The Drosophila mus101 Gene, Which Links DNA Repair, Replication and Condensation of Heterochromatin in Mitosis, Encodes a Protein With Seven BRCA1 C-Terminus Domains

Corresponding author: Daryl S. Henderson, Department of Genetics, University of Cambridge, Downing St., Cambridge CB2 3EH, United Kingdom., dsh25{at}mole.bio.cam.ac.uk (E-mail)

Communicating editor: R. S. HAWLEY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101^lcd. To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIß. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.

MORE than 30 genes of Drosophila melanogaster implicated in DNA repair, replication, recombination, and/or cell cycle checkpoint control have been identified through mutations conferring larval hypersensitivity to DNA-damaging agents (reviewed by DUSENBERY and SMITH 1996 ; SEKELSKY et al. 1998 ; HENDERSON 1999A ). The X-linked locus mus101 was among the very first such mutagen-sensitive (mus) genes to be identified. The two original alleles, mus101^D1 and mus101^D2, confer hypersensitivity to methyl methanesulfonate (MMS), nitrogen mustard, and -rays, but not to UV radiation or N-acetyl-2-aminofluorene, a UV mimetic (BOYD et al. 1976 ). Other alleles of mus101 causing different phenotypes were later isolated: the mus101^K451 (fs(1)K451) mutant is both female sterile (KOMITOPOULOU et al. 1983 ; ORR et al. 1984 ) and MMS sensitive (this report); mus101^lcd and mus101^SM are late larval lethals (this report; A. SCHALET, personal communication); and mus101^ts1 is a temperature-sensitive lethal (GATTI et al. 1983 ; SMITH et al. 1985 ).

The mus101^D1 mutant is partially defective in postreplication repair (PRR; BOYD and SETLOW 1976 ; BROWN and BOYD 1981 ), as shown by a reduction in the average size of the newly synthesized DNA molecules compared to wild type after UV treatment. Nonirradiated cells from this mutant accumulate high molecular weight DNA molecules more slowly than control cells, suggesting a defect in S phase (BOYD and SETLOW 1976 ). In common with other PRR mutants, mus101^D1 disrupts "magnification" of ribosomal DNA genes in the male germline, a little-understood process that restores the copy number of 18S and 28S rDNA tandem repeats lost through deletion (HAWLEY et al. 1985 ).

The female sterile mus101^K451 mutant was isolated in a screen for mutants affecting eggshell formation (KOMITOPOULOU et al. 1983 ; ORR et al. 1984 ). The eggshell abnormalities observed in this mutant result from defective amplification of clusters of chorion protein genes, which is a form of DNA replication specific to follicle cells (for review see CALVI and SPRADLING 1999 ). The recent finding that the Drosophila homolog of the yeast origin recognition complex 2 (ORC2) subunit is also required for chorion gene amplification (LANDIS et al. 1997 ) raises the intriguing possibility that Mus101 protein may physically interact with DmORC2 at replication orgins of chorion gene clusters.

Animals hemizygous for mus101^ts1 and heterozygous for the autosomal recessive cell marker mwh exhibit high levels of chromosomal instability, evidenced by the frequent occurrence of mwh clones (SMITH et al. 1985 ). This chromosome instability observed genetically is also observed cytologically: neuroblast cells from hemi- or homozygous mus101^ts1 show a striking failure to condense heterochromatic regions of chromosomes (GATTI et al. 1983 ; SMITH et al. 1985 ). Similar genetic and cytological phenotypes have been observed for other mus101 alleles (BAKER and SMITH 1979 ; SMITH et al. 1985 ; this report).

The phenotypes associated with the various mus101 alleles summarized above suggest roles for the mus101⁺ gene product in different aspects of chromosome metabolism. It is evident that the molecular cloning of this gene should bring important new insights into the function of its encoded protein. Here we describe the cloning of mus101, achieved through a strategy of chromosome walking and mutation mapping in region 12B. We report that mus101 encodes a protein with seven BRCT (BRCA1 C terminus) domains, a motif originally noted as a repeated region in the carboxy-terminus of the human ovarian and breast cancer protein BRCA1, in the budding yeast checkpoint protein Rad9, and in a human protein that binds to p53, named 53BP1 (KOONIN et al. 1996 ). More recent additions to the BRCT domain superfamily include the human proteins XRCC1, poly (ADP-ribose) polymerase (PARP), DNA ligases III and IV (ALTSCHUL et al. 1997 ; BORK et al. 1997 ; CALLEBAUT and MORNON 1997 ), and TopBP1 (YAMANE et al. 1997 ) among others. Mus101 is most similar to human TopBP1, a protein identified in a two-hybrid screen through its association with DNA topoisomerase IIß (YAMANE et al. 1997 ). Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control (FENECH et al. 1991 ; SAKA and YANAGIDA 1993 ), suggesting that the two proteins may be functional homologs.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks and culture conditions:
The Drosophila stocks, balancer chromosomes, and phenotypic markers described in this work are catalogued in LINDSLEY and ZIMM 1992 or FLYBASE 1999 , except as noted below. Flies were maintained on standard yeast-cornmeal-agar medium at 25° unless mentioned otherwise.

mus101 lethal alleles:
mus101^lcd was recovered from a collection of X-linked lethal mutations induced by hybrid dysgenesis (SIMMONS et al. 1984 ) that we screened for mitotic mutants. Of 101 lethal lines, 37 produced mutant males dying as late larvae or pupae. These were examined for mitotic defects in squash preparations of brain ganglia from third-instar larvae, essentially as described in GONZALEZ and GLOVER 1993 . Two stocks, including mus101^lcd, showed obvious differences from wild type in these preparations.

A second late larval lethal allele, mus101^SM, was generously provided to us by A. Schalet (Yale University). This mutation arose spontaneously from a cross of wild-type (Amherst) males to females carrying a mei-9 mutation [In(1)dl-49, y^31d w^a mei-9^L1 rb v^Of f] (A. SCHALET, personal communication). mus101^SM fails to complement the larval lethality of mus101^lcd, and both mus101^lcd and mus101^SM fail to complement the mutagen sensitivity of mus101^D1.

Molecular biology:
Standard molecular biology techniques were employed following AUSUBEL et al. 1989 and SAMBROOK et al. 1989 . The plasmid vectors pBluescript I and II (Stratagene, La Jolla, CA) were used for all subcloning. The mus101^lcd genomic DNA library was constructed in the vector EMBL4 (FRISCHAUF et al. 1983 ) according to AXTON 1990 . Microdissection of polytene chromosome bands 12B1,2 was performed according to SAUNDERS et al. 1989 . Cosmids comprising the chromosome walk were recovered from the European Drosophila Genome Project (EDGP) gridded library (SIDEN-KIAMOS et al. 1990 ) by filter hybridization. Initial localization of the gene garnet (g) to a 6.5-kb EcoRI fragment of the chromosome walk was made through the identification of restriction fragment length polymorphisms (RFLPs) on Southern blots containing genomic DNAs of three garnet mutants [g^S2, g^S3, and In(1)g^w] probed with this fragment. This location was later confirmed by the hybridization of this EcoRI fragment to a garnet cDNA (gift of V. Lloyd, Dalhousie University, Halifax, Nova Scotia, Canada).

Creation of Df(1)w^LCD by -irradiation:
In(1)z⁺64b9 is a rearranged X chromosome carrying an inversion between 3C1 and 12B9, with w⁺ at 3C1 not included within the inverted segment (SORSA et al. 1973 ). In(1)z⁺64b9 males were irradiated with 45 Gy from a ⁶⁰Co source and mated to a fourfold excess of balancer-bearing virgin females [either FM7, y w^a sn^x2 v g/DFB, y w^a or FM7, y w^a sn^x2 v g/FM6, or y ml(1)]. Balanced lines were established by allowing the F₁ progeny to mate inter se, since the irradiated chromosomes were balanced in the F₁ females and all the F₁ males were FM7 (the DFB and FM6 balancer chromosomes carry lethal mutations). Irradiated chromosomes balanced over FM7 and DFB were scored for garnet and/or white phenotypes in the F₁ generation. For irradiated chromosomes balanced over FM6 (25% of the F₁), the F₂ generation was examined for visible mutations. The lethality of Df(1)w^LCD is not complemented by the duplications Dp(1;f)LJ9 and y⁺ g⁺ na⁺ Y. It also fails to complement the lethality of the LS allele of crm, a gene adjacent to w, indicating that the deficiency extends into one or more lethal loci in 3C.

Creation of new deficiencies in region 12B by transposase-mediated P-element excision:
Deficiencies in region 12B were created by "imprecise excision" of a single P-element transposon in the strain P[w⁺, ry⁺E]2 (LEVIS et al. 1985 ) that we had localized to the distal part of the walk (12B1,2). This P element resides within a 12-kb BamHI fragment found in wild-type DNA (Oregon-R and Canton-S), detected using a 4.7-kb BamHI fragment (B4.7) from the end of the insert in the cosmid 29E9.

To mobilize this element, females homozygous for the P insert were crossed en masse to +/Y; 2-3(99B), Sb/+ males to generate P/Y; 2-3(99B), Sb/+ transposase-expressing males in the F₁ generation. Such F₁ males were crossed to crm/FM7 virgin females and the F₂ progeny examined for the presence of phenotypically white, Bar, non-Stubble females (i.e., p*/FM7; +/+, where the asterisk indicates deletion or mutation of the w⁺ gene of the P insert). Such mutants were crossed individually to FM7 males to establish stocks. Only those chromosomes bearing lethal mutations (i.e., lines in which white-eyed F₃ males were not recovered) were kept for further study. A total of 23 lethal mutants were isolated from 1698 F₂ crosses. These were analyzed in complementation tests with the lethal mutant mus101^SM. Virgin p*/FM7 females were crossed with mus101^SM/y⁺g⁺na⁺Y males, and their heterozygous mutant female progeny were examined for viability. These mutants can be divided in two classes: 11 mutants that complement mus101^SM and 12 mutants that fail to complement mus101^SM. All 12 of the P-element excision mutants that delete mus101 were found to delete garnet as well. This result indicated that the deletions in these mutants extend from the distal part of the walk to the garnet locus or beyond and therefore were not informative with respect to mapping the mus101 locus, since they remove too large a portion of the walk. Five of the 11 strains that complement mus101^SM were also tested for their ability to complement garnet. These strains (including p116D, p205A, p281A, and p490D), when heterozygous with g⁴, have wild-type eye color. The 11 lethal mutants that complement mus101^SM were analyzed at molecular level in Southern-blotting experiments to be able to exclude those regions of the walk not containing mus101. In 6 of these mutants, no deletion had occurred proximal to the site of the P insertion. In a 7th mutant, a chromosomal rearrangement appeared to have occurred. None of these mutants were characterized further. The 4 remaining mutants (p116D, p205A, p281A, and p490D) are discussed in RESULTS.

Isolations of cDNA clones:
cDNAs prefixed with an "x" were recovered from a 0–3-hr embryonic library constructed in the vector gt10 (POOLE et al. 1985 ). A ZAP 2–14-hr embryonic cDNA library (Stratagene) probed with the mus101 cDNA x99 yielded a single partial-length cDNA. An ovary library constructed in gt22a (STROUMBAKIS et al. 1994 ) was found to be a rich source of mus101 cDNAs.

We have isolated two further cDNAs corresponding to distinct genes, in addition to those reported in RESULTS (Fig 2). Both cDNAs were obtained from a 0–4-hr embryonic cDNA library (BROWN and KAFATOS 1988 ). cDNA F3 was isolated using the 4.1-kb BamHI fragment (B4.1) from cosmid 29E9. F3 is 1.5 kb and has no significant homology to any sequence in the database. This cDNA also hybridized to genomic fragment B6.2, immediately distal to B4.1. cDNA F5 hybridized to three independent genomic probes: an 8-kb PstI fragment (P8) from cosmid 110D5; a 15-kb BamHI fragment (B15) from cosmid 165E3; and a 5-kb XbaI fragment (X5) from phage A26-7. cDNA F5 is 3.5 kb and hybridized to genomic fragments spanning an 30-kb region of the genomic walk, suggesting that the gene corresponding to this cDNA has several introns. cDNA F5 has similarity to a hypothetical 80-kD protein of Saccharomyces cerevisiae (accession no. 731675) and a Caenorhabditis elegans expressed sequence tag (accession no. 3874484).

View larger version (92K): In this window In a new window Download PPT slide   Figure 1. Mitotic chromosome defects in mus101lcd neuroblasts. (A) Wild-type XX metaphase. (B) Chromosomes from a mus101lcd/Y larva showing undercondensed pericentric heterochromatin (arrows). (C) Extreme phenotype more typical of mus101lcd/Y, with undercondensed or broken chromosomes. Blocks of chromatin are probably chromatid arms. Brains in A and B were incubated in 10 µM colchicine for 1 hr followed by hypotonic treatment. Bar, 10 µm.

View larger version (20K): In this window In a new window Download PPT slide   Figure 2. Molecular map of region 12B. Genomic region 12B is represented by 150 kb of cloned DNA. The main map (center) shows the restriction enzyme recognition sites for EcoRI and BamHI, indicated by vertical lines above and below the horizontal line, respectively. Subcloned restriction fragments mentioned in the text are labeled (e.g., B15). EDGP cosmids that comprise the chromosome walk are indicated above the map as overlapping horizontal solid lines. A26-7 represents a phage clone. EV8, P8, and X5 represent subcloned restriction fragments (see MATERIALS AND METHODS). The dashed lines above the cosmids represent genomic regions deleted in the indicated deficiency strains; the arrowheads mark the approximate positions of the breakpoints defined molecularly. The triangle in the distal part of the walk (region 12B1,2) represents the site of integration of the P element in the strain P[w+, ry+E]2. The positions of the genes garnet and Yp3 are indicated. Not all of the 30-kb interval between these two genes is represented on the map; this discontinuity is indicated by the double slash. cDNAs F3 and F5 are referred to in MATERIALS AND METHODS. An expanded view of the region of the walk containing mus101 is shown at the bottom right. The fragments used to search for transcription units and RFLPs are labeled (e.g., B10). The arrows below this map show the identified transcription units and direction of transcription (see text for details). The fragments X9 and B10 were used in germline transformation rescue experiments.

DNA sequencing:
Sequencing reactions were performed using the ABI PRISM big dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). mus101 accession no. is AF257463.

P-element-mediated germline transformation:
Embryos of the genotype w¹¹¹⁸/w¹¹¹⁸; +/+; 2-3(68C)/+ were used for injection. A 9-kb XhoI fragment from cosmid 44F7 was subcloned into the transformation vector pW8 (KLEMENZ et al. 1987 ) to give the rescue construct P[w⁺, X9]. The 10-kb BamHI fragment from cosmid 22F12 was subcloned into the transformation vector pCaSpeR (PIRROTTA 1988 ) to form the negative control construct P[w⁺, B10].

Mutagen sensitivity tests:
These tests were carried out as described in HENDERSON 1999B . The mutagen sensitivity of the female sterile mutant mus101^K451 has not been documented previously. We have found this mutant to be more sensitive to MMS than either mus101^D1 or mus101^D2. A dose as low as 0.02% v/v MMS is sufficient to kill all mus101^K451 males, compared to doses of 0.04 and 0.07% required to kill mus101^D1 and mus101^D2 animals, respectively.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of a new lethal allele of mus101 showing defective condensation of pericentric heterochromatin:
The isolation and cytological characterization of the temperature-sensitive (ts) lethal mutant mus101^tsl revealed that mus101 is an essential gene whose wild-type product is necessary for condensation of heterochromatin in mitosis (GATTI et al. 1983 ). We isolated a further (non-ts) lethal allele, mus101^lcd, from a collection of X-linked lethal mutations induced by hybrid dysgenesis (SIMMONS et al. 1984 ), in a screen for P-induced alleles that would facilitate the molecular cloning of the gene through the recovery of sequences flanking the P element. mus101^lcd mutants die as late third-instar larvae or shortly after pupariation and have very small imaginal discs (hence the superscript lcd for lethal-compact-disc). Examination of neuroblasts of mus101^lcd/Y third-instar larvae revealed numerous chromosomes with striking undercondensation of pericentric heterochromatic regions (Fig 1B), reminiscent of the mitotic phenotype observed in brains of mus101^tsl mutants shortly after they are shifted to the nonpermissive temperature (GATTI et al. 1983 ). This phenotype may be the earliest to appear in mus101^lcd neuroblasts, since most of the mitoses in mus101^lcd brains show more extreme defects, characteristically grossly undercondensed chromatin or chromosome breakage (it is difficult to distinguish between these two phenotypes in orcein-stained preparations). A typical example is shown in Fig 1C. These mitotic phenotypes are also observed in neuroblasts of mus101^lcd/Df(1)HA92 hemizygous females (not shown). Table 1 summarizes the frequency of observed chromosome condensation defects in mus101^lcd larval brains.

Refinement of the cytological map position of mus101:
Although mus101^lcd had been induced in a dysgenic cross, in which the strain 2 provided both P-element "ammunition" and a source of transposase, an observed P element in this mutant at region 12C1 was found to be separable by recombination from the mus101^lcd mutation. Furthermore, introduction of a transposase source [2-3(99B)] into this strain failed to revert the mus101^lcd mutation to wild type. Therefore, as an alternative means to isolate the gene we adopted a positional cloning strategy.

The mus101 gene was originally localized to between bands 12A6,7 and 12D3 on the basis of the failure of mus101 mutants to complement Df(1)HA92 (MASON et al. 1981 ). We obtained a further refinement of the mus101 location by using available T(1;Y) stocks to synthesize interstitial duplications and deficiencies (AXTON 1990 ) and by creating a small deficiency, Df(1)w^LCD, that is missing the cytological interval 12B2-9 and uncovers mus101. These mapping experiments, taken together with the recombination studies of A. Schalet, which had positioned mus101 0.1 cM distal to garnet (g; A. SCHALET, personal communication), established the cytological location of mus101 as 12B2-6.

Molecular cloning of region 12B by chromosome walking:
Molecular entry into the 12B region was gained using several approaches. Although the P element at 12C1 in the mus101^lcd line was not responsible for the mus101 mutation (see above), its molecular cloning nonetheless provided the first step toward the isolation of the mus101 gene. Characterization of a recovered clone revealed that the P element at 12C1 had inserted adjacent to the Yolk protein 3 (Yp3) gene. This flanking genomic DNA was used in turn as a probe to identify cloned wild-type genomic segments (as phages and cosmids) from 12C1 and, more distally, into region 12B6,7. Recovered EDGP cosmids belonging to this contig (12.2) include 7C5, 22F12, and 189B8 (Fig 2). Microdissection of polytene chromosome bands 12B1,2 and PCR amplification of the recovered DNA provided probes corresponding to the distal part of 12B. EDGP cosmids belonging to this contig (12.6) include 29E9, 36G4, 94D8, and 110D5 (Fig 2). Contig 12.2 was extended distally with the isolation of the cosmid 44F7, and the two contigs were joined and the relative orientation of contig 12.6 established by the isolation of the bridging cosmid, 165E3.

The locations of two important landmarks on the chromsome walk were identified: the distal breakpoint of Df(1)w^LCD near the distal end of the walk and the garnet locus in its proximal part (Fig 2). These two landmarks define the boundaries of the genomic region containing mus101 (see above), an interval of 90 kb.

Creation of new deficiencies in region 12B by excision of a marked P element:
We sought to create new deficiencies in region 12B through transposase-mediated excision of a single P element located in the distal part of the walk in the strain P[w⁺, ry⁺E]2 (LEVIS et al. 1985 ; Fig 2). To enrich for such deletion mutants, we screened for loss of the w⁺ marker of P[w⁺, ry⁺E]2 associated with organismal lethality. Four of 23 mutants generated in this way (p116D, p205A, p281A, and p490D) were informative with respect to narrowing the location of mus101; each had a deletion extending proximally from the site of insertion of the P element into the region covered by the chromosome walk (Fig 2). The most proximal breakpoint, that of deficiency p490D (Df(1)p490D), is near the distal end of the BamHI fragment B15 (Fig 2). This result positioned mus101 to within the interval defined by this breakpoint distally and the garnet locus proximally, 30 kb of cloned DNA.

Pinpointing the location of mus101:
To further narrow the location of mus101 we sought to determine whether any of the mus101 alleles showed a RFLP in the 30-kb interval between the breakpoint of Df(1)p490D and garnet. To achieve this we used subcloned BamHI and EcoRI fragments to probe Southern blots of genomic DNAs of mus101 mutants. Of the various probes used, only the fragment B10 detected RFLPs in the lethal mutant mus101^lcd and in the mutagen-sensitive strains mus101^D1 and mus101^D2 (not shown). The presence of RFLPs in three independent mus101 mutant alleles detected by the B10 probe thus identified this genomic fragment as a putative mus101 locus. This possibility was further strengthened because B10 partially overlaps a 6.5-kb EcoRI fragment that we had shown to contain garnet, which mapping experiments had placed proximal but very near to mus101 (see above).

Identification of transcription units in the mus101 region:
We used two methods to identify transcription units in the mus101 region: Northern blotting and isolation of cDNAs. Northern blots of total RNA extracted from 0- to 4-hr wild-type embryos were probed separately with the contiguous genomic fragments E2.9, B0.9, and B10 (Fig 2). Probe E2.9 detected an 3-kb transcript (corresponding to transcription unit "A" in Fig 2). Probe B10 detected two transcripts, of 5 kb and 3.5 kb (corresponding to mus101 and garnet, respectively). Probe B0.9, distal to B10, also recognized the 5-kb transcript, indicating that this transcription unit spans the B0.9 and B10 restriction fragments.

We used a 9-kb XhoI fragment (X9) that partially overlaps fragments E2.9, B10, and E6.5 and contains the entire B0.9 fragment (Fig 2) to recover cDNAs corresponding to the three transcripts detected by Northern analysis. Six partial cDNAs belonging to three different classes were isolated: garnet (x91, x94, x95, and x96) (OOI et al. 1997 ), transcription unit "A" (x93), and mus101 (x99). A homology search using the BLASTX program (ALTSCHUL et al. 1997 ) revealed that the partial sequence of the cDNA x93 is similar to the human clone KIAA0544 (NAGASE et al. 1998 ; accession no. 3043612) and to the C. elegans locus CEC11H1 (accession no. Z70205), identified in the genome project. Although no function has been assigned to these loci, KIAA0544 has significant similarity to human cell growth regulators and apoptosis inhibitors (NAGASE et al. 1998 ).

A BLASTX search using the x99 sequence revealed similarity to a human cDNA (NAGASE et al. 1996 ), which in turn is similar to the Schizosaccharomyces pombe rad4⁺/cut5⁺ gene (FENECH et al. 1991 ; SAKA and YANAGIDA 1993 ). rad4/cut5 mutants show sensitivity to a variety of DNA-damaging agents, and the Rad4/Cut5 protein is essential for S phase (SAKA et al. 1994 ) and important in a DNA replication checkpoint. The similar phenotypes presented by mus101 and rad4/cut5 mutants suggested that a gene corresponding to cDNA x99 was a very strong candidate for mus101. Therefore we sought and recovered additional cDNAs using x99 as a probe (see MATERIALS AND METHODS).

Identification of the mus101 gene by P-element-mediated germline transformation:
To confirm the cloning of mus101, we designed two genomic constructs for use in P-element-mediated germline transformation experiments. The X9 fragment referred to above was selected as a rescue fragment (Fig 2). This fragment was predicted to contain a complete mus101 gene together with truncated versions of transcription unit "A" and garnet. The B10 fragment, which is missing 2 kb of promoter elements and coding region from the 5' end of mus101, was selected as a negative control. The construct P[w⁺, X9] fully rescued the phenotypes of all mus101 alleles tested (Table 2). In constrast, the construct P[w⁺, B10] rescued none of the mus101 mutant phenotypes. These experiments prove that mus101 corresponds to the identified gene between transcription unit "A" and garnet depicted in Fig 2.

Genomic organization of mus101:
We determined the complete genomic sequence of mus101 (5.3 kb) by sequencing both the distal 6 kb and very proximal end of the X9 restriction fragment (700 bp). The distal end of X9 overlaps transcription unit "A" (620 bp), and the proximal end overlaps garnet. Conceptual translation of the mus101 sequence revealed that it encodes a polypeptide with a predicted molecular weight of 158 kD and a calculated pI of 6.4. The gene contains no introns. There are three in-frame methionines at the N terminus of the predicted protein at positions 1, 5, and 7. The region upstream of the first ATG has the best match to the Drosophila consensus sequence for translation initiation (CAVENER and RAY 1991 ). However, it is also possible that the ATGs corresponding to the methionines at positions +5 and +7 are used to initiate translation, since the region upstream of these ATGs also has considerable similarity to the CAVENER and RAY 1991 consensus sequence.

Further upstream, at positions -377 to -370 and -352 to -345 with respect to the first ATG, are two DRE (DNA replication-related element) motifs. DRE is a cis-acting positive regulatory element (TATCGATA) present in the promoters of a variety of genes, including those encoding proteins with functions in DNA replication, transcription, translation, signal transduction, and cell cycle control (MATSUKAGE et al. 1995 ). We have not carried out extensive searches for other regulatory motifs in this region.

mus101 encodes a protein with seven BRCT domains and is similar to the human protein TopBP1:
Searches of the databases using the PSI-BLAST program (ALTSCHUL et al. 1997 ) for proteins similar to Mus101 revealed the highest scores for a human protein first described as having similarity to the fission yeast Rad4/Cut5 gene product (NAGASE et al. 1996 ). This human protein is identical to TopBP1, a protein identified through its in vitro binding to DNA topoisomerase IIß (YAMANE et al. 1997 ). In addition to TopBP1 and Rad4/Cut5, proteins showing significant similarity to Mus101 are C. elegans clone F37D6.1, Arabidopsis thaliana clone T10M13.12, the Drosophila protein Pebble (Pbl) required for cytokinesis (PROKOPENKO et al. 1999 ), the mouse transforming protein Ect2, and the human DNA repair protein XRCC1.

All these proteins have BRCT domains, a module composed of 100 amino acids with a characteristic hydrophobic profile, easily identified in a hydrophobic cluster analysis plot (CALLEBAUT and MORNON 1997 ). In fact, the regions of highest similarity that these proteins show with Mus101 are in the BRCT domains, with the exception of TopBP1 and the C. elegans clone F37D6.1, where the regions of similarity extend beyond the BRCT domains. As an example, Fig 3A shows the similarities between the highly conserved BRCT domain I in Mus101, TopBP1, C. elegans clone F37D6.1, Rad4/Cut5, Pbl, and Ect2. The number and distribution of BRCT domains in these proteins are compared in Fig 3B.

View larger version (29K): In this window In a new window Download PPT slide   Figure 3. A comparison of proteins structurally related to Mus101. (A) Alignment of the primary structures of BRCT domain I from Mus101, TopBP1, C. elegans clone F37D6.1, fission yeast Rad4/Cut5, Drosophila Pebble (Pbl), and mouse Ect2. The boxed residues are highly conserved and the boldface residues conserved among BRCT domains, according to CALLEBAUT and MORNON 1997 . (B) Comparison of the number and distribution of BRCT domains in Mus101, TopBP1, F37D6.1, Rad4/Cut5, Pbl, and Ect2. BRCT domains are represented by solid ovals. The diamond labeled TCS and the circle labeled PSC represent regions of homology shared by Mus101 and human TCOF1 and Drosophila posterior sex comb proteins, respectively. The open ovals labeled DH and the boxes labeled PH of Pbl and Ect2 represent Dbl and pleckstrin homology domains, respectively (PROKOPENKO et al. 1999 ).

The pairwise alignment of Mus101 and TopBP1 generated using the BLAST2 program (TATUSOVA and MADDEN 1999 ) is shown in Fig 4. Both proteins have similar size: Mus101 is 1425 and TopBP1 is 1522 amino acids long. The human TopBP1 protein has eight BRCT domains, the greatest number of domains so far observed in a single protein (YAMANE et al. 1997 ). Mus101 has seven BRCT domains, and their distribution is similar to those of TopBP1, with only the central BRCT domain (VI) of TopBP1 not represented in Mus101 (Fig 3B). The N-terminal and central part of Mus101 has 24% identity and 42% similarity to the N terminus of TopBP1. This region contains the Mus101 and TopBP1 BRCT domains I, II, III, IV, and V. The C terminus of Mus101 has 33% identity and 51% similarity with the C terminus of TopBP1. This region contains the Mus101 BRCT domains VI and VII and the TopBP1 BRCT domains VII and VIII.

View larger version (61K): In this window In a new window Download PPT slide   Figure 4. BLAST2 alignment of Mus101 and TopBP1. (A) Alignment of the N-terminal-to-central regions of Mus101 and TopB1. Identity, 206/828 (24%); positives, 361/828 (42%); gaps, 95/828 (11%). Residues comprising each BRCT domain are boxed. Domains were predicted on the basis of CALLEBAUT and MORNON 1997 and through comparisons of multiple sequence alignments of BRCT domain-containing proteins. Actual domains may include up to five or six additional residues at each end. (B) Alignment of the C-terminal regions of Mus101 and TopBP1. The BRCT domain names VI and VII refer to the Mus101 protein; the homologous domains in TopBP1 are VII and VIII, respectively. Identity, 67/200 (33%); positives, 104/200 (51%); gaps, 19/200 (9%).

The region between BRCT domains V and VI of the Mus101 protein that does not have similarity to TopBP1 contains two distinct subregions with similarity to other proteins. Residues 825–909 of Mus101 share 27% identity and 46% similarity with the C terminus of human treacle protein (TCOF1), a putative nucleolar trafficking phosphoprotein that is defective in patients with Treacher Collins syndrome, a craniofacial developmental disease (WISE et al. 1997 ). Residues 1046 to 1129 of Mus101 are 26% identical (38% similar) to the C terminus of Drosophila posterior sex combs (Psc) protein. Psc is a product of one of the Polycomb group genes (Pc-G). Pc-G genes are needed to maintain patterns of expression of homeotic genes during Drosophila development by repression of target genes (for review see PIRROTTA 1995 ).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have molecularly cloned the mus101 gene and found it to encode a predicted protein of 1425 amino acids containing seven BRCT modules distributed throughout its length. The number and position of BRCT domains vary in different proteins. For example, BRCA1, Rad9, and XRCC1 each have two domains in their C termini, Ect2 has two domains in its N terminus, and Rad4/Cut5 has two domains in its N terminus and two domains in the central part of the protein (BORK et al. 1997 ; CALLEBAUT and MORNON 1997 ). The majority of proteins described so far have on average two to four BRCT domains, although some have a greater number, like the C. elegans clone F37D6.1, of unknown function, which has six domains (BORK et al. 1997 ; CALLEBAUT and MORNON 1997 ) and TopBP1, which has eight (YAMANE et al. 1997 ). It has been suggested by CALLEBAUT and MORNON 1997 and BORK et al. 1997 that BRCT domains may be sites for interaction between proteins implicated in the maintenance of the genome in response to damage. The knowledge of such interactions is beginning to shed light upon poorly understood repair mechanisms. Examples are accumulating showing that BRCT domain-containing proteins can participate not only in different aspects of DNA repair and cell cycle checkpoint control, but also in other essential cellular processes, like transcription (ANDERSON et al. 1998 ; GOWEN et al. 1998 ; KLEIMAN and MANLEY 1999 ; XU et al. 1999 ; ZHONG et al. 1999 ).

The protein most closely resembling Mus101 in searches of the current databases is TopBP1, which interacts with DNA topoisomerase IIß (Topo IIß). This interaction is mediated through the C terminus of TopBP1, where two consecutive BRCT domains are located (YAMANE et al. 1997 ). Topo IIß is a member of the type II subfamily of topoisomerases that generates transient double-strand breaks to catalyze topological changes in DNA (AUSTIN and MARSH 1998 ). YAMANE et al. 1997 have speculated that TopBP1 may function in the repair of strand breaks resulting from a failure of the DNA rejoining reaction of Topo IIß. This hypothesis is supported by their more recent finding that regions of TopBP1 containing BRCT domains are able to bind nicked circular and linear DNA molecules, but not intact circular DNA (YAMANE and TSURUO 1999 ).

D. melanogaster has only a single Topo II isoform, present in at least three separate functional pools: one for chromosome condensation, one for chromosome segregation, and one pool that remains associated with the chromosome throughout the cell cycle (SWEDLOW et al. 1993 ). Drosophila Topo II associates with Barren, a member of the condensin/cohesin family of proteins necessary for segregation of sister chromatids at mitosis (BHAT et al. 1996 ). Drosophila Topo II was also recently identified as a component of the chromatin-remodeling factor CHRAC (chromatin-accessibility complex; VARGA-WEIZ et al. 1997 ), suggesting a structural role for this protein in chromatin organization. The C-terminal region of TopBP1 that interacts with Topo IIß has 33% identity and 51% similarity with Mus101. It is possible that Mus101 is a functional homolog of TopBP1 and that Mus101 also binds Topo II. Such a putative interaction between Mus101 and Topo II, if it were specific to heterochromatic regions, could explain the failure of heterochromatin to condense properly in the lethal mutants mus101^lcd and mus101^ts1.

Mus101 BRCT domains I and II are very similar to BRCT domains I and II of the fission yeast replication checkpoint protein Rad4/Cut5 and the oncogene product Ect2, regions that are important to the roles of these proteins in cell cycle control. Truncation of the N-terminal region of Ect2 to remove these two BRCT domains increases its transforming activity, suggesting that this region has a negative effect on cell division (MIKI et al. 1993 ). The N terminus of Rad4/Cut5 containing these BRCT domains is essential for complementation of the temperature-sensitive phenotype in rad4/cut5 mutants. Moreover, its overexpression blocks cell division (SAKA and YANAGIDA 1993 ). The localization of the mutations in three independent rad4/cut5 alleles revealed the same amino acid substitution T45M in the conserved stretch VTHLIA in BRCT domain I (SAKA et al. 1997 ; see Fig 3A). This mutation prevents the interaction of Rad4/Cut5 with the DNA replication and DNA damage checkpoint protein Crb2 (SAKA et al. 1997 ).

There are many similarities between mus101 and rad4⁺/cut5⁺. Both are essential genes and both are required for DNA repair. They also both encode members of the BRCT superfamily, with similar BRCT domains I and II. Rad4/Cut5 is a component of the replication checkpoint control system (SAKA and YANAGIDA 1993 ) and together with Crb2 forms a complex with the checkpoint protein Chk1. It has been suggested by SAKA et al. 1997 that these proteins may form a checkpoint sensor-transmitter pathway to arrest cell cycle progression. Is Mus101 also necessary for the DNA replication checkpoint? In S. pombe, the replication checkpoint has been categorized into two classes: one activated by a defect in the replication machinery (mutations in polymerases and ligases) and the other activated by limitation of normal nucleotide supply [effected by hydroxyurea (HU) treatment or mutation of cdc22]. Rad4/Cut5 is necessary for both checkpoints, whereas Crb2 is necessary only for the nucleotide supply checkpoint (SAKA et al. 1997 ). Tests of mus101^D1, mus101^D2, and mus101^K451 larvae have revealed wild-type levels of sensitivity to HU (BANGA et al. 1986 ; R. R. YAMAMOTO and D. S. HENDERSON, unpublished results), suggesting that the nucleotide supply checkpoint is still largely intact in these mutants and perhaps highlighting a functional distinction between Rad4/Cut5 and Mus101.

	ACKNOWLEDGMENTS

We thank A. Schalet, A. Carpenter, M. Gatti, B. Baker, V. Lloyd, D. Sinclair, E. Frei, and S. Krishnan for providing fly stocks or cloned DNA and helpful information. We are grateful to F. Cullen, L. Campbell, C. Salles, D. Callister, and A. Bain for expert technical assistance, and past and present members of the lab for support and helpful discussion, especially P. Deak, E. Wojcik, H. Ohkura, R. Wiegand, and S. Brand. R.R.Y. was funded by the Brazilian agency CNPq. This work was made possible by grants from the Cancer Research Campaign.

Manuscript received February 22, 2000; Accepted for publication June 8, 2000.

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