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Genetics, Vol. 176, 2151-2163, August 2007, Copyright © 2007
doi:10.1534/genetics.107.073551
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* Stowers Institute for Medical Research, Kansas City, Missouri 64110, Comparative Genomics Centre, James Cook University, Townsville, 4811 Australia and Department of Physiology, University of Kansas Medical Center, Kansas City, Kansas 66160
1 Corresponding author: Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110.
E-mail: cml{at}stowers-institute.org
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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All MCM members belong to the AAA+ ATPase family, which has a distinct ATPase domain that spans 
200 bases (WALKER et al. 1982). This domain, referred to as the MCM box, consists of a Walker A ATPase motif, a Walker B ATPase motif, and an arginine finger motif (R-finger). Conserved sequences within the Walker B motif (IDEFDKM) and R-finger (SRDF) define the MCM family (KOONIN 1993). Six of these members are conserved in all eukaryotes and form a heterohexameric complex known as Mcm2-7, which has been studied extensively for its role in DNA replication (KEARSEY and LABIB 1998; TYE 1999). Mcm2-7 is required for licensing and initiating origins of replication, and it acts during elongation as a helicase at the replication forks (LABIB et al. 2000; PATEL and PICHA 2000). Because of this function and studies in yeast, Arabidopsis and Drosophila, members of the Mcm2-7 complex, are thought to be essential. In addition, two other MCM family members, Mcm8 and Mcm9, have recently been identified and are thought to be a distinct subgroup of MCM proteins (MAIORANO et al. 2006). Mcm8 has been reported in vertebrates and Drosophila, but not in fungi and nematodes, and although it retains some sequence similarities in the Walker B and R-finger, its Walker A ATPase motif contains sequences more like the canonical ATPases (GOZUACIK et al. 2003; BLANTON et al. 2005). Mcm9 is also found in similar organisms with the exception that it is missing in Drosophila, and it is unique to the family in that it lacks the carboxy-terminal ATPase domain including the Walker B motif (BLANTON et al. 2005; LUTZMANN et al. 2005).
Recently, studies have indicated that in addition to the role in DNA replication, certain members within the Mcm2-7 complex, as well as other MCM family members, have functions outside of DNA replication (FORSBURG 2004). Specifically, some of these functions include a role in transcriptional activation (YANKULOV et al. 1999; DAFONSECA et al. 2001), chromosome condensation (CHRISTENSEN and TYE 2003), cohesion (RYU et al. 2006), and recombination (BLANTON et al. 2005; SHUKLA et al. 2005). The existence of multiple functions is consistent with studies in yeast, which showed that MCM proteins are far more abundant than would likely be required for the number of replication origins that exist, and this abundance cannot explain the fact that slight decreases in amounts of MCM proteins lead to the inability to complete S-phase and progress through the cell cycle (LIANG et al. 1999; BAILIS and FORSBURG 2004). Moreover, in addition to the heterohexameric Mcm2-7 complex, subcomplexes of MCM family members have been identified (SU et al. 1996; LEE and HURWITZ 2000), which fuel the speculation that these complexes could be functionally distinct subgroups that possess functions beyond those involved in DNA replication.
Limited functional studies have been done on the Drosophila orthologs of the Mcm2-7 complex. Although genes for each of these members have been identified in Drosophila (FEGER 1999), only mcm2 (TREISMAN et al. 1995), mcm4 (FEGER et al. 1995), and mcm6 (SCHWED et al. 2002) have been shown to be required for mitotic DNA replication. Null alleles of each inhibit proliferation of cells of the central nervous system (CNS) and imaginal discs, which leads to a reduction in brain size and lack of discs within the developing larvae. These larvae begin pupariation but never develop into adults. In addition to a role in mitotic DNA replication, two other functions for mcm6 have been identified that were not observed in mcm2 or mcm4 mutants. Mcm6 is required for endo-reduplication which is a process of reoccurring rounds of DNA replication in the absence of cell division that occurs within the developing larvae and is responsible for most of the larval growth and is also required for chorion gene amplification (SCHWED et al. 2002).
Until now, there have been no genetic studies that analyzed the roles of mcm5 in Drosophila. Although it is speculated that mcm5 is required for DNA replication in Drosophila, specific functions in this process as well as other functions it may have remain unknown. The fortuitous identification of an allele of mcm5 in a screen for new meiotic mutants stimulated us to begin a thorough genetic dissection of this gene to determine the functions of mcm5 in Drosophila.
In this study we show that the mcm5 locus is essential, in that homo- or hemizygotes for a null mutation in mcm5 die prior to eclosion. They do, however, survive to third instar larvae with rudimentary imaginal discs and small brains, suggesting a defect in facilitating mitotic DNA replication. This defect in mitotically dividing cells does not extend to endo-reduplicating tissues, since the highly polytene chromosomes of the salivary gland appear normal. These findings are similar to what has been identified for mutants in mcm2 and mcm4, but differ from findings in mcm6, which is also considered essential for endo-reduplication.
In addition to the null allele of mcm5, we have also identified an EMS-induced allele that is not required for the essential functions of Mcm5, which is to say that homo- and hemizygotes for this mutant are viable and fertile, but rather has a function in the meiotic recombination pathway. We demonstrate that the decrease in recombination is not due to a failure to form either synaptonemal complex or double-strand breaks (DSBs) or to a general inability to repair DSBs that are induced by DNA damaging agents in somatic cells. This observation suggests that we have identified a residue or domain in the Drosophila mcm5 gene that is specifically required for meiotic recombination.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Excision:
A P-element insertion mutant, KG05409, bearing a mutation between art1 and mcm5 was obtained from the Bloomington Drosophila Stock Center (BL 14003). A null allele of mcm5 was generated by imprecise excision of the P element using the 
-2-3-mediated mobilization standard protocol. Homozygous lethal excisions were analyzed by PCR. Genomic DNA was isolated from single males that were generated by crossing yw; excision/TM3, Sb1 virgin females to yw/Y; P{SUP or P}KG05409 males. PCR analysis of a precise excision event with primer pair 5'-AGCATCTCCTCGTGGATCCCG-3' (located in the art1 gene) and 5'-CACCACGCGTTCACAGAG-3' (located in the mcm5 gene) would result in an amplicon size of 1.6 kb. This primer pair would fail to amplify the chromosome containing the P{SUP or P}KG05409. PCR analysis of mcm5exc222 resulted in an amplicon size of 600 bases. The excision event removed 195 bases upstream of the 5'-UTR of mcm5, the entire 5'-UTR of 137 bases, and 650 bases of the mcm5 gene, thereby creating a null allele of mcm5.
Generation of transgenes:
The genomic rescue construct was constructed by cloning a 4-kb SacII–XbaI fragment from Bac clone RP98-29B6 (BacPac Resources). This fragment, which contains the entire gene region of mcm5 plus 578 bases 5' to mcm5 (the 5'-UTR of Art1 and 164 bases of coding region of art1) and 809 bases 3' to the mcm5 gene, was cloned into pCasPeR4 that was previously digested with SacII and XbaI. pCasPeR4-mcm5, denoted as p{mcm5+}, was sequenced and then introduced into Drosophila by standard P-element-mediated transformation protocols. Three independent transformants (P{mcm5+} 7, 33, and 39), which all mapped to the X chromosome, were tested for the ability to complement the mcm5A7 meiotic phenotype by analyzing nondisjunction frequency of the X chromosome.
The germline expression construct was generated by amplifying the full-length mcm5 cDNA sequence with primers 5'-GTACGGTACCATGGAAGGCTTCGACG-3' and 5'-GTCGCGGCCGCTTAGCAAATTCGATAG-3' from clone RE67590 (FlyBase). The PCR product was digested with KpnI and NotI and cloned into the pUASp vector (gift from Pernille Rørth; RøRTH 1998) digested with the same enzymes. Sequence was verified on both strands. p{UASp-mcm5+} was introduced into Drosophila by standard P-element-mediated transformation protocols. Germline expression was achieved by expressing the p{UASp-mcm5+} construct under the control of the nos-Gal4::VP16 driver (RøRTH 1998). Virgin females of the genotype p{UASp-mcm5+}; mcm5A7/TM3, Sb1 were crossed to either p{nos-Gal4::VP16}/y+Y; mcm5A7/TM3, Sb1 or yw/y+Y; mcm5A7/TM3, Sb1 males. Virgin females of the appropriate genotype were crossed individually to tester males and the frequency of X nondisjunction was determined (see below).
Larval analysis:
Non-GFP-expressing third instar larvae from the genotype yw/X; mcm5exc222/TM3, P{GAL4-twi.G}2.3, P{UAS-2xEGFP}AH2.3, Sb, Ser or yw/X; mcm5A7/TM3, P{GAL4-twi.G}2.3, P{UAS-2xEGFP}AH2.3, Sb, Ser were identified and analyzed by standard techniques. Brains and associated imaginal discs were visualized under a standard dissecting scope. Salivary gland preparations were prepared by dissection into saline solution, fixed in 45% acetic acid solution for 2 min, squashed onto Sigma-coat coverslips, and set in vapors of liquid nitrogen for 30 min. The coverslips were removed, and the slides were placed in methanol, air dried, and stained with 1.0 µg/ml 4',6-diamidino-2-phenylindole (DAPI) solution.
Overview of the isolation of an EMS-induced mutant in mcm5:
A genetic screen was designed to recover new meiotic mutants that did not exclude essential genes (details of screen in PAGE et al. 2007). Briefly, we adapted the FLP–FRT system to design a screen that required a meiotic nondisjunction event of an autosome to survive the screen. We tested each potential mutant as a germline clone for defects in meiosis. We screened 25,571 chromosome arms on 3R and isolated 10 meiotic mutants. This article describes the analysis of one mutant isolated from this screen called A7. A7 was identified as a novel meiotic mutant in that it fully complemented all known meiotic mutants on 3R, as well as all mutants isolated from the screen. After the initial isolation of mutant A7, a secondary test was done to confirm the meiotic phenotype. A7 displays a strong meiotic phenotype showing 27.5% X nondisjunction and 8% nullo fourth nondisjunction.
At the time of the screen, we were not aware that the original FRT P{ovoD1} chromosome had an associated lethal on the 3L arm. Through the process of the FLP-induced recombination the mutant A7 stock contained this lethal, which was subsequently removed. The 3L-lethal-removed version was retested and showed similar levels of meiotic nondisjunction (data not shown), and therefore all future studies in this article were done with the lethal-removed version of A7. Upon the removal of the associated lethal, it was determined that A7 is a homozygous viable and fertile mutation.
Mapping and identification of an allele of mcm5:
The EMS-induced lesion in A7 was mapped using indel and deficiency mapping (for details see PAGE et al. 2007). Briefly, recombinants are made between the unmarked FRT-bearing chromosome containing A7 and a chromosome containing a distal marker element known as EP. The location of each recombination event was determined by PCR for indel markers. Selected recombinants were tested for the presence of the A7 mutation by analyzing each for a meiotic nondisjunction phenotype. Indel mapping placed the lesion between the interval 85B3–87C7. This was in agreement with mapping by deficiencies. In parallel, the lesion in A7 was mapped by testing for complementation of the meiotic nondisjunction phenotype using the 3R set of Bloomington deficiencies and additional deficiencies from the Exelixis kit. Three deficiencies [Df(3R)BSC38, Df(3R)Exel6169, and Df(3R)Exel7305] that overlap in the interval 86C6–86C7 failed to complement A7. Since the breakpoints of two of these deficiencies have been molecularly characterized, we conclude that the interval is defined by the sequence coordinates 6715093 and 6698001 on 3R (Bloomington Database). To verify that these deficiencies did not uncover a gene that was haplo-insufficient, we tested all three deficiencies that failed to complement A7 with the original FRT chromosome the mutation was induced on. All three deficiencies fully complement the original FRT chromosome, and thus no dominant effects were observed (data not shown).
Nondisjunction and recombination assays:
To measure the frequency of meiotic nondisjunction of the X and fourth chromosomes, virgin females of the listed genotype are crossed individually to attached-XY, y+ v f B; C(4)RM, ci eyR males (X and fourth, Table 3) (for details see ZITRON and HAWLEY 1989; HAWLEY et al. 1992; HARRIS et al. 2003) or y sc cv v f y+/B[S]Y males (X only) (ZIMMERING 1976; MATSUBAYASHI and YAMAMOTO 2003). The frequency of recombination on the X chromosome was measured by crossing y w/y sc cv v f y+; FRTmcm5A7 or y w/y sc cv v f y+; FRT single female virgins to y sc cv v f y+/B[S]Y males. Only female progeny resulting from the above cross were analyzed for the markers y sc cv v f and y+ (for details see PAGE et al. 2000; PAGE and HAWLEY 2001).
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Tilling alleles:
Additional alleles were obtained through TILLING of mcm5 at the Fred Hutchinson Cancer Research Center (FlyTILL). Mutations were verified by sequencing the mcm5 gene with the above primer pairs (data not shown). The following Zucker stocks were obtained for this study: Z3-PMM-0509 (I471I), Z3-2022 (R491Q, SIFT score 0.01), Z3-2146 (intron), Z3-3224 (M513I, SIFT score 0.15), Z3-PMM-0153 (E427E), Z3-5239 (R344R), Z3-5602 (E550E), Z3-1138 (E498K, SIFT score 0.22), Z3-0852 (P545P), Z3-1334 (S689L, SIFT score 0.00), Z3-3278 (M431I, SIFT score 0.02), Z3-4809 (F630I), and Z3-2156 (F630I).
Western blotting:
Females of the appropriate genotype were mated with males and fed yeast for 3 days. Ovaries were dissected in PBS–Triton (PBS + 0.1% Triton-X-100). Twenty ovaries were homogenized in 50 µl of 1x sample loading buffer (50 mM Tris-HCl pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) by a small pestle. The extract was heated to 100° for 5 min and 20 µl of each genotype were loaded on a 10% SDS–PAGE gel. Protein was transferred onto PVDF membrane, blocked in PBS–Tween-20 (PBS + 0.1% Tween-20) containing 4% dry powdered milk, and probed with primary antibodies overnight at 4°. The membrane was washed in PBS–Tween-20 and probed with secondary antibody for 2 hr at room temperature. The washed membranes were reacted with alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit antibody. The bound antibodies were detected by reacting with substrate solution containing 50 µg 5-bromo-4-chloro-indolyl-phosphate/ml and 100 µg 4-Nitro Blue Tetrazolium chloride/ml of rinse buffer (100 mM Tris pH 8.8, 1 mM MgCl2). All Blue Precision Plus Protein Standards were used as a marker (Bio-Rad, Hercules, CA).
Immunostaining:
Approximately 30–40 females, which had eclosed 2–3 days previously, were mated to 10–15 males and fed on yeast for 2–3 days prior to egg chamber dissection. These females were anesthetized and the abdomens were ruptured one by one with forceps. For early egg chambers, oocytes were fixed using previously published methods (PAGE and HAWLEY 2001) with minor exceptions. Basically ovaries were dissected in PBS and immediately fixed for 20 min in 200 µl of PBS containing 2% formaldehyde (Ted Pella) and 0.5% Nonidet P-40 plus 600 µl heptane. Fixed ovaries were washed three times for 15 min each in PBS–Tween-20. Ovarioles were teased apart with forceps and blocked for 1 hr in PBS–Tween-20 containing 1% bovine serum albumin (BSA) (Calbiochem, La Jolla, CA) at room temperature. Primary antibody was incubated overnight at 4° and washed as before, and secondary antibodies were applied for 4 hr at room temperature. Ten minutes prior to washing a final concentration of 1.0 µg/ml DAPI was added. Ovarioles were washed as before and mounted in ProLong Gold (Invitrogen, Carlsbad, CA). Late-stage oocytes were fixed as previously described (GILLILAND et al. 2005). Whole ovaries were dissected in 1x Robb's media (55 mM sodium acetate, 8 mM potassium acetate, 20 mM sucrose, 2 mM glucose, 0.44 mM MgCl2, 0.1 mM CaCl2, and 20 mM HEPES, pH 7.4) containing 1% BSA and individually ovarioles were teased apart with forceps. Ovaries were fixed in solution containing 1x fix buffer (100 mM potassium cacodylate, 100 mM sucrose, 40 mM sodium acetate,and 10 mM EGTA) and 8% formaldehyde (Ted Pella) for 5 min. Ovaries were washed in PBS–Triton three times for 15 min each, vitelline membrane was removed by rolling ovaries between frosted slides, and then ovaries were blocked 1 hr in PBS–Triton containing 5% normal goat serum. Antibody labeling was done as described above except washes were done in PBS–Triton.
Microscopy was conducted using a DeltaVision microscopy system (Applied Precision, Issaquah, WA) equipped with an Olympus 1x 70 inverted microscope and high-resolution CCD camera. Images were deconvolved using the SoftWoRx v.25 software (Applied Precision). Images are shown as maximum-intensity projections of the complete germarium, or as a subset of sections, or as a single deconvolved section (noted in each figure legend). His2Av foci were counted by manually examining all optical sections of anti-His2Av staining nuclei.
Antibodies:
Mouse anti-Orb antibodies (4H8 and 6H4) obtained from the Iowa Hybridoma Bank (LANTZ and SCHEDL 1994) were used together at a 1:30 dilution. Mouse anti-C(3)G antibody (ANDERSON et al. 2005) was used at a dilution of 1:500. Guinea pig anti-C(3)G antibody was used at 1:500 dilution (PAGE and HAWLEY 2001). Rabbit anti-His2Av (gift from Kim McKim; MEHROTRA and MCKIM 2006) was used at 1:500. Rabbit anti-Mcm5 (gift from Paul O'Farrell; SU et al. 1996) was used at a dilution of 1:666. Rat anti-tubulin antibodies, MAS078P (Harlan Ser-Lab) and MAS1864 (Chemicon), were used together at a dilution of 1:250. Mouse anti-
tubulin antibody (Sigma, St. Louis) was used for Western blotting at 1:1500. Secondary goat anti-mouse or rabbit Alexa-488 and Alexa-555 conjugated antibodies (Molecular Probes, Eugene, OR) were used at 1:500, and donkey anti-rat Cy3 conjugated antibody was used at 1:200 (Jackson ImmunoReseach). Anti-rabbit and anti-mouse alkaline phosphatase-conjugated antibodies (Sigma) were used at 1:5000.
MMS and X-ray treatment:
Sensitivity to different DNA-damaging agents was determined for two developmental stages of Drosophila. Two- to 3-day-old virgin yw; mcm5A7/TM3 Sb1 females were mated to yw/y+Y; mcm5A7/TM3 Sb1 males. For chemical treatment, flies were set up in standard food vials (5 females and 5 males per vial testing 5 vials per concentration), and for X-ray treatment flies were set up in cages with grape plates (100 females and 30 males), and each allowed to lay eggs for 24 hr. After an additional 24 or 48 hr, the embryos or larvae were treated with various concentrations of 0.25 ml methyl methanesulfonate (MMS) (Sigma) in water or X-rayed with an 1000-R dose (shelf 6, 115 kV, and 5 mA for 2 min) (Faxitron Cabinet X-ray Systems). Untreated larvae of the same parents were used as controls. After X-ray treatment, embryos or larvae from grape plates were transferred to bottles containing standard food. The offspring were analyzed starting at day 10 and continued to be analyzed until day 18. Zero, 0.02%, and 0.04% MMS-treated embryos and larvae began to hatch at day 10; 0.08% on day 11; and 0.16% on day 12. In both treatments, the eclosed adult flies were counted to determine viability. In controls, the relative viability of homozygotes is 33% of total viable progeny.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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30% X chromosome nondisjunction. Mapping of the mutant, denoted mcm5A7, indicates the gene responsible for this elevated level of nondisjunction is mcm5. Indeed, the mcm5A7 allele results from a single A 
T transversion (A2081T) in the first codon of the last exon that corresponds to an aspartic acid to valine change (D694V) (Figure 3A). This acidic residue, which is located in the C-terminal region outside of the conserved MCM box, is highly conserved from yeast to humans (Figure 3B).
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We also obtained 12 additional point mutations in both the MCM box and C-terminal region in mcm5 from the Seattle Fly-TILLING project (Figure 3C). Of these, 5 were noncoding, 1 was in an intron, and 6 resulted in amino acid substitutions. Of these 6, 3 were predicted to cause deleterious changes on the basis of SIFT scores of <0.05. However, none of these additional alleles was lethal over the deficiency Df(3R)BSC38, which uncovers the mcm5 gene or showed a meiotic phenotype when analyzed over this deficiency (data not shown).
Defects observed in mcm5A7 are not due to reduced levels of Mcm5 protein:
One possible explanation for the lack of an effect of the mutant on viability might be that the process of oogenesis requires higher levels of Mcm5 protein than do the mitotic divisions required for growth and viability, and that the mcm5A7 reduced the level of Mcm5 protein in the ovaries below some threshold required for meiotic function, but not to a sufficient degree to be lethal. To verify that the ovaries homozygous for the mcm5A7 mutant expressed a Mcm5 protein of predicted size and at wild-type levels, we compared the expression of Mcm5 from mcm5A7 and control ovaries. By Western blotting, it was confirmed that ovaries from females homozygous for mcm5A7 produced levels of Mcm5 protein that were similar or identical to wild-type protein (Figure 4).
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Oocytes homozygous for the mcm5A7 mutant are proficient in synapsis:
In Drosophila, formation of the synaptonemal complex (SC), a structure that is required for synapsis and meiotic recombination, occurs only after premeiotic S-phase is complete (GRELL 1984). The transverse filament protein, C(3)G (the yeast Zip1 homolog), is a marker often used for visualization of SC structure. Previous localization studies by PAGE and HAWLEY (2001) show that C(3)G is first present within multiple cells of the germline cyst in region 2A. This staining is reduced to two adjacent cells in region 2B and is lost by region 3 in all cells but the oocyte, which can be detected by cytoplasmic Orb staining (LANTZ and SCHEDL 1994). As the oocyte enters the vitellarium stage (stage 2) the C(3)G thread-like staining persists early, gradually breaking down and most is lost by stage 6. Comparison of the localization of C(3)G in mcm5A7 to previous studies and to FRT controls (data not shown) indicates that there is no observable defect in the timing of C(3)G expression, the ability to form SC, or in the SC structure by immunocytology (Figure 5), which indicates the mutant is able to complete synapsis.
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HIS2Av (gift from Kim McKim; MEHROTRA and MCKIM 2006). Using this antibody, McKim and colleagues show that DSBs first appear after SC formation in region 2A where the highest numbers of foci are detected. Most His2Av foci disappear by region 2B where only a few foci are occasionally detected, and all foci are gone by region 3, presumably as repair is being completed. On the basis of His2Av staining pattern, we found no significant difference in the number of DSBs formed in mcm5A7 compared to control (in region 2A, average number DSBs in mcm5A7 is 10.1, N = 22 and 9.7, N = 13 in controls) or in the ability to repair breaks by region 3 that had formed in region 2A (Figure 6).
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Although the DSBs created in mcm5A7 mutant oocytes are not left unrepaired, they produce significantly reduced levels of crossovers and chiasmata (the physical manifestation of exchange). Homozygotes for mutants that greatly reduce the level of meiotic recombination usually also display defects in the ability to arrest meiotic progression at metaphase I (MCKIM et al. 1993). This failure to arrest reflects a requirement for at least one chiasmata, the physical manifestation of an exchange event, to hold at least a pair of homologs together at the midspindle and thus create a metaphase plate. Accordingly, we analyzed meiotic spindles from both mcm5A7 and wild-type ovaries to determine if there was a failure to arrest at metaphase I. Although we did not observe any defects in prometaphase figures (Figure 7, compare A and B to E and F), we did observe a failure to arrest at metaphase I in 42% (n = 54) of mcm5A7 oocytes (Figure 7, compare C and D to G and H). Indeed, the predicted frequency of cells without a chiasmata on all five chromosome arms in mcm5A7 (E0 frequency of 0.88) is 52%, and thus we would expect this high frequency of a failure to arrest at metaphase I (MCKIM et al. 1993). In addition to the anaphase I-like figures seen in Figure 7, we also less commonly observed (11%) figures that contained greater than two spindles.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We have also identified a function of Mcm5 in the maturation of DSBs into crossovers and shown that this meiotic function is separable from the role of Mcm5 in mitotic replication. We have identified a residue or domain in Mcm5 that is specifically required for this meiotic function. The observation that mcm5A7 homozygotes and mcm5A7/Df heterozygotes demonstrate similar levels of X chromosome nondisjunction argues that the mutant constitutes a null allele with respect to the role of this protein in meiotic recombination, while the fact that this mutant so strongly affects meiosis without affecting viability demonstrates that the mcm5A7 mutant is a clear separation-of-function allele in terms of the role of the Mcm5 protein in mitosis and meiosis.
Recombination-deficient mutants in Drosophila can coarsely be grouped into four classes: those like mei-W68 (which encodes the fly homolog of SPO11) that block the formation of DSBs, those like mei-9 and mus312 that are involved in the resolution of recombination intermediates, those like spn-A and spn-B that are involved in the repair of DSBs, and a class of mutants, often referred to as precondition mutants, that appear to simply alter the probability that DSBs will be processed into crossover events (CARPENTER and SANDLER 1974; LINDSLEY and SANDLER 1977; BHAGAT et al. 2004). Precondition mutants are characterized by the fact that they not only decrease the total number of exchange events, but also alter the mechanisms that normally control the distribution of exchanges, such that exchanges occur more commonly in proximal regions than in distal regions (BAKER and HALL 1976; LINDSLEY and SANDLER 1977). They also usually ablate crossover interference, the process that serves to distribute crossover events along the arms of chromosomes. The mcm5A7 mutant, as well as mutants in the rec/mcm8 and mei-218 genes, are all precondition mutants. As shown in Figure 8, the three genes defined by these mutants encode either bona fide MCM proteins (Mcm5 or Mcm8) or a protein with a MCM domain (Mei-218) (J. SEKELSKY and K. MCKIM, personal communications).
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So then how might these mutants suppress crossing over by a mechanism that is unrelated to their traditional roles in replication? One possibility is that at least Mcm5 is known to regulate transcription via a physical interaction with Stat1 (ZHANG et al. 1998; SNYDER et al. 2005). Thus it is at least possible, even though the mcm5A7 mutation lies well outside the putative Stat1 interacting domain of Mcm5 (DAFONSECA et al. 2001), that the change created by this mutation impairs the interaction of Mcm5 with Stat1 or some other transcriptional regulator, and in doing so prevents the expression of one or more genes that function in the maturation of DSBs to crossovers.
However, on the basis of a recent finding by Gasser and her collaborators (SHIMADA and GASSER 2007) that origin recognition complex proteins in yeast function in the process of establishing and maintaining sister chromatid cohesion, in a fashion that is independent of their role in replication initiation, we propose that MCM proteins in flies might play a similar role in meiosis. We imagine that like the fly Ord and C(2)M proteins, which are thought to be involved in conversion of the cohesion complex into the lateral elements of the synaptonemal complex (for review of this process see PAGE and HAWLEY 2004), the Mcm5, Rec, and Mei-218 proteins also play a role in the function of axial and/or lateral elements and that it is this defect, rather than a problem in replication per se, that underlies their meiotic defects.
We should note that this adaptation of the Mcm5 protein for a meiotic function may not be universal. Forsburg and her collaborators have created the corresponding mutation to that found in mcm5A7 in S. pombe (see Figure 2B) and failed to observe any defect in meiotic recombination (S. FORSBURG, personal communication). This may reflect the rather unusual constellation of repair and recombination proteins found in Drosophila (SEKELSKY et al. 2000). Notably lacking from the fly genome are obvious homologs of the Dmc1 protein, which at least in other organisms is required to promote interhomolog exchange events and suppress sister chromatid exchange events. Although ABDU et al. (2003) have proposed that the missing Dmc1 function might be provided by fly Rad51 homologs, Spn-B and Spn-D, one could imagine that the MCM proteins also play such a role in flies, and thus measurement of meiotic sister chromatid exchange in these mutants would be of real interest. Alternatively, it has recently been demonstrated that the mechanism of recombination in S. pombe is fundamentally different from the double Holliday junction mechanism that prevails in S. cerevisiae (BISHOP 2006; CROMIE et al. 2006), leaving the possibility open either that flies are more like the budding yeast in their mechanism of recombination (in a fashion that makes Mcm5 nonessential for recombination in S. pombe) or that perhaps there are even more than two variations on a theme with respect to the process of meiotic recombination, such that flies have their own unique set patterns of nucleic acid needlework with which to perform crossing over.
Finally, it is worth noting that SHIMA et al. (2007) have recently identified a hypomorphic viable allele of mcm4 that causes chromosome instability and identified a unique function of this protein in tumor suppression in mice. Thus, it is obvious that core MCM proteins play roles outside of DNA replication and that the identification of separation-of-function mutants is going to be essential in elucidating the multiple roles of MCM proteins.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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