Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase ζ

Mutation Research 485 (2001) 237–253

Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase ζ J.C.J. Eeken a,b,∗ , R.J. Romeijn a,b , A.W.M. de Jong a,b , A. Pastink a,b , P.H.M. Lohman a,b a

b

Department of Radiation Genetics and Chemical Mutagenesis, MGC, Leiden University Medical Center, P.O. Box 9503, 2300 RF, Leiden, The Netherlands J.A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection, Leiden, The Netherlands Received 17 September 2000; received in revised form 14 December 2000; accepted 19 December 2000

Abstract In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS). Addition of this agent to the medium results in an increased larval mortality of the mutants. Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated. One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant. In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation. The localisation of mus205 to this region was confirmed by deficiency mapping. The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene. The REV3 gene encodes the catalytic subunit of DNA polymerase ζ , involved in translesion synthesis. The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein. The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation. In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents. In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations. This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Translesion synthesis; Drosophila; mus205; REV3

1. Introduction The maintenance of the integrity of the genetic information contained in the DNA is a vital function in every organism. This integrity is continuously threatened by internal and external factors damaging DNA ∗ Corresponding author. Tel.: +31-71-5276144/5276158; fax: +31-71-5221615. E-mail address: [email protected] (J.C.J. Eeken).

in numerous ways. All organisms from bacteria to humans have several mechanisms of defence, including a number of DNA-repair pathways. Structural homology of the genes involved as well as functional similarities, indicate that the pathways of DNA-repair have been conserved through evolution. The main pathways handling DNA changes include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), recombinational repair (RR), nonhomologous end-joining (NHEJ) and postreplication

0921-8777/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 ( 0 1 ) 0 0 0 6 2 - 3

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repair. However, some DNA adducts may be refractory to repair and still be present when the cell enters S-phase. The high fidelity DNA polymerases of the replication mechanism cannot deal with damaged DNA and replication is stopped. To avoid eventual cell death, the DNA has to be replicated past the damage. It is becoming clear that the cell has several ways to deal with this problem, one of which is the availability of special DNA polymerases that indeed can synthesise across lesions (translesion synthesis). Some are able to pass for example TT dimers relatively error free (DNA pol η, homologous to bacterial UmuC and DinB and yeast Rev1p) whereas others are error prone (DNA pol ζ , encoded in yeast by the REV3 and REV7 genes). In Drosophila, the majority of putative DNA repair mutants have been isolated based on the hypersensitivity to larval killing by the alkylating agent methyl methane sulfonate (MMS), the mus (mutagen sensitive) mutants. In a number of studies, mus mutants were recovered on all the three major chromosomes; chromosome I (X-chromosome): mus101–mus107 and mus109–mus111; chromosome II: mus201–mus212; chromosome III: mus301–mus311. The effect of some of these mutants on specific genetic endpoints after mutagenic treatment has been reported in many publications. However, in order to relate the genetic effects of the mus mutants to particular DNA pathways and to compare Drosophila DNA repair processes to those in other organisms, it is imperative to characterise the genes involved at the molecular level. The following Drosophila DNA repair genes have been cloned so far: Rrp1, the homologue of (HSA)HAP1 [39]; the homologues of XPA [41], XPB/haywire [20,34], XPG/mus201 [7] and XPC [17]; the homologue of (SCE)RAD1/(HSA)XPF/ mei-9 [40]; the homologue of ATM/ATR/mei-41 [15]; mus209, PCNA [48]; mus308, with homology to prokaryotic DNA polymerase I genes [16]: mus309, encoding the Drosophila Ku70 homologue [3]; a homologue of (SCE)RAD51 [1]; the homologue of RAD54 [21] and the homologue of RAD6 [19]. No mutants have been obtained yet for the Rrp1, XPA, XPC, RAD51 and RAD6 genes. We started a screen to isolate MMS-sensitive P-insertion mutations located on chromosome II of Drosophila melanogaster. The inserted P-element in the recovered mutations can be used to isolate the affected gene. Here we report the isolation and genetic

characterisation of the D. melanogaster MMSsensitive mutant mus205. 2. Materials and methods 2.1. Drosophila melanogaster strains To induce P-insertion mutations in D. melanogaster, a dysgenic cross was made using males of the P-strain, MR-h12/Cy [13,14]. This particular strain carries one complete P-element in region 38A of the second (MR) chromosome in addition to several incomplete P-elements that are inserted randomly in the genome [8]. This strain is able to induce on average three to four new insertions/chromosome in a dysgenic male [9]. The chromosome used to induce new P-insertion mutations in, is devoid of P-elements (M-strain) and marked with the recessive eye colour markers cn bw. Two mutagen sensitive strains were used for complementation studies: cn bw mus201D1 [5] and cn bw mus205A1 [43]. For the localisation of mus205, crosses were made to strains carrying the following deletions: Df(2R)NCX8, bwD /In(2LR)O, Cy dplvI pr cn2 (CyO), Df(2R)NCX13 bwD /CyO and Df(2R)ST1 (cn− ), Adhn5 pr/CyO (18). Unless stated otherwise, all cultures were maintained at 25◦ C. A description of mutants can be found in FlyBase [11]. For details of genetic tests with D. melanogaster the reader is referred to FlyBase [11] section GreyBook [50]. 2.2. Recovery of MMS-sensitive II-chromosomes The procedure followed is adapted from the scheme used by Snyder and Smith [43]. The hybrid dysgenesis cross and screening procedures are shown in Fig. 1. Males of the MR-h12/Cy strain were crossed to virgin females of the laboratory stock cn bw (this stock is an M-strain and devoid of P-elements). The F1 dysgenic males, cn bw/MR-h12, were mass mated to In(2LR)O, Cy dp pr cn2 /Sco l(2)91DT S females. In the F2 the P-element mutagenised cn bw chromosomes were recovered in the males cn bw P-insertion/In(2LR)O, Cy dp pr cn2 (these males can be distinguished from their MR-12/In(2LR)O, Cy dp pr cn2 brothers because they are homozygous for the recessive eye colour marker cn). These males were crossed individually to three to five virgins of the In(2LR)O, Cy dp pr cn2 /Sco l(2)91DT S stock. The crosses were

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Fig. 1. Mating scheme for the isolation of MMS-sensitive P-insertion mutations on chromosome II. Asterisk (∗ ) shows the chromosome potentially carrying P-insertions.

immediately placed at 29◦ C. After 2 days, the parent flies were removed and the crosses maintained for 1 more day at 29◦ C, after which they were placed at 25◦ C. Due to the presence of l(2)91DT S , a dominant temperature sensitive lethal, only F3 males and females of the genotype cn bw P-insertion/In(2LR)O, Cy dp pr cn2 will survive. These surviving F3 flies were transferred to fresh vials. After 48 h, they were sub-cultured and the alkylating agent MMS was added to the first vial (0.2 ml 0.07% in phosphate buffer pH 6.8/vial). The cultures were scored for the absence of cn bw P-insertion/cn bw P-insertion (white eyed flies) in the MMS-vial but present in the subculture (putative P-insertion MMS-sensitive mutants). Each suspected MMS-sensitive mutant (MMS-sensitive) was retested in first instance with 0.07% MMS. For the retest, female virgins of the genotype cn bw MMS-sensitive/O, Cy dp pr cn2 were crossed to cn bw MMS-sensitive/cn bw MMS-sensitive males and vice

versa. Putative P-insertion MMS-sensitive mutants (designated MMS-1,2 etc.) were crossed to mus201D1 and mus205A1 and tested for allelism by determining the larval sensitivity to MMS. The MMS-sensitive mutants recovered in this scheme are likely caused by the insertion of a P-element. In order to verify the P-element nature of the induced mutation, reversion experiments were performed. The principle of the crosses to recover reversions is exactly the same as those used to induce the mutations (Fig. 1), except that in the P cross, instead of homozygous cn bw females, homozygous MMS-sensitive cn bw females were used. 2.3. Molecular analysis of genomic and cDNA clones Genomic DNA of MMS-7 mutant flies was extracted, restricted with BamHI and loaded on a preparative agarose gel. A region containing the desired fragment length of approximately 11 kb was cut out

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of the gel, electro-eluted and fragments were cloned in ␭EMBL3. Filters were screened with a P-element probe and positive clones were isolated and purified. Similarly, the wild-type BamHI fragment was isolated from BerlinK wild-type flies. In a subsequent screen using the wild-type BamHI fragment, a 4.6 kb BglII and a 10 kb SalI fragment was isolated. These fragments were analysed with several restriction enzymes and subsequently several smaller subclones constructed in pUC18 for sequencing. The SalI–EcoR1, EcoR1–EcoR1, EcoR1–BamHI, BamHI–BglII, BglII–EcoR1, EcoR1–BglII, BglII– BglII, BglII–SalI fragments of the 10 kb genomic SalI fragment were subcloned and sequenced. The smaller fragments were sequenced in both directions using plasmid specific primers. For the larger fragments, new sequence-specific primers were chosen at the end of each sequence run. Both strands were, in this way, sequenced entirely and repeatedly in those cases where the complements did not match. Screening several head cDNA libraries resulted in the isolation of a number of clones, all ending with a poly A tail, the longest of which was 5.5 kb. All these clones were used for sequencing from plasmid primers as well as internal sequence specific primers. To identify the putative 50 end of the gene, several consecutive 50 RACE experiments were performed using adult RNA as a template as specified by the manufacturer (GibcoBRL). Nucleotide sequencing was carried out by the dideoxy chain termination method using T7 polymerase according to the manufacturer (PharmaciaLKB). In several cases, sequence reactions were carried out using an AutoRead sequencing kit (PharmaciaLKB) and analysed on an ALF Automated Sequencer (PharmaciaLKB). 2.4. In situ hybridisation All recovered MMS-sensitive mutants were hybridised in situ with a P probe according to the method described by Engels [10]. Several isolated genomic fragments of the mus205 gene were also used to hybridise to polytene chromosomes of wild-type flies. 2.5. Somatic recombination test In a preliminary experiment, cn bw females were mated to cn males. After 2 days, the parental flies

were transferred to fresh vials. To the first set of vials 0.2 l of an 0.08% MMS solution (phosphate buffer pH 6.8) was added. The second set of vials were used as controls. In the treated group, numerous white spots were observed with equal frequencies in males and females (100 spots/92 eyes; spots ranging from 2 to 64 ommatidia). In the control flies, only 16 spots were observed in 1378 eyes scored. It should be realised that in this system (cn bw/cn bw+ ) white spots can arise (1) from all somatic recombination events that occur in heterochromatin and euchromatin between the centromere and bw, which is located near the tip of the right arm of the chromosome, and (2) mutations in the bw+ gene. Suppression of recombination events is expected in cn bw/CyO flies due to the fact that the CyO chromosome carries multiple inversions. As a result of these inversions, the majority of the recombinational events should be inviable due to the formation of dicentrics in combination with fragments and duplications/deletions derivatives. The balancer chromosome used, CyO, carries a paracentric inversion In(2LR)30E/F;50C10/D1 superimposed on the pericentric inversion In(2L)22D1/2;33F5/34A1 on the left arm and the pericentric inversion In(2R)42A2/3;58A4/B1 on the right arm of the chromosome. The new order of the chromosome is: 20-22D1|33F5-30F|50D1-58A4|42A2-centromere-34A1|22D2-30E|50C10-42A3|58B1-60 [29]. As a result, not only are parts of the chromosome reallocated relative to the centromere, but also in a number of regions, the orientation is inverted. In predicting the products of recombinational events, especially the inverted character of a particular region has to be appreciated. In addition it should be noted that the cn gene is located in 43E and the bw gene in 59E1-2. Recombination in the region 20-22D1 can occur, but they have no effect on the markers cn and bw and will not lead to white spots. Recombination in regions 22D2-30E, 30F-33F5, 42A3-50C10 and 50D1-58A4 leads to the formation of a dicentric chromosome and an acentric fragment; no white spots can be generated in this way. Recombination in region 34A1-42A2 leads to two chromosome derivatives. One chromosome with duplication of the regions 20-22D1 and 30F-33F5 in combination with deletion of the regions 42A3-50C10 and 58B1-60. This chromosome, when viable, is deficient for the cn gene (42E) and the bw gene (59E1-2) and therefore, in principle, could give

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rise to white spots. It is known that these deletions are not viable in germline transmission. It appears therefore unlikely, but it is not excluded, that this chromosome derivative contributes to observed white spots in CyO heterozygotes. The other chromosome derivative is, in a way, the reverse of the first; it carries duplications for the regions 42A3-50C10 and 58B1-60 and deletions for the regions 20-22D1 and 30F-33F5. This chromosome, however, carries a wild-type bw gene and therefore can not result in a white spot. Finally, recombinations in region 58B1-59E1-2 (the location of the bw gene), will lead to the formation of white spots in CyO heterozygotes. Crosses were made (1) between cn MMS-7/CyO females and cn MMS-7 bw/cn MMS-7 bw males, cn/CyO females and cn MMS-7 bw/cn MMS-7 bw males and later (2) between cn MMS-7 bw/cn MMS-7 bw and cn bw/cn bw females and Df(2R)NCX8 (cn− )/CyO males. Vials with offspring were treated as before, with 0.00875, 0.0175, 0.035 and 0.07% MMS and various doses of X-rays (dose rate 1 Gy/min, Andrex machine at 4 mA and 200 kV, 1 mm Al filter built in). Each vial produced two kinds of offspring, one of which in all cases was heterozygous for the CyO chromosome, which in principle should suppress somatic recombination, and serves as an internal control in all the crosses. 2.6. Sex-linked recessive lethal test The effect of MMS-7 on forward mutations induced by X-rays and MMS was measured using the sex-linked recessive lethal (SLRL) test. Males of the genotype In(1)scS1L sc8R + In(1)S, wa B (Muller-5) were X-irradiated (Andrex machine at 4 mA and 200 kV, 1 mm Al filter built in) and mated to homozygous MMS-7 females. The induced lesions in the sperm of the male are repaired in the oocyte with the repair systems provided by the MMS-7 female. Mutations thus formed in essential genes on the X-chromosome, are recovered in a balanced state in the F1 females. The offspring of each single F1 female is subsequently screened for the presence or absence of males of the In(1)scS1L sc8R + In(1)S, wa B genotype. Absence of these males indicates the induction of a mutation in any one of the essential genes present on the X-chromosome. In the same way, the effect of the alkylating agent MMS was measured. Muller-5

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males were collected, starved for 3 h and subsequently fed for 24 h on MMS dissolved in phosphate buffer (pH 6.8) supplemented with sucrose (5%). The effect of NQO (4-nitroquinoline-1-oxide) on the other hand was tested in MMS-7 male spermatogonial stem cells. Larvae were treated chronically by addition of NQO to the food (add 4 ml of 12.5 mM NQO in 100% Tween– ethanol (1:3) to 100 ml Drosophila medium). Newly emerged males were collected and immediately mated to Muller-5 females. The first sperm sampled in this way represents the treated spermatogonial stem cells. 3. Results 3.1. Isolation and characterisation of a P-insertion mus205 mutant From the crosses as shown in Fig. 1, nine independent MMS-sensitive mutants were isolated from 10 separate experiments, in which a total of 13840 chromosomes were tested. Seven of these mutants were recovered as clustered events, as might have been expected from the mutation induction by P-elements in dysgenic males. Two mutants appeared as single events, three as clusters of 2, one as a cluster of 3, two as clusters of 4 and one as a cluster of 5. Two of the mutants are in addition to the MMS sensitivity also semi-lethal and will not be considered here. Of the remaining seven mutants, four (mutants 1–4) are only slightly sensitive, whereas the remaining three (mutants 5–7) are highly sensitive (Table 1). To measure the sensitivity, two types of crosses were used. Cross A: homozygous MMS-sensitive females (carrying in addition to the MMS-sensitive mutant the recessive markers cn and bw, phenotypically recognised by white eyes) crossed to heterozygous MMS-sensitive/Cy (not MMS-sensitive) balancer males (homozygous for the recessive marker cn and heterozygous for the recessive marker bw and therefore phenotypically bright red eyed in addition to their Curly wings due to the dominant marker Cy of the Cy-balancer). Cross B: heterozygous MMS-sensitive/Cy (not MMS-sensitive) balancer females crossed to homozygous MMS-sensitive males. The two genotypic F1 offspring of both crosses, homozygous MMS-sensitives and heterozygous MMS-sensitives over the Cy-balancer should appear in a ratio of 1:1 in untreated controls, whereas this

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Table 1 The relative survival of seven viable MMS-sensitive mutations induced by P-element mutagenesis in a cn bw II-chromosome Mutant

Crossa

Controlb

cn bw

A B A B A B A B A B A B A B A B

1056/1213

cn bw MMS-1 cn bw MMS-2 cn bw MMS-3 cn bw MMS-4 cn bw MMS-5 cn bw MMS-6 cn bw MMS-7

0.007% MMSc

0.02% MMS

0.07% MMS

6/197 (4%) 84/442 (23%) 0/529 (0%) 23/611 (5%) 4/144 (3%) 46/154 (40%)

92/247 (44%) 231/390 (66%) 58/332 (22%) 487/949 (65%) 57/284 (25%) 433/804 (67%) 20/126 (24%) 336/523 (100%) 8/68 (13%) 114/204 (64%) 0/207 1/366 0/266 0/636 1/65 0/155

2224/2824 2484/3133 1024/1594 427/488 1056/1243 1549/1883 872/1126 1895/2438 306/336 303/404

52/615 (10%) 625/785 (97%) 0/279 (0%) 70/152 (59%) 125/185 (70%) 22/42 (70%)

a Cross A: homozygous MMS-sensitive females × heterozygous MMS-sensitive/Cy males. Cross B: heterozygous MMS-sensitive/Cy females × homozygous MMS-sensitive males. b F homozygous MMS-sensitive flies/F heterozygous MMS-sensitive/Cy flies. 1 1 c In brackets: relative survival of homozygous MMS-sensitive flies: ratio MMS-treated flies/ratio control × 100%.

ratio will decrease when MMS is applied. The sensitivity of the mutant can be expressed as the relative survival by dividing the ratio of the treated sample by the ratio of the control (untreated sample) expressed as a percentage. Initially, 226 chromosomes were retested in addition to many control experiments using the original cn bw chromosome. All these controls clearly showed that the cn bw chromosome used, is, by itself, already slightly MMS-sensitive in comparison to the Cy heterozygotes (Table 1). In cross B, the relative survival at the concentration of 0.07% MMS (as used in the selection scheme) is for the cn bw control flies only 66%. In cross A, the relative survival is even lower, 44%. The fact that the relative survival after MMS treatment is higher when the parental cross is started with heterozygous females (cross B) than with homozygous MMS-sensitive females, indicates a maternal effect of the MMS-sensitive mutant, suggesting the deposition of its product in the oocyte. Similar maternal effects were also obvious in all the MMS-sensitive mutants recovered. MMS-sensitive mutants 1–4 are only slightly sensitive (Table 1) and actually only detected when homozygous sensitive females are used in the test cross (cross A). The mutants 5–7 were clearly more sensitive (Table 1) and these were tested for allelism

with the known MMS-sensitive second chromosome mutants mus201 and mus205. Mutant MMS-5 appears an allele of mus201 and MMS-7 an allele of mus205. Here we report the cloning of mutant MMS-7. 3.2. Reversion of mus205P In order to verify the P-insertion nature of the isolated mus205 mutant MMS-7, we determined whether this mutant could be reverted (see Section 2). In 739 chromosomes tested, two clusters of reversions were found (one cluster of 2, Revertant 5,6 and one cluster of 6, Revertant 7–12). To measure the sensitivity, heterozygous MMS-sensitive/Cy (not MMS-sensitive) balancer females were crossed to homozygous MMS-sensitive males and the offspring and the offspring was tested for survival after MMS treatment. The relative survival of the revertants is given in Table 2. This result indicates that MMS-7 is likely to be caused by a P-element insertion. 3.3. Cloning the mus205 gene using the P-tagged mutant MMS-7 The insertion of a P-element in the gene affected in MMS-7, opens the possibility to clone the particular

J.C.J. Eeken et al. / Mutation Research 485 (2001) 237–253 Table 2 The relative survival of revertants of the MMS-7 (mus205) mutant Mutant revertants

Crossa

Controlb

0.07% MMSc

cn bw (original stock) cn bw MMS-7 MMS-7 revertant 5 MMS-7 revertant 6 MMS-7 revertant 7 MMS-7 revertant 8 MMS-7 revertant 9 MMS-7 revertant 10 MMS-7 revertant 11 MMS-7 revertant 12

B B B B B B B B B B

1056/1213 303/404 359/452 419/523 145/186 261/339 377/446 239/389 383/470 199/224

231/390 (66%) 0/155 (0%) 76/153 (62%) 262/317 (100%) 33/58 (73%) 126/220 (74%) 60/138 (51%) 31/85 (59%) 108/177 (75%) 14/44 (36%)

a Cross B: heterozygous MMS-sensitive/Cy females × homozygous MMS-sensitive males. b F homozygous MMS-sensitive flies/F heterozygous MMS1 1 sensitive/Cy flies. c In brackets: relative survival of homozygous MMS-sensitive flies: ratio MMS-tretated flies/ratio control × 100%.

gene involved. First, however, we had to make sure that the only P-element present in the MMS-7 stock is the one affecting the gene causing the MMS-sensitive phenotype. In a preliminary experiment, a Southern blot with digested genomic DNA of homozygous MMS-7, heterozygous MMS-7/Cy flies, together with several revertants, was hybridised with a labelled P-element probe. In all these stocks, the hybridisation signals indicated the presence of several P-elements (result not

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shown). To eliminate the background P-elements, the MMS-7 chromosome was re-isolated into an M-strain background lacking P-elements. In the ‘cleaned’ MMS-7 stock, only one P signal could be detected. In addition, the signal of this particular P-element was clearly weaker in the MMS-7/Cy flies when compared to the homozygous MMS-7 flies, confirming its localisation on chromosome II. The size of the P-element containing fragment after BamHI digestion is approximately 11 kb, after BglII 6.6 kb and after SalI 11.5 kb. These three fragments from the MMS-7 flies were cloned in ␭EMBL3 and used to generate a restriction map of the region (Fig. 2) and to clone the homologous fragments from wild-type flies (Berlin K). The wild-type BamHI fragment was subsequently used as a probe on Southern blots of BamHI and BglII digested DNA of MMS-7 and its revertants. This analysis shows that the P-element present in the MMS-7 fragment is absent in the revertants (Fig. 3). The size difference in the fragments from MMS-7 and its revertants indicates that the inserted P-element in the MMS-7 mutant is approximately 2 kb. The wild-type BamH1 fragment (without the P-insert) of 9 kb (Fig. 2) was used to probe a Northern blot of RNA from adult flies. A strong signal was observed of approximately 1.6 kb and subsequently a cDNA of 1124 bp was isolated from a Drosophila head cDNA library. Sequence analysis showed homology to the Drosophila calcineurin B gene located

Fig. 2. Physical map of the genomic location of mus205.

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Fig. 3. Southern blots of DNA isolated from stock MMS-7 and its revertants. (A) DNA digested with BamH1 (probe: BamH1 fragment); Lane 1: P-insertion mutant; Lanes 2–10: revertants. (B) DNA digested with BglII (probe: BamH1 fragment); Lane 1: P-insertion mutant, Lanes 2–10: revertants.

in region 43E [46]. In our calcineurin B sequence, we observed a BglII site and subsequent sequencing of BglII subclones of the genomic BamHI fragment enabled us to locate the gene on the physical map (Fig. 2). The dCnB2 gene is physically linked directly to the neighbouring cinnabar gene [47]. Northern blots of polyA+ mRNA from adult flies were subsequently probed with the wild-type genomic SalI fragment, as well as with subclones 32, 64 and 43 derived from this SalI fragment (Fig. 2). With probe 32 (as well as with the SalI fragment itself) a transcript of 1.6 kb was detected. Using probe 32, a cDNA was isolated that is identical to the GAPDH-1 gene [44]. Finally, after extremely long exposures, a weak signal of about 7 kb was found with the subclones 64 and 43 as probe (Fig. 4). The Northern blot probed with subclone 43 showed in addition, a very weak signal of about 4 kb. Using subclones 64 and 43, a cDNA clone was obtained of 5.5 kb. Sequence analysis of this clone and of several 50 RACE products resulted in a composite cDNA of 7153 bp (Acc. No. AF 298215). A polyadenylation signal (AATAAA) can be found in this sequence at bp 7090–7076.

Fig. 4. Northern blots of polyA+ mRNA extracted from adult flies of wildtype (BerlinK, BK) and MMS-7 (C1). Left panel: probe 64 (see Fig. 2); right panel: probe 43 (see Fig. 2).

In addition, 9459 bp of the wild-type genomic SalI fragment containing the complete wild-type mus205 gene (Acc. No. AF 298216) was sequenced. The 50 end of the 7153 bp cDNA maps at position 690 of this fragment. Sequence comparison showed the presence of 11 small introns of 57–69 bp between nucleotides 1266–1323, 1554–1610, 1856–1912, 2161– 2218, 2664–2732, 2850–2911, 3317–3373, 3459– 3523, 3611–3668, 6357–6421 and 7517–7580 of the genomic sequence. In mutant MMS-7, a P-element of approximately 2 kb is inserted between bp’s 1171 and 1172 of the genomic SalI sequence while duplicating the sequence between 1163 and 1172 (GTCTACTC). Only one RT-PCR product of the relevant part of the mutant mRNA has been obtained. The sequence shows that an aberrant transcript is produced with an additional intron starting at nucleotide 20 of the P-element and ending 34 bp’s after the P-element in the mus205 sequence, resulting in a mRNA in the MMS-7 mutant that is 15 bp longer than that of the wild-type. As a result of the insertion of the first part of the inverted repeat of the P-element in the mRNA, a stopcodon is introduced nine nucleotides after codon 13 of the wild-type product.

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Fig. 5. Comparison of (HSA)POL δ with the DmREV3, (SCE)REV3, (HSA)REV3 proteins.

Within the 7153 bp cDNA, a putative ORF between nucleotide 445 and 6835 can be identified. Blast searches [2] indicated significant homology between the Drosophila mus205 protein and the REV3 gene product of Saccharomyces cerevisiae as well as with the recently identified REV3 homologs in man [12] and mouse [45]. 3.4. Comparison of the DmREV3, (SCE)REV3, (HSA)REV3 and (HSA)POL δ proteins The protein products of DmREV3, (SCE)REV3, (HSA)REV3 are clearly related to DNA polymerase δ. A schematic representation of the structure of these proteins is shown in Fig. 5. The overall homology in the 50 region and the 30 DNA polymerase ‘core region’ is indicated (in percentage identical amino acids). A comparison of (HSA)POL δ with (SCE)POL δ, SpPOL δ and a number of herpes virus DNA polymerases [49] showed several specific regions of homology, including in the N-terminal part the regions

N1–N5 and in the C-terminal part the DNA polymerase specific boxes I–VI, regions A–C and ends with motifs CT1–3 and two zinc finger motifs. If we compare DmPOL δ with (HSA)POL δ, the overall homology in the first 250 amino acids is very high (44% identical amino acids). This region includes the conserved boxes N1, N2 and N3. The percent identical amino acids in the 30 part of DmPOL δ and (HSA)POL δ is 65.7% (including N5 and the polymerase specific boxes I–VI). Homology in the N-terminal part between (HSA)POL δ and (SCE)REV3, DmREV3 and (HSA)REV3 exists, but is clearly less (identical amino acids respectively 20.0, 28.3 and 29.2%). The homology in the N-terminal parts between (SCE)REV3, DmREV3 and (HSA)REV3 is in fact more extensive as shown in Fig. 6. In the N-terminal part, the overall homology between (SCE)REV3/DmREV3, (SCE)REV3/ (HSA)REV3 and DmREV3/(HSA)REV3 amounts to respectively 33.3, 35.0 and 50.5% identical amino acids (Fig. 5). The homology in the C-terminal, the DNA polymerase ‘core region’ again is more

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Fig. 6. Homology in the N-terminal part of the (SCE)REV3, DmREV3 and (HSA)REV3 proteins.

conserved among the REV3 and DNA polymerase δ proteins than between REV3 and DNA polymerase δ. These data show that, although the REV proteins compare to DNA polymerase δ, the REV3 proteins and DNA polymerase δ’s are two distinct families. In region N2 of DNA polymerase δ, a conserved glycine repeat motif (G-x4-G-x2-G-x8-G-x3-YFY) can be observed that is also present in (SCE)REV3, DmREV3 and (HSA)REV3. This type of glycin repeat motifs have been implicated in nucleotide binding. The Exo I, II and III sites are important for 30 –50 exonuclease activity, involved in ‘proof reading’. ExoI in POL δ (as well as POL , and T4 and T5 bacteriophage DNA polymerases) is located in region N5 and has an essential motif, DIEC-xn-FP ((HSA)POL δ: SFDIECAGRKGIFPEP). In (SCE)REV3, DmREV3 and (HSA)REV3 this motif is degenerate and the homologous sequences read: TLEIHANTRSDKIPDP, TLEVFVSTRGDLQPDP and SVELHARTRRDLEP DP. In box IV, the second Exo-site is located and defined by the highly conserved motif ITGYN-IQN-FD

LP. This motif is in (SCE)REV3, DmREV3 and (HSA)REV3 respectively LSGFE-IHN-FSWG, YAGYE-IEM-SSWG and, LLGYE-IQM-HSWG. The third Exo-site is located between box IV and II. The motif of this site is characterised by the motif Y-X3-D-X2-L. In (HSA)POL δ, the sequence is AVY-CLK-D-AY-LPL. The sequence of the homologous regions in (SCE)REV3, DmREV3 and (HSA)REV3 is, respectively, LNY-WLS-R-AQ-INI, MEY-YLE-R-VR-GTL and VDH-YVS-R-VR-GNL. Most remarkable is that the (SCE)REV3, the DmREV3 and the (HSA)REV3 increase progressively in size over DNA polymerase δ due to the insertion of, respectively, 264, 969 and 1943 amino acids, which are apparently unrelated to each other, with the exception of a small 50–60 aa region. 3.5. Localisation of mus205 According to the genetic localisation of mus205 (2-54.9, 43) we expected the mus205/DmREV3 gene

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247

Fig. 7. Genetic localisation of the mus205 gene.

on the left arm of chromosome II approximately in region 38 close to the centromere (region 40/41), since purple (2-54.5) is located in region 38B/C and lethals as l(2)41Aa (2-55.1) and l(2)41Ab (2-55.1) in region 41. However, the in situ hybridisation of a P-element probe to salivary gland polytene chromosomes of the MMS-7 mutant, as well as in situ hybridisation of the BamHI and BglII fragments from the wild-type mus205/DmREV3 region (see Fig. 2) to salivary gland chromosomes of wild-type flies, showed a prominent signal in region 43E on the right arm of chromosome II. In order to confirm the localisation of mus205 in region 43E, a number of allelism tests were performed with multilocus deletions in this region. The results, as shown in Fig. 7, confirmed the localisation results of the in situ hybridisation. The fact that Df(2R)ST1 uncovers cinnabar but not mus205/Dmrev3 leads to the order and linking of the cinnabar, calcineurin B2, GAPDH-1 and mus205/DmREV3 genes as indicated in Fig. 7, which essentially is an extension in the distal direction of the map presented by Heizler [18]. These data were recently confirmed by the sequence data of the Drosophila Genome Project (GADFLY, Genome Annotation Database of Drosophila, http://flybase.bio.indiana.edu/annot/).

3.6. Effect of mus205/Dmrev3/MMS-7 on mutation and recombination in somatic cells after treatment with MMS The chromosome carrying the MMS-7 mutation, is also marked with the recessive eye colour markers cn and bw. This combination of mutations changes the red eye colour of wild-type flies into white when homozygous. Treating larvae homozygous for cn but heterozygous for bw with mutagens may induce clones of cells that have become hemi- or homozygous for bw, resulting in white spots in the otherwise bright red (cn) eyes. These spots may be the result of induced bw mutations, chromosome loss and somatic recombination. We used this system to investigate the effect of the MMS-7 mutant on MMS and X-ray induced mitotic recombination. Initially, two crosses were made: (1) cn MMS-7/CyO females with cn MMS-7 bw/cn MMS-7 bw males (test-cross) and (2) cn/CyO females and cn MMS-7 bw/cn MMS-7 bw males (control-cross). From each cross, two genotypes result, cn MMS-7/cn MMS-7 bw, CyO/cn MMS-7 bw and cn/cn MMS-7 bw and CyO/cn MMS-7 bw. Obviously, the CyO/cn MMS-7 bw flies in both crosses are identical and can be

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used as internal controls in comparing the effects in cn MMS-7/cn MMS-7 bw and cn/cn MMS-7 bw flies. The number of induced white spots were scored and, in addition, classified to size. The majority of the spots are small (2 ommatidia). Part of the spots are larger than 2 ommatidia (up to 64) and can be more reliably scored. The number of spots given in Tables 3 and 4 are only those larger than 2 ommatidia. Clearly, the number of homozygous MMS-7 flies relative to their heterozygous Cy-sibs is progressively reduced after treatment with MMS, but not X-rays. This shows that MMS-7 larvae are hypersensitive to MMS, but hardly to X-rays. The number of induced somatic events (primarily somatic recombination) by MMS as well as X-rays is dose dependent and systematically higher in cn MMS-7/cn MMS-7 bw larvae than in cn/cn MMS-7 bw. Rather suprisingly, also in the Cy flies a dose dependant increase, although lower as in the other flies, can be detected. This is possibly due to recombinational events in the regions 34A1-42A2 and 58B1-59E1-2 as discussed in Section 2. In view of the unexpected effect of MMS-7 (mus205/DmREV3) on somatic recombination, and the fact that the original cn bw chromosome by itself appeared slightly MMS-sensitive (see Section 3.1), the experiments were repeated in a modified form, starting with the following crosses: (1) cn MMS-7 bw/cn MMS-7 bw females and Df(2R)NCX8 (cn− mus205− )/CyO males and (2) cn bw/cn bw females and Df(2R)NCX8 (cn− mus205− )/Cy males. The offspring, cn bw

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MMS-7/Df(2R)NCX8 and cn bw/Df(2R)NCX8 larvae are now heterozygous for any interfering factor present on the original cn bw chromosome. They were treated together with their respective sibs cn bw MMS-7/CyO and cn bw/CyO with several doses of the methylating agent MMS. The relative survival of the cn bw MMS-7/Df(2R)NCX8 is clearly decreased compared to the survival of the cn bw/Df(2R)NCX8 larvae. However, in these experiments, the frequency of spots in the cn bw MMS-7/Df(2R)NCX8 and cn bw/Df(2R)NCX8 flies now is nearly the same (Table 4), showing that DmREV3 has no effect on the somatic recombination and that the detected earlier effect may have been caused by another factor present on the original cn bw chromosome. 3.7. Effect of mus205/Dmrev3/MMS-7 on mutation induction after treatment with X-rays and NQO and MMS In order to study the effect of mutant mus205 on the recovery of mutations, Muller-5 males were treated with X-rays and crossed to homozygous MMS-7 females. The DNA lesions induced in the sperm of the treated male, will be repaired by the repair mechanisms present in the oocyte and therefore under the control of the maternal genotype of the females used in the matings. This test measures forward mutations to recessive lethals in all essential genes on the X-chromosome (approximately 700). The results are shown in Table 5.

Table 5 Induction of sex-linked recessive lethals in mus205 after exposure to X-rays Genotype cn cn cn cn cn cn cn cn cn cn

(control)a

bw/cn bw bw MMS-7/cn bw MMS-7 bw/cn bw (control) bw MMS-7/cn bw MMS-7 bw/NCX8 (control) bw MMS-7/NCX8 bw/NCX8 (control) bw MMS-7/NCX8 bw/NCX8 (control) bw mms-7/NCX8 a

Treatment

No. of chromosomes

No. of lethals

Frequency (%)

No No 20 Gy 20 Gy No No 10 Gy 10 Gy 20 Gy 20 Gy

1895 1894 1918 1815 1184 1064 2274 2282 1943 1888

8 4 118 100 7b 2 74 102 118 153

0.4 0.2 6.1 5.5 0.6 0.2 3.2 4.8 6.1 8.1

Control cn bw data from [42]. b Included two clusters, one of 5 and one of 7 mutations. c Not significantly different.

(NS)c (NS)

(NS) (NS)

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Table 6 Induction of sex-linked recessive lethals in mus205 after exposure to NQO Genotype

Treatment

No. of chromosomes

No. of lethals

Frequency (%)

cn bw/NCX8 (control) cn bw MMS-7/NCX8

0.5 mM 4NQO 0.5 mM 4NQO

1878 2036

11 11

0.6 0.5 (NS)a

a

Not significantly different.

Since the possibility exists, that on the same, cn bw (control), chromosome where the MMS-7 was induced, a pre-existing MMS-sensitive mutation was present, we repeated the experiments not using homozygous cn bw MMS-7/cn bw MMS-7 female flies, but heterozygous flies with the genotype cn bw MMS-7/Df(2R)NCX8 (cn− mus205− ). These results are also given in Table 5. Obviously, there is no difference in the results obtained between the original experiments using the homozygous cn bw MMS-7/cn bw MMS-7 flies and those using cn bw MMS-7/Df(2R)NCX8. Similarly, the effect of NQO was measured. However, NQO in Drosophila is (slightly) mutagenic only if stem cell spermatogonia are treated. Therefore, we treated cn bw/NCX8 (control) and cn bw MMS-7/NCX8 males chronically as larvae and mated them, immediately after eclosion, to Muller-5 females (Table 6). No effect of MMS-7/mus205/Dmrev3/ on the forward mutation frequency could be detected. In view of the fact that the mus205 gene of Drosophila proved to be a homolog of the yeast REV3 gene, the reported data showing hypermutability of the alkylating agent MMS on mutation induction in mus205 [42] needed reaffirmation. Muller-5 males

were treated with 0.3 mM MMS and mated to cn bw MMS-7/cn bw MMS-7 and cn bw MMS-7/Df(2R)NCX8 females (Table 7). A hypermutability as reported could only be found in cn bw MMS-7/cn bw MMS-7 and not cn bw MMS-7/Df(2R)NCX8. These results indicate again that most likely a second mutation is present in the original cn bw stock affecting mutation induction in a similar way as mus201 (deficient in the NER gene XPG, 7).

4. Discussion The Drosophila mus205 gene was isolated using a P-insertion mutation. The gene could be mapped to region 43E and the physical linkage of mus205 to the genes GAPDH-1, calcineurin B2 and cinnabar was shown. The P-element is inserted in the first exon of the gene. Transcription of the mutant leads to a mRNA of approximately the same size as the wild-type gene. However, due to the presence of the P-element, new splice donor and acceptor sites are introduced leading to a truncated protein of only 15 aa’s. The wild-type gene encodes a putative protein of 2130 aa’s that shows homology to the catalytic

Table 7 Induction of sex-linked recessive lethals in mus205 after exposure to MMS Genotype

Treatment

No. of chromosomes

No. of lethals

Frequency (%)

cn cn cn cn cn cn cn cn

No No 0.3 mM 0.3 mM No No 0.3 mM 0.3 mM

2076 1553 2395 2105 1184 1064 2092 2053

10 3 56 92 7a 2 60 61

0.5 0.2 2.3 4.4 (S)b 0.6 0.2 2.9 3.0 (NS)c

bw/cn bw (control) bw MMS-7/cn bw MMS-7 bw/cn bw (control) bw MMS-7/cn bw MMS-7 bw/NCX8 (control) bw MMS-7/NCX8 bw/NCX8 (control) bw MMS-7/NCX8 a

MMS MMS

MMS MMS

Included two clusters, one of 5 and one of 7 mutations. Significantly different. c Not significantly different. b

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subunit of DNA polymerase ζ of yeast [32] and mammals [12,45]. Striking is the resemblance of these polymerases to DNA polymerase δ. Not only is there high homology in the C-terminal polymerase domains, but also in the N-terminal 200–300 aa’s. The main difference is caused by an internal region in the protein which increases in size from 264 aa’s in yeast, to 969 aa’s in Drosophila and 1943 aa’s in human. The homology in this region between yeast, Drosophila and human is restricted to only a small, approximately 60 aa, region. In addition, the REV3 protein in all three cases lacks amino acids essential for the 30 –50 exonuclease activity of DNA polymerase δ, implicated in ‘proofreading’ function [33]. The catalytic subunit of DNA polymerase ζ , encoded by the (SCE)REV3 gene, is one of the main factors in the RAD6 dependent translesion synthesis pathway [32,35] responsible for the majority of spontaneous [37,38] and induced mutations [22,27]. In yeast, the effect of mutations in the genes involved in this translesion synthesis pathway is the reduction in spontaneous and induced mutations. The initial tests used to characterise the Saccharomyces cerevisiae rev mutants (including rev-3), involved the reversion of ochre-suppressible, amber, initiation, proline missense and frameshift alleles after UV [23–26]. However, also the forward mutation rate to auxotrophy by UV, NQO and X-rays is reduced [28,30,31,36]. The biochemical characterisation of mus205 showed that in this mutant, the removal of UV-induced cyclobutane pyrimidine dimers was reduced by approximately 50% and that the capacity to synthesize full length DNA, measured 4 h after irradiation, was clearly reduced [4,6]. These data pointed in the direction of a defect in the removal of UV-induced dimers as well as a defect in postreplication repair. Unscheduled DNA synthesis (UDS) after UV, however, is not completely abolished, indicating residual capacity to repair UV lesions. After treatment with alkylating agents and X-rays, UDS is completely absent. The genetic effect of REV3/mus205 in Drosophila, tested in the original EMS-induced allele mus205A1 by Smith and Dusenbery [42], appears rather different from the data as obtained with yeast. Using a general forward mutation assay (sex-linked recessive lethal, SLRL test), the mutation frequency after treatment with the ethylating agent ENU is not reduced and even increased after treatment with EMS (1.2×),

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MNU (2.1×) and MMS (3.7×). This result was explained by the assumption that possibly apurinic sites cannot be repaired and consequently give rise to enhanced mutagenesis. Our data clearly indicate that the cn bw chromosome, that was used to induce the mus205, already must carry a mutation that affects the NER in a similar way as mus201 (the Drosophila homolog of XPG). In fact, the increase in mutation frequency after EMS, MNU and MMS in mus205 is about half that observed in mus201, (completely excision repair deficient), where the removal of UV-induced dimers in mus205 is only reduced to 50%. Using cn bw MMS-7/Df(2R)NCX8 flies, the interference of factors possibly present in the original cn bw chromosome could be circumvented. In these flies, no effect could be found on forward mutations induced by X-rays, NQO and MMS, nor was there any effect in a somatic mutation and recombination assay, although the survival is clearly reduced after treatment with MMS. UV was not tested since this agent is hardly mutagenic in flies. The difference with results obtained with yeast, may be explained if damaged cells in Drosophila go into apoptosis rather easily since the function of these cells can readily be replaced by neighbouring cells in same developing tissue.

Acknowledgements This work was supported by the J.A. Cohen Institute, Interuniversity Research Institute for Radiopathology and Radiation Protection (IRS: project 4.4.14) and the Dutch Cancer Foundation, KWF (RUL 96-1320). The authors wish to thank Drs. W. Ferro, M. Nivard, P. Van Sloun and N. de Wind for their critical comments during the preparation of the manuscript.

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