higher levels than the wild-type haywire product also suggests the possibility of turnover of haywire/ERCC3 as a normal part of its function. We would like to raise the possibility that the haywire protein might normally be degraded as part of the functional cycle of TFIIH in transcription initiation. It is also possible that the 68-kD polypeptide that accu-
mulates in hay nc2 flies is the product of some other gene that is upregulated in this particular mutant background. If so, however, the product of this hypothetical upregulated gene would have to cross-react with the affinity-purified, anti-haywire antiserum. In addition, upregulation of the other hypothetical gene appears to depend strictly on the poison product effect of the hay nc2 allele, as the polypeptide was not detected in any of the six revertants of hay nc2. The hay nc2 allele genetically interacts with mutations in the B2t locus (Regan and Fuller 1988), which encodes a testis-specific b-tubulin isoform (Kemphues et al. 1979). Levels of b2-tubulin are critical for spermatogenesis in flies. Flies carrying a deficiency for B2t are already close to a threshhold requirement of b2-tubulin, since B2t null heterozygotes are fertile at 258 but sterile at 188 (Fuller et al. 1989). Further decreasing the level of transcription by altering the function of a basal transcription factor might bring the level of expressed b2tubulin below the critical threshold required for the many microtubule-mediated tasks necessary for spermatogenesis. It is feasible that either full-length haywire protein with the Arg652-to-Cys mutation or accumulation of a 68-kD fragment of the haywire protein in hay nc2 flies could cause such a deleterious effect on transcription, causing the hay nc2 mutation to act as a dominant enhancer of B2t mutations. We thank Barbara Robertson and Kim Schuske, who conducted genetic screens to identify new revertant alleles of hay nc2, and Cricket Wood for technical assistance. We thank Stig Hansen and Robert Tjian for help and advice in producing affinity-purified antibodies to the haywire protein. We acknowledge the protein and nucleic acid (PAN) facility, Stanford University, for synthesizing oligonucleotides used in this work. This work was supported by National Institutes of Health grants HD-18127 and HD-29194 to M.T.F.
LITERATURE CITED Ashburner, M., 1989 Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Biggin, M. D., T. J. Gibbon and G. F. Hong, 1983 Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc. Natl. Acad. Sci. USA 80: 3963–3965. Drapkin, R., J. T. Reardon, A. Ansari, J.-C. Huang, L. Zawel et al., 1994 Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. Nature 368: 769–772. Feaver, W. J., J. Q. Svejstrup, L. Bardwell, A. J. Bardwell, S. Buratowski et al., 1993 Dual roles of a multiprotein complex from S. cereviseae in transcription and DNA repair. Cell 75: 1379– 1387. Fuller, M. T., C. L. Regan, L. L. Green, B. Robertson, R. Deuring et al., 1989 Interacting genes identify interacting proteins involved in microtubule function in Drosophila. Cell Motil. Cytoskeleton 14: 128–135. Gorbalenya, A. E., E. V. Koonin, A. P. Donchenko and V. M. Blinov, 1989 Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17: 4713–4730. Guzder, S. N., P. Sung, V. Bailly, L. Prakash and S. Prakash, 1994 RAD25 is a DNA helicase required for DNA repair and RNA polymerase II transcription. Nature 369: 578–581. Hays, T., R. Deuring, B. Robertson, M. Prout and M. T. Fuller, 1989 Interacting proteins identified by genetic interactions: a missense mutation in a-tubulin fails to complement alleles of
haywire Alleles the testis-specific b-tubulin gene of Drosophila. Mol. Cell. Biol. 9: 875–884. Kemphues, K. J., R. A. Raff, T. C. Kaufman and E. C. Raff, 1979 Mutation in a structual gene for a b-tubulin specific to testis in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 76: 3991–3995. Kretz, K. A., G. S. Carson and J. S. O’brien, 1989 Direct sequencing from low-melt agarose with Sequenase. Nucleic Acids Res. 17: 5864. Laemmli, U. K., 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685. Linder, P., P. F. Lasko, M. Ashburner, P. Leroy, P. J. Nielsen et al., 1989 Birth of the D-E-A-D box. Nature 337: 121–122. Lindsley, D. L., and G. G. Zimm, 1992 The Genome of Drosophila melanogaster. Academic Press, San Diego. Ma, L., A. Westbroek, A. G. Jochemsen, G. Weeda, A. Bosch et al., 1994a Mutational analysis of ERCC3, which is involved in DNA repair and transcription initiation: identification of domains essential for the DNA repair function. Mol. Cell. Biol. 14: 4126– 4134. Ma, L., E. D. Siemssen, M. H. M. Noteborn and A. J. van der Eb, 1994b The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity. Nucleic Acids Res. 22: 4095–4102. Mounkes, L. C., and M. T. Fuller, 1998 The DUG gene of Drosophila melanogaster encodes a structural and functional homolog of the S. cerevisiae SUG1 predicted ATPase associated with the 26S proteasome. Gene 206: 165–174. Mounkes, L. C., R. S. Jones, B.-C. Liang, W. Gelbart and M. T. Fuller, 1992 A Drosophila model for xeroderma pigmentosum and Cockayne’s syndrome: haywire encodes the fly homolog of ERCC3, a human excision repair gene. Cell 71: 925–937. O’Connell, P., and M. Rosbash, 1984 Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene. Nucleic Acids Res. 12: 5495–5513. Olsen, O. A., and M. M. Green, 1982 The mutagenic effects of diepoxybutane in wild-type and mutagen-sensitive mutants of Drosophila melanogaster. Mutat. Res. 92: 107–115. Park, E., S. N. Guzder, M. H. Koken, D. I. Jaspers, G. Weeda et al., 1992 RAD25 (SSL2), the yeast homolog of the human xeroderma pigmentosum group B DNA repair gene, is essential for viability. Proc. Natl. Acad. Sci. USA 89: 11416–11420. Pirotta, V., 1988 Vectors for P-mediated transformation of Drosophila, pp. 437–456 in A Survey of Molecular Cloning Vectors and Their Uses, edited by R. L. Rodreguez and D. T. Denhardt. Butterworths, Boston. Qui, H., E. Park, L. Prakash and S. Prakash, 1993 The Saccharomyces cerevisiae DNA repair gene RAD25 is required for transcription by RNA polymerase II. Genes Dev. 7: 2161–2171. Regan, C. L., and M. T. Fuller, 1988 Interacting genes that affect microtubule function: the nc2 allele of the haywire locus fails to complement mutations in the testis-specific b-tubulin gene of Drosophila. Genes Dev. 2: 82–92.
297
Regan, C. L., and M. T. Fuller, 1990 Interacting genes that affect microtubule function in Drosophila melanogaster : two classes of mutation revert the failure to complement between haync2 and mutations in tubulin genes. Genetics 125: 77–90. Robertson, H. M., C. R. Preston, R. W. Phillis, D. M. JohnsonSchlitz, W. K. Benz et al., 1988 A stable genomic source of P element transposase in Drosophila melanogaster. Genetics 118: 461–470. Rubin, G. M., and A. C. Spradling, 1982 Genetic transformation of Drosophila with transposable element vectors. Science 218: 348– 353. Sambrook, J., E. F. Fritsch and T. Maniatis, 1989 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Sanger, F., S. Nicklen and A. R. Coulson, 1977 DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463. Schaeffer, L., R. Roy, S. Humbert, V. Moncollin, W. Vermeulen et al., 1993 DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science 260: 58–63. Sweder, K. S., and P. C. Hanawalt, 1994 The COOH terminus of suppressor of stem loop (SSL2/RAD25) in yeast is essential for overall genomic excision repair and transcription-coupled repair. J. Biol. Chem. 269: 1852–1857. Talesa, V., E. Culetto, N. Schirru, H. Bernardi, Y. Fedon et al., 1995 Characterization of a null mutation in ace-1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans. FEBS Lett. 357: 265–268. Towbin, H., T. Staehelin and J. Gordon, 1979 Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350–4354. Vermeulen, W., R. J. Scott, S. Rudger, H. J. Muller, J. Cole et al., 1994 Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3. Am. J. Hum. Genet. 54: 191–200. Walker, J. E., M. Saraste, M. J. Runswick and N. J. Gay, 1982 Distantly related sequences in the a- and b-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1: 945–951. Weeda, G., R. C. A. van Ham, W. Vermeulen, D. Bootsma, A. J. van der Eb et al., 1990 A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne’s syndrome. Cell 62: 777–791. Weeda, G., E. Eveno, I. Donker, W. Vermeulen, O. ChevallierLagente et al., 1997 A mutation in the XPB/ERCC3 DNA repair transcription gene, associated with trichothiodystrophy. Am. J. Hum. Genet 60: 320–329. Williams, L. D., and B. R. Shaw, 1987 Protonated base pairs explain the ambiguous pairing properties of O6-methylguanine. Proc. Natl. Acad. Sci. USA 84: 1779–1783. Communicating editor: R. S. Hawley
