Five spindle genes, spn-A, spn-B, spn-C, spn-D and spn-E were originally identified in a screen for maternal-effect mutants on the third chromosome because homozygous mutant females lay ventralized eggs (Tearle, 1987). spn-A has been identified as the Drosophila Rad51-like (Rad51) gene, whose sequence among the five known Drosophila Rad51-like genes is most closely related to the Rad51 homologs of human and yeast. Rad51 is a conserved protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells and during meiosis in germ cells. Yeast Rad51 mutants are viable but show meiosis defects. In the mouse, RAD51 deletions cause early embryonic death, suggesting that in higher eukaryotes Rad51 is required for viability. Drosophila Rad51/spn-A null mutants are viable but oogenesis is disrupted by the activation of a meiotic recombination checkpoint. The meiotic phenotypes result from an inability to effectively repair DSBs. In Drosophila the Rad51-dependent homologous recombination pathway is not essential for DNA repair in the soma, unless exposed to DNA damaging agents. It is therefore proposed that under normal conditions a second, Rad51-independent, repair pathway prevents the lethal effects of DNA damage (Staeva-Vieira, 2003).

Chromosomal integrity is essential for proper embryonic and postembryonic development, prolonged survival and successful reproduction. Highly conserved repair mechanisms exist in all organisms, from bacteria to mammals, to recognize and repair DNA damage. The repair of double-stranded DNA breaks (DSBs) is a necessary mechanism for recombining parental genomes during meiosis and is used as a defense mechanism after DNA damage caused by irradiation or chemical agents. The presence of DNA damage activates a cell cycle checkpoint. This allows time for the cell to correct the damage so as not to propagate the defect or affect normal cellular functions. In Saccharomyces cerevisiae mutations in the same genes show increased sensitivity to ionizing radiation and meiotic phenotypes, such as chromosome non-disjunction and/or rearrangements. This suggested a functional relationship between the mechanisms of mitotic DNA repair and meiotic recombination (Staeva-Vieira, 2003).

Genetic studies in S.cerevisiae led to the discovery of the Rad52 epistasis group of DSB repair genes. A core protein in this pathway is Rad51, which is related to the bacterial RecA protein. Rad51 has DNA-dependent ATPase activity and catalyzes strand exchange between homologous DNA molecules. Rad51 and Rad51-related proteins are found from yeast to humans (for review see Sung, 2003). In yeast, the Rad51 null mutant is viable but shows sporulation defects (Shinohara, 1992). The mouse Rad51 knockout is embryonic lethal (Lim, 1996; Tsuzuki, 1996), thus the role of Rad51 in mouse meiosis could not be studied. In both mouse and yeast, a meiosis-specific Rad51-related gene, Dmc1 (Bishop, 1992; Pittman, 1998), has been identified and shown to be required for chromosome synapsis and strand exchange during prophase of meiosis I (Staeva-Vieira, 2003).

In contrast to yeast, Drosophila members of the Rad52 epistasis group were not identified on the basis of meiosis defects or mutagen sensitivity. Rather, mutations in the Drosophila RAD51-related gene, spindle-B (spnB), and the Rad54 homolog, okra, were discovered as maternal-effect mutants with altered patterning of the eggshell, the so-called spindle phenotype (Morris, 1999). It was shown that this phenotype, observed in spnB and okra mutants, was due to reduction in the levels of the morphogen Gurken, a TGFalpha-like protein that controls both dorso-ventral patterning of the egg and antero-posterior polarity of the embryo (Ghabrial, 1998). Ghabrial suggested that the activation of a meiotic checkpoint, which resulted in defective Gurken translation, was the result of a failure to repair DNA breaks in mutants for okra, spnB and spindle-D (spnD), another Rad51-related protein (Ghabrial, 1999; Abdu, 2003). Accordingly, the spindle phenotype was suppressed by mutants for the Spo11 homolog, mei-W68 -- these mutants are defective in double-stranded break formation and thus are unable to activate the checkpoint (Ghabrial, 1999). Spn mutants were also suppressed in combination with mutants of known transducers of cell cycle checkpoints, such as Drosophila mei-41, an ATR/ATM phosphatidylinositol 3-kinase-like protein, and the Drosophila homolog of Chk2 kinase, chk2/mnk/loki (Ghabrial, 1999; Abdu, 2002). A target for the meiotic checkpoint in Drosophila is the ATP-dependent helicase Vasa (Styhler, 1998; Tomancak, 1998), which is phosphorylated upon checkpoint activation and may regulate Gurken translation (Ghabrial, 1999). Sequence analysis indicates that there are at least five Drosophila genes that show significant homology to yeast and human Rad51. It remained unclear whether these genes have distinct functions in DSB repair and whether the activation of the meiotic checkpoint was a consequence of the failure to repair DSBs. Furthermore, while mutations in spnB show meiotic defects, they do not affect DNA repair in somatic cells (Ghabrial, 1998), raising the possibility that in Drosophila distinct sets of Rad51-like genes may control DSB repair either in the germline or in the soma (Staeva-Vieira, 2003).

spnA mutants exhibit the spindle eggshell phenotype. In spnA oocytes synapse of homologous chromosomes is correctly initiated during meiosis but its resolution is delayed and unrepaired double-stranded breaks persist longer than in wild type causing the activation of a meiotic recombination checkpoint. spnA null mutants are viable but show sensitivity to irradiation, suggesting that SpnA acts in the soma but that other repair mechanisms compensate in the absence of SpnA. Analysis of the expression pattern of the five known Drosophila Rad51 homologs together with the analysis of the mutant phenotype of three of these genes suggest that the Drosophila Rad51 genes act in concert during oogenesis and that only a subset of them are used for repair in the soma (Staeva-Vieira, 2003).

Screens in Drosophila have recovered many mutations that cause disruption to normal meiotic chromosome behavior. They were identified based on the ability to recognize abnormal events, such as chromosome loss, non-disjunction or a change in recombination frequency. Mutagen sensitivity screens, similar to those performed in yeast, have also been conducted in Drosophila to identify genes necessary for DNA repair. As would be expected, some of these mutagen-sensitive mutants showed meiotic defects as well. Interestingly, none of the Rad52 epistasis genes of Drosophila were recovered from these types of screens. Instead, due to downstream effects on D/V patterning through the activation of a meiotic checkpoint, the spindle oogenesis phenotype has proven to be an effective assay by which to uncover these genes. Thus far, four members of the Rad52 epistasis group in Drosophila have been found through this approach (Staeva-Vieira, 2003 and references therein).

In Drosophila, there are five members of the Rad51 family. This analysis confirms that Spindle-A is the structural and functional homolog of the yeast and mammalian Rad51 protein. Biochemical analysis of in vitro purified Rad51 has shown that it has strand exchange capabilities (Alexiadis, 2002). The other Rad51 paralogs show greater sequence homology to Rad51 accessory proteins, which have been shown to promote Rad51 foci formation on DNA. Both rad51D and spnD, in the adult, are expressed specifically in the germline. Therefore, it is suggested that they are Rad51 accessory proteins involved in meiotic recombination, compensating for a lack of a Drosophila Dmc1 homolog. Initial studies on spnB revealed a striking similarity to Dmc1, namely its importance in meiotic recombination and its resistance to the effects of MMS (Ghabrial, 1998). However, spnB RNA is expressed in the soma as well as the germline. Moreover, evidence is presented that spnB mutant larvae are less tolerant than their wild-type siblings to the DNA damaging effects of ionizing radiation. Based on its sequence homology to XRCC3, it is possible that SpnB functions as a necessary partner for Drosophila Rad51 during meiotic recombination and takes on a supporting role in Rad51 stabilization (Liu, 1998; Brenneman, 2002) during DSB repair of the soma (Staeva-Vieira, 2003).

Analysis of Rad51 function in vertebrate development has b een difficult due to the early embryonic lethality of RAD51-/- mice. Vertebrate cell culture studies have suggested an essential role of RAD51 in the repair of breaks generated during DNA replication (Sonoda, 1998), thus providing some explanation for the embryonic lethality in mice. Drosophila Rad51 null animals can survive to adulthood. Therefore, the requirement for Rad51 in the repair of DNA breaks occurring during DNA replication may not be conserved. However, other possibilities exist. (1) Maternal Rad51 may persist to repair DSBs occurring throughout embryogenesis. However, female flies doubly mutant for mei-W68 and spnA produce embryos that survive to adulthood, suggesting that neither maternal nor zygotic Drosophila Rad51 function are essential for viability. (2) Another possibility is that the Rad51 genes may have partially overlapping, redundant functions. However, neither of the other family members shows strong homology to Rad51. (3) Flies doubly mutant for the spnA and its closest relative, spnB, are viable (Gonzalez-Reyes, 1997) and the next closest paralog, spnD, is expressed specifically in the germline, though only adult animals have been tested. An alternative explanation, and the one that is favored, is the existence of an alternative repair pathway that can compensate in the event of homologous recombination failure. Homologous recombination has been considered the major DNA repair pathway in Drosophila. Recent evidence in Drosophila has shown that when the homologous recombination pathway is compromised, the error-prone non-homologous end joining (NHEJ) pathway can compensate and prevent a lethal outcome (Adams, 2003). Therefore, in Drosophila, it would be predicted that an efficient cooperation must exist between the homologous recombination and NHEJ pathways to prevent the lethal effects of DNA DSBs, presumably with homologous recombination being the primary choice and NHEJ playing a backup role (Staeva-Vieira, 2003).

During meiotic recombination, crossing over between homologous chromosomes guarantees their proper segregation. Defects in the proper formation of recombination intermediates result in the activation of a pachytene, or meiotic recombination, checkpoint. In mice, if defects in chromosomal synapsis or meiotic recombination persist, the result is the activation of the pachytene checkpoint and removal of the arrested germ cells most probably by apoptosis. In this study, it is shown that a meiotic recombination checkpoint is activated in response to a loss of SpnA function. spnA mutant females do not show an appreciable defect in egg deposition, suggesting that the apoptotic pathway is not activated in response to the meiotic recombination checkpoint. Moreover, the p53 protein, a strong inducer of apoptosis during the mitotic cell cycle, has been shown not to be involved in the Drosophila meiotic recombination checkpoint (Abdu, 2002). Instead, as the data indicate, the unsuccessful processing of meiotic-induced DSBs results in a Chk2-dependent delay of the meiotic cell cycle. Concomitant with this delay, a defect in the EGFR/TGFalpha signaling pathway is observed, that results in the production of eggs with dorsal/ventral patterning defects. Thus, these results show a coupling between progression through the meiotic cell cycle and oocyte patterning and development. The ATP-dependent helicase Vasa has been implicated in mediating at least two aspects of meiotic checkpoint activation, Gurken translation and karyosome formation. It remains unclear if Vasa is directly activated by the checkpoint transducer kinase Chk2/Mnk and how defects in DSB repair lead to checkpoint activation. The spindle eggshell phenotype has proven to be an efficient assay to identify genes that lead to the activation of the meiotic checkpoint, making Drosophila an excellent genetic system to identify additional components that regulate the interplay between DNA repair, cell cycle progression and cell differentiation during meiosis and possibly, as these studies suggest, also mitosis (Staeva-Vieira, 2003).

GENE STRUCTURE

cDNA clone length - 2083

Bases in 5' UTR - 682

Exons - 1 or 2

Bases in 3' UTR - 337

PROTEIN STRUCTURE

Amino Acids - 336

Structural Domains

The RecA protein is the central enzyme in prokaryotic recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination. The RecA-like proteins Rad51 and Dmc1 of yeast are both required for meiotic recombination and the former is also necessary for repair of double-strand breaks in vegetative cells. Genes encoding Rad51 homologs have been isolated recently from several higher eukaryotes. This paper describes the isolation and molecular characterization of a genomic DNA fragment from Drosophila melanogaster containing the coding sequence for a RecA-like protein. This protein exhibits strong sequence homology with the Rad51 proteins of budding yeast, fission yeast, chickens, mouse and humans, and slightly less (but still strong) homology with yeast Dmc1. Both in situ hybridization and Southern analysis indicate that the Rad51 gene is present only once per genome in Drosophila (at 99D on chromosome arm 3R). However, there are at least three other fragments that cross-hybridize strongly at low stringency (McKee, 1996).

DmRad51/spnA is predicted to encode a 336 amino acid protein. It shows 55.1% identity to S. cerevisiae Rad51 over the entire protein (McKee, 1996). Even higher conservation is observed in sequence alignment with the mouse and human Rad51 (64.6% identical and ~80% similar). There were four members of the Drosophila Rad51 family identified from the Genome Project, rad51-like (CG7948; spnA), spnB (CG3325), rad51C (CG2412) and rad51D (CG6318) (Sekelsky, 2000; Abdu, 2003) and a Rad51C-like protein (CG31069), which was shown to be encoded by spindle-D. Alignment comparison of all five Drosophila Rad51 family members reveals that SpnA protein is the most similar to yeast and human Rad51. This similarity is not only restricted to the RecA core domain (72.2% identical to HsRAD51) but extends to the N-terminus (49.6%) and C-terminus (74.1%). Phylogenetic analysis shows that the other Drosophila members, SpnB, SpnD, Rad51C and Rad51D, are more similar to the Rad51 accessory proteins HsXRCC3, HsRAD51C, HsRAD51D and ScRad55 (or HsXRCC2), respectively (Sekelsky, 2000; Abdu, 2003). Based on genome annotation and sequence similarity, it is concluded that Spindle-A is the structural homolog of the yeast and mammalian Rad51 protein (Staeva-Vieira, 2003).

date revised: 25 March 2004

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