RNA blotting analysis detects a single Rad51 transcript of about 1.35 kb that is present throughout development at low levels. Transcript levels are induced at least tenfold in ovaries, as measured by RNase protection analysis, suggestive of a role in female meiosis. Transcript levels are significantly lower in testes than in bulk RNA of adult males, however, indicating that Rad51 may be repressed in meiosis of Drosophila males (McKee, 1996).
Twenty-two new alleles of spindle-A have been identified by mutagenesis screening. All new spnA alleles are viable in trans to the original spnA alleles (spnA003, spnA050 and spnA057) and in trans to a deficiency for the region, suggesting that they are not affecting a function essential for viability. Like the original spnA alleles, the new alleles show ~100% maternal-effect embryonic lethality. All mutant lines produce a spectrum of eggshell ventralization phenotypes similar to those described for known mutations affecting the Egfr signaling pathway, ranging from fused dorsal appendages to complete ventralization (Gonzalez-Reyes, 1997). For most spindle-class mutants, the eggshell phenotype has been attributed to a defect in RNA and protein localization or protein synthesis of the Egfr ligand Gurken (Grk) (Gonzalez-Reyes, 1997). In spnA mutants, the level and distribution of Grk protein is disrupted (Gonzalez-Reyes, 1997). In one example, instead of the normal crescent of Grk protein along the dorsal-anterior side of the wild-type oocyte nucleus facing the somatic follicle cells, Grk protein distribution is less coherent and only found in a few spots along the mutant oocyte nucleus. Another common feature shared among the spindle-class mutants is a disruption in oocyte nuclear morphology (Gonzalez-Reyes, 1997; Ghabrial, 1998). Mature wild-type oocytes contain highly compact chromatin called the karyosome. In spnA mutant oocytes, as well as in other spindle-class mutants, the DNA is less organized and diffuse. In contrast to the wild type, where DNA is found as a condensed sphere in the center of the nucleus, DNA in spnA mutant oocytes clusters along the periphery of the nucleus adjacent to the nuclear membrane (Staeva-Vieira, 2003).
spnA was mapped to the cytological region 99D01-99E01. The Berkeley Drosophila Genome Project predicted a Rad51-like gene (CG7948) to reside within this region of the genome. CG7948 shows strong sequence similarity to the yeast and mammalian Rad51 gene. Since two other spindle-class genes, spnB and okra, encode members of the Drosophila Rad51 family and Rad54, respectively (Ghabrial, 1998), CG7948 was a likely candidate for spnA. Sequence analysis of spnA alleles revealed unique missense mutations within CG7948. Thus, spindle-A encodes a Drosophila Rad51-like gene (Staeva-Vieira, 2003).
The molecular characterization of all 25 spnA alleles revealed 22 missense mutations (spnA009A and spnA032B contained the same mutation) and two stop codon mutations. Each missense mutation affects an amino acid conserved from yeast to human Rad51. Western analysis using an antibody that was raised against the entire Drosophila Rad51 protein revealed that spnA093A, which has an early stop codon at amino acid 70, produces no detectable protein and classifies as a protein null. The other nonsense allele spnA048B introduces a late stop at amino acid 265 and produces a truncated protein. Three missense alleles, spnA087A and spnA010A, which have missense mutations in the Walker-A box and Walker-B box, respectively, and spnA057, which has a change within the DmRad51 core domain, were also analyzed. Western analysis revealed that the missense alleles produce stable proteins that are of similar size to the wild-type protein. Since all alleles of spnA including the null allele spnA093A are viable in trans to the deficiency, Df(3R)X3F, it is concluded that SpnA function is required for oogenesis but is not essential for normal cell viability (Staeva-Vieira, 2003).
Since spnA and spnB are expressed in the soma as well as the germline, it was of interest to investigate a possible somatic function for these genes. To determine if these proteins function in the mitotically active cells of the soma, spnA, spnB and spnB;spnA doubly mutant embryos and larvae were exposed to 20 Gy of ionizing radiation (X-rays) and examined for survival. The progeny of heterozygous parents were irradiated at either 0-24 h, 24-48 h or 48-72 h after egg laying (AEL). The survival of the irradiated progeny was compared to that of their unirradiated siblings and a survival ratio was established. At the irradiation dosage chosen, there was no apparent difference in the survival of irradiated and unirradiated control flies (survival ratio close to 1) for all irradiation times, indicating an insensitivity to this dose of irradiation. At the same dose, spnA, spnB and spnB;spnA doubly mutant embryos and larvae show an age-dependent sensitivity to ionizing radiation. When irradiated during embryogenesis (0-24 h AEL), there was little difference in survival between mutant and control. However, during later larval stages (48-72 h AEL) there was a significant difference between the survival of spnA mutant larvae and their heterozygous control siblings. This increase in sensitivity to irradiation with age is likely due to maternal proteins present in the developing embryos; as the animals get older less maternal protein is present due to degradation. Heterozygous control progeny experience the same degradation of maternal product but survive due to their ability to synthesize necessary gene product de novo. While spnA mutants show a striking sensitivity to irradiation at late third instar (48-72 h AEL), spnB mutants show only a modest sensitivity to ionizing radiation. This is in contrast to the insensitivity observed when spnB mutants were exposed to MMS (Ghabrial, 1998). spnB;spnA double mutants do not show a synergistic sensitivity to ionizing radiation, rather, they behave similar to spnA alone, suggesting that the two genes are part of the same non-redundant pathway. Most importantly, these results show that SpnA does indeed play a role in the soma to protect against chromosomal damage inflicted by DNA damaging agents (Staeva-Vieira, 2003).
To better characterize the role of SpnA during oogenesis, the meiotic defect of the spnA null allele was analyzed in detail. It was asked whether meiotic chromosome synapsis is affected in the mutants, whether DNA breaks occur during meiosis and can be repaired in the mutant, and finally whether spnA mutants indeed cause activation of a meiotic checkpoint. In Drosophila, each germline stem cell, at the anterior tip of each ovariole, divides asymmetrically to produce a new stem cell and a differentiating cystoblast. The cystoblast undergoes four rounds of mitotic division with incomplete cytokinesis to generate a cyst of 16 cells. The cells within a cyst remain interconnected by cytoplasmic bridges called ring canals. The initiation of meiosis is indicated by the appearance of the synaptonemal complex (SC), which assembles in region 2a of the germarium in the four cells of the cyst that form first and thus contain either three- or four-ring canals. The two four-ring canal cells will become the pro-oocytes as defined by the persistence of the SC and their accumulation of oocyte specific markers in region 2b. By stage 1 of oogenesis, the SC, as observed by immunostaining for the Drosophila SC component C(3)G, and oocyte markers are restricted to a single cell, the future oocyte. As the egg matures, C(3)G begins to lose association with chromatin and the SC is no longer observed (Staeva-Vieira, 2003).
The distribution of C(3)G was followed in spnA mutant germline cysts. As in the wild type, C(3)G expression is first detected in region 2A. However, C(3)G restriction to the oocyte and its dissolution from the chromatin are delayed. At stage 1 of oogenesis, while the SC is always restricted to just the oocyte in wild type, it persists from time to time in both pro-oocytes in spnA mutants, similar to what was observed previously (Huynh, 2000). Furthermore, when C(3)G staining decreases in the maturing stage 5 oocyte, it remains in the mutant. By stage 7, the staining is no longer detected in the oocyte of either wild-type or spnA mutant egg chambers. These results suggest that synapse formation is appropriately initiated in spnA mutants but the failure to repair broken DNA causes a delay in the resolution of synapsis, first in the cyst that will not become the oocyte and subsequently in the oocyte as it progresses through meiosis (Staeva-Vieira, 2003).
The meiotic phenotype of spnA suggests that there may be a delay in proper meiotic chromosome dynamics due to the failure to repair DSBs. To visualize DSBs cytologically, an antibody was used that recognizes gamma-H2AX, a phospho-epitope of the human histone H2A variant, H2AX, which becomes phosphorylated upon DSB formation. The phospho-epitope is conserved in Drosophila histone variant HIS2AV and becomes phosphorylated in the event of DSBs, whether induced exogenously or during meiosis (Madigan, 2002; Jang, 2003). During wild-type meiosis, few gamma-HIS2AV foci were observed, presumably due to the rapid repair of DSBs and the formation of viable recombination intermediates. When observed, gamma-HIS2AV foci were found in only one cell in region 2a of the germarium. This early appearance of gamma-HIS2AV, before the restriction of other oocyte markers to a single cell, suggests that the regulation of DSB formation and persistence may be a critical event in oocyte specification. gamma-HIS2AV foci were not observed from region 2B onwards. Thus, DSBs are rapidly processed and recombination intermediates are formed concomitant with oocyte specification. In contrast, spnA mutant germaria show a more robust HIS2AV activation in one or two cells of a growing cyst in region 2a, suggesting that in spnA DSBs form at the normal time but their resolution is delayed. Furthermore, gamma-HIS2AV localization is more extensive along the DNA rather than in distinct foci as observed in the wild type, possibly due to the accumulation of unresolved breaks along the chromosomes. HIS2AV activation persists in the oocyte nucleus through later stages of oogenesis suggesting a failure to properly repair DNA breaks (Staeva-Vieira, 2003).
If all the defects observed in spnA mutants are due to the activation of a checkpoint upon failure to repair DSBs, one would predict that mutations that prevent break formation in the first place would suppress the spnA phenotype. This rationale is suggested by results in yeast where mutations in spo11 suppress the meiotic sporulation defects of dmc1 mutations (Roeder, 1997; reviewed in Bishop, 1999). Subsequent to the yeast work, it was shown that the eggshell phenotype of two Drosophila Rad51 family members, spnB and spnD, as well as the Rad54 homolog, okra, is suppressed in the absence of mei-W68, the Drosophila homolog of Spo11 (Ghabrial, 1998). Therefore double mutants between mei-W68 and spnA were generated and their ability to produce properly patterned eggs was examined. Control females that were efficient at producing DSBs during meiosis [mei-W68/+; spnA093A/Df(3R)X3F] but were defective in SpnA function, produced progeny with spindle eggshells. In contrast, in flies defective in DSB production and SpnA function [mei-W68/mei-W68; spnA093A/Df(3R)X3F], the spindle phenotype was rarely observed (<1%). Furthermore, the oocyte nuclear morphology and Gurken protein localization and distribution appeared normal in the double mutants. mei-W68 also suppresses the embryonic lethality associated with loss of maternal SpnA. In this situation, embryos from doubly mutant females survived to adulthood with a frequency similar to that observed in mei-W68 progeny alone. The fact that all phenotypes associated with spnA mutants are suppressed by mei-W68 suggests that it is indeed the role of SpnA in repair of meiotic-induced DSBs that is essential for normal oogenesis and survival (Staeva-Vieira, 2003).
The data show that DSBs are readily detectable by gamma-HIS2AV staining and persist during oogenesis in spnA mutants. Unrepaired DSBs or unresolved recombination intermediates lead to the activation of an ATM/ATR-dependent cell cycle checkpoint in mitosis and meiosis, which often causes delays in cell cycle progression in order to repair DNA damage (Roeder, 2000). To test whether unrepaired DSBs or unresolved recombination intermediates in spnA mutants trigger a cell cycle checkpoint that leads to defects in oocyte development, it was desirable to inactivate the checkpoint response (Staeva-Vieira, 2003).
Two genes have been implicated in checkpoint function, the Drosophila ATR homolog Mei-41 and the Chk2 homolog DmChk2/Mnk/Loki. Since Drosophila mei-41 mutants also show a defect in meiotic recombination, it is difficult to assess the exact step in meiosis that is affected in this mutant. Focus was therefore placed on the checkpoint protein, DmChk2. On its own, chk2 mutants do not appear to have a meiotic phenotype. Females doubly mutant for chk2 and spnA produced progeny with wild-type egg shape (100 versus 6%), even a reduction in the copy number of chk2 (chk2/+, spnA057/spnA093A) partially suppresses the spindle phenotype (22 versus 94%). In addition, the karyosome appears normal in the oocytes from females doubly mutant for chk2 and spnA, suggesting that the abnormal nuclear morphology observed in spnA mutant oocytes is not the result of fragmented DNA, since the DNA breaks should persist in these double mutants. In contrast to the mei-W68;spnA doubles, deletion of chk2 did not suppress the maternal-effect embryonic lethality of spnA. Thus, it is concluded that the spnA phenotype results from the activation of a Chk2-dependent meiotic checkpoint (Staeva-Vieira, 2003).
Rad51 is a highly conserved protein throughout the eukaryotic kingdom and an essential enzyme in DNA repair and recombination. It possesses DNA binding activity and ATPase activity, and interacts with meiotic chromosomes during prophase I of meiosis. Drosophila Rad51, Spindle-A (SpnA) protein has been shown to be involved in repair of DNA damage in somatic cells and meiotic recombination in female germ cells. In this study, DNA binding activity of SpnA is demonstrated by both agarose gel mobility shift assay and restriction enzyme protection assay. SpnA is also shown to interact with meiotic chromosomes during prophase I in the primary spermatocytes of hsp26-spnA transgenic flies. In addition, SpnA is highly expressed in embryos, and the depletion of SpnA by RNA interference (RNAi) leads to embryonic lethality implying that SpnA is involved in early embryonic development. Therefore, these results suggest that Drosophila SpnA protein possesses properties similar to mammalian Rad51 homologs (Yoo, 2006).
Rad51 protein has been demonstrated to possess DNA binding activity, ATPase activity, and strand transfer activity. These properties are essential to homologous recombination since the primary function of Rad51 protein is to bring two homologous DNA molecules into close proximity to facilitate the formation of heteroduplex DNA and to mediate strand exchange between them. Thus, the presumptive first step of homologous recombination is binding of Rad51 protein to the DNA molecule. In the present study, the DNA binding activity of SpnA protein is demonstrated in a Mg2+-dependent but ATP-independent manner. A substantial body of biochemical evidence indicates that the binding reaction of Rad51 protein is affected by nucleotide cofactors, Mg2+ concentration, and salt concentration. In contrast to Mg2+ which is an indispensable factor for DNA binding assay, the precise role of ATP is still controversial. It has been reported that yeast Rad51 protein binds to both ds- and ss-DNA only in the presence of ATP and that neither ADP nor the nonhydrolyzable ATP analogues can substitute the function of ATP. In contrast, the binding of yeast Rad51 protein to DNA in the absence of nucleotide cofactor has been demonstrated at low pH condition. A study using human Rad51 protein also represented that both ds- and ss-DNA binding activities are not dependent upon a nucleotide cofactor. Therefore, it is not surprising that ATP is not an essential factor for DNA binding of SpnA protein. Recently, the Drosophila Rad51 homolog, SpnA protein is also shown to be involved in the strand exchange step. Although the precise biochemical properties of SpnA protein remain to be determined, these results strongly support the central role of SpnA in repair of DSBs by homologous recombination (Yoo, 2006).
Several studies using mouse spermatocytes and oocytes have reported that Rad51 foci appear as early as premeiotic S phase before the initiation of synapsis. These foci are increased in number and become organized during leptotene, and then, dramatically decreased as pachytene progresses. In this study, SpnA foci were also observed during early prophase I and rapidly disappeared at late prophase I of the spermatocytes of hsp26-spnA transformants. Although it is not a physiological condition, the time course of appearance of SpnA and its distribution in spermatocytes are similar to mammalian Rad51 homologs. The failure to detect SpnA foci in wild type spermatocytes suggest that SpnA might be absent or, if any, at very low level in the spermatocytes. A previous study also reported no role of SpnA in male meiotic chromosome pairing, a diagnostic phenotype in meiotic recombination. Taken together with the observation that spnA null mutant males are fertile, SpnA may be dispensable during Drosophila spermatogenesis unlike mammalian Rad51 proteins (Yoo, 2006).
The disruption of rad51 gene in mice results in early embryonic lethality, indicating a crucial role of Rad51 in embryogenesis. Since the cells are rapidly dividing during embryogenesis, Rad51 might be required for the cell proliferation, cell cycle control, DNA replication, or transcription possibly by interacting with p53, Brca1, or Brca2. However, the precise role of Rad51 protein during embryogenesis is not understood yet. In contrast to mammals, Drosophila spnA null mutants are viable although they are defective in recombination and DNA repair. The survival of Drosophila mutants might be explained by the presence of maternal SpnA proteins for early embryonic development. The depletion of maternally stored spnA transcript may demonstrate a possible role of SpnA in rapidly dividing cells during early development. Double mutants for spnA and mei-W68 escape the complete sterility of spnA mutant females caused by defects in DNA repair during oogenesis. mei-W68 is the Drosophila homolog of Spo11 that induces the DSB formation. Since spnA phenotypes are suppressed by mei-W68 mutation, the double mutants are fertile and their progeny survive to adulthood, suggesting that SpnA may not be essential for viability. In this study, in an attempt to examine the phenotype caused by the depletion of SpnA solely, the maternal effect of SpnA was eliminated by employing RNAi technique. Interestingly, the results showed that both dsRNA and siRNA targeting spnA severely interfered with normal development, implying a role of SpnA during embryogenesis. It is possible that SpnA functions in the repair of mis-incorporated nucleotides during DNA replication or in the removal of nucleotide wastes during embryogenesis. Although many SpnA foci associate with unknown material stained with DAPI during mitosis, the role of SpnA in embryonic development is unclear and need to be addressed in the future experiments (Yoo, 2006).
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date revised: 15 August 2008
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