Targets of Activity

The effects of okra, spnB, and spnD on the signaling process were examined directly to more precisely establish their respective roles in grk-Egfr signaling. Specifically, because the three genes are required in the germline, the effect of the mutants was examined on the expression and localization of GRK mRNA and Grk protein. In okr mutant ovaries, GRK mRNA is correctly localized to the oocyte in early stages. In mid-oogenesis, however, instances of persistent mis-localization of the RNA are found in an anterior-cortical ring. The spnB and spnD mutant phenotypes show an even more severe mis-localization. At stage 9, 85% of the mutant egg chambers show persistent mis-localization of GRK mRNA in the anterior-cortical ring (see also, Gonzalez-Reyes, 1997). At stage 10, the spnB and spnD mutant egg chambers show a range of phenotypes including cases in which the RNA is normally localized, others in which it is only partially localized, others in which it is still present in an anterior cortical ring (but not localized), and others in which the level of RNA is reduced or undetectable (Ghabrial, 1998).

In addition to their effects on GRK mRNA localization in the oocyte, okr, spnB, and spnD also affect the accumulation of Grk protein. In wild-type egg chambers, Grk is restricted to the oocyte, and in mid-oogenesis, it is localized to a dorsal-anterior patch. In okr mutant ovaries, levels of Grk are variably reduced throughout oogenesis. Within a single ovariole, egg chambers expressing Grk can alternate with egg chambers that do not. In spnB and spnD mutant egg chambers, the early accumulation of Grk in the oocyte is normal. By mid-oogenesis, however, the level of Grk in the oocytes is reduced and is often undetectable (Ghabrial, 1998).

The effects of okr, spnB, and spnD on Grk accumulation in the oocyte place these genes upstream of grk in the genetic hierarchy controlling dorsal-ventral patterning in the egg chamber. To assess the relationship between okr, spnB, and spnD and a different class of genes in the patterning hierarchy that are required for the localization of grk RNA and which produce dorsalizing phenotypes, the phenotypes produced by double mutants with K10 were examined. For all three double-mutant combinations, it was found that rather than producing all ventralized or all dorsalized eggs, the mutant females produce a broad spectrum of phenotypes ranging from completely dorsalized to completely ventralized. Given that these experiments do not reveal a simple epistatic relationship between okr, spnB, spnD, and K10, the three genes must affect Grk activity by a pathway that is at least partially independent of K10. Significantly, as this same spectrum of phenotypes is produced by K10 mutant females that have only one wild-type copy of grk, these results are consistent with a role for these genes in directly affecting the accumulation of Grk in the oocyte. Given that the mislocalization of grk mRNA that is observed in okr, spnB, and spnD mutant egg chambers is similar to that observed in K10 mutant egg chambers, a defect in the accumulation of K10 protein was sought in okr, spnB, and spnD ovaries. In all three mutant genotypes, there is a reduction in the level of K10 in the oocyte nucleus. Thus, okr, spnB, and spnD are necessary for normal accumulation of both K10 and Grk in the oocyte. The failure to accumulate wild-type levels of K10 leads to the mislocalization of grk mRNA in mid-oogenesis, whereas the failure to accumulate wild-type levels of Grk leads to ventralization of the eggshell and embryo. The former defect may also account for the production of rare dorsalized eggs by okr, spnB, and spnD mutant females. Moreover, the fact that both ventralized and dorsalized eggs are found suggests that the two effects are to some degree independent: Ventralized eggs arise from cases in which Grk levels are reduced and K10 levels are normal or reduced, whereas dorsalized eggs arise from cases in which Grk levels are fairly normal and K10 levels are reduced (Ghabrial, 1998).

The genes okra and spindle-B act during meiosis in Drosophila to repair double-stranded DNA breaks (DSBs) associated with meiotic recombination. spn-B (DMC1/RAD51-like) and okr (Dmrad54) are homologous to genes in the yeast RAD52 epistasis group that function in the recombinational repair of DSBs; in Drosophila, mutations in these genes lead to mitotic and/or meiotic defects, consistent with a requirement in DNA repair. RAD51, which promotes homologous pairing and strand exchange during double strand break repair, is a single-stranded DNA binding protein and possesses DNA-stimulated ATPase activity. Unexpectedly, mutations in spn-B and okr cause dorsoventral patterning defects during oogenesis. These defects result from a failure to accumulate Gurken protein, which is required to initiate dorsoventral patterning during oogenesis. The block in Gurken accumulation in the oocyte cytoplasm reflects activation of a meiotic checkpoint in response to the persistence of DSBs in the nucleus. Vasa is a target of this meiotic checkpoint, and so may mediate the checkpoint-dependent translational regulation of Gurken (Ghabrial, 1999).

Given that yeast genes homologous to okr and spn-B are known to function in the nucleus, where their encoded proteins bind to DNA and catalyse DNA-strand exchange as part of the recombination-repair process, it is surprising that, in Drosophila, mutations in these genes cause defects in Grk accumulation in the oocyte cytoplasm. A test was performed to see whether the production of eggs with dorsoventral patterning defects by mutant females reflects a defect in repair of DSBs, or reveals a new function for these genes. In Saccharomyces cerevisiae, mutations that prevent DSB repair during meiotic recombination can be suppressed by mutations in SPO11, which encodes a topoisomerase-II-like protein that is required to make the DSBs that initiate meiotic recombination. In Drosophila, mei-W68 has been identified as the SPO11 homolog. Accordingly, flies were made doubly mutant for mei-W68 and either okr, spn-B or spn-C to determine whether spindle-class dorsoventral eggshell patterning defects would be produced in the absence of DSB formation. In double-mutant egg chambers, Grk protein accumulates in the same way as in wild-type egg chambers. Moreover, patterning of the eggshells, which is very sensitive to the levels of Grk protein, proceeds normally in double-mutant females. Other spindle-class defects are also suppressed by mei-W68: oocyte nuclear morphology in double-mutant egg chambers appears normal, and fertility of double-mutant females is comparable to that observed for females mutant only for mei-W68. Similarly, mutations in mei-P22 and c(3)G that (like mei-W68 mutations) eliminate meiotic recombination are able to suppress okr mutant phenotypes. Thus, in the absence of DSBs, the developmental defects observed in okr, spn-B and spn-C mutants are suppressed, indicating that these phenotypes are indeed caused by the presence of improperly processed DSBs during meiosis (Ghabrial, 1999).

Next, attempts were made to determine the mechanism through which the persistence of unrepaired DSBs during oogenesis causes patterning defects. In S. cerevisiae, mutations in the DSB-repair genes RAD51 and DMC1 cause cells to arrest in prophase of the first meiotic division. Meiotic arrest is also observed in Dmc1-deficient mice. In S. cerevisiae, this meiotic arrest is checkpoint dependent -- a failure to repair DSBs does not cause meiotic arrest directly; instead, recognition of the presence of DSBs triggers the activation of a pathway that halts the cell cycle until the damaged DNA is repaired. One of the checkpoint genes required to arrest meiosis in response to the presence of unrepaired DSBs is MEC1, which encodes a member of the ATM/ATR subfamily of phosphatidylinositol-3-OH-kinase-like proteins. In otherwise wild-type yeast, mec1 mutations lead to occasional premature meiosis I, as indicated by the persistence of foci of Rad51 (believed to represent sites of DSB repair) on metaphase-I chromosomes. These results indicate that Mec1 may normally act to delay the cell cycle in the presence of repair intermediates. In Drosophila, mei-41 encodes a homolog of Mec1, and mei-41 mutants show meiotic non-disjunction as well as maternal-effect defects in the timing of mitotic cell cycles in the early embryo (Ghabrial, 1999).

To test whether the production of patterning defects by mutations in the spindle-class genes is due to the engagement of an analogous meiotic checkpoint, flies were made doubly mutant for okr, spn-B or spn-C and mei-41. Mutations in mei-41 are indeed able to suppress the dorsoventral patterning defects caused by mutations in the spindle-class genes. In double-mutant flies, a dramatic increase is observed in the accumulation of Grk protein, as indicated by whole-mount antibody staining and by restoration of anteroposterior and dorsoventral patterning in the eggshell. Significant suppression of the oocyte nuclear-morphology defect is also observed. Suppression of the spindle-class defects by mei-41 is not as complete as that by mei-W68, raising the possibility that there may be some functional redundancy between mei-41 and a putative Drosophila ATM homolog, as appears to be the case for yeast MEC1 and TEL1. From these results, it is concluded that the patterning defects observed in mutants of the spindle class are caused by the activation of a mei-41-dependent checkpoint pathway in response to the persistence of unrepaired DSBs during meiosis (Ghabrial, 1999).

Vasa is a target of the meiotic checkpoint. These results raise the question of how the mei-41-dependent checkpoint pathway affects accumulation of Grk. One candidate for a downstream target and effector of the mei-41-dependent pathway is the product of the vasa (vas) gene. vas encodes a protein similar to the translation-initiation factor eIF4A, produces mutant phenotypes similar to those observed in okr, spn-B and spn-C mutants, and has been implicated in the translational control of Grk and certain other oocyte-specific proteins. However, unlike okr, spn-B and spn-C mutations, mutations in vas are not suppressed by mutations in mei-41. The karyosome phenotype of vas mutants is also not suppressed by mei-41 mutations. This difference between vas and the spindle-class mutants indicates that Vas may act downstream of this mei-41-dependent meiotic checkpoint. To address this question more directly, Vas expression was studied in spindle-class-mutant backgrounds. At the level of whole-mount antibody staining, Vas does not appear to be affected. However, the mobility of Vas, as assessed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), is altered: Vas migration appears to be retarded in spn-B mutant ovaries as compared with wild-type ovaries or ovaries heterozygous for spn-B. These results indicate that Vas might be post-translationally regulated by the mei-41-dependent checkpoint pathway. In support of this interpretation, the mobility of Vas from ovarian lysates prepared from flies doubly mutant for spn-B and mei-W68 or mei-41 is restored to that observed in wild-type lysates. Taken together, these data support a model in which activation of the mei-41-dependent checkpoint pathway occurs in response to the presence of DSBs and leads to the modification of Vas, resulting in the downregulation of its activity and a consequent decrease in Grk translation (Ghabrial, 1999).

In yeast, activation of the recombination checkpoint downregulates transcription of genes that are targets of transcription factor Ndt80, including cyclins required for progression through meiosis and a set of sporulation genes required for the morphological changes that normally accompany yeast meiosis. It is proposed that, in Drosophila, activation of this checkpoint pathway results in the modification of Vas and downregulation of the translation of Vas targets such as Grk. In this regard, phosphorylation has been suggested to be a mechanism for downregulating the activity of several translational activators, including Vas-like proteins (Buelt, 1994), plant eIF4A and vertebrate p68 RNA helicase (Ghabrial, 1999 and references therein).

Strand pairing by Rad54 and Rad51 is enhanced by chromatin

The role of chromatin in the catalysis of homologous strand pairing by Rad54 and Rad51 was investigated. Rad54 is related to the ATPase subunits of chromatin-remodeling factors, whereas Rad51 is related to bacterial RecA. In the absence of superhelical tension, the efficiency of strand pairing with chromatin is >100-fold higher than that with naked DNA. In addition, Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. These findings indicate that Rad54 and Rad51 have evolved to function with chromatin, the natural substrate, rather than with naked DNA (Alexiadis, 2002).

Homologous recombination occurs in the repair of DNA double-strand breaks as well as during meiosis. Genetic studies in Saccharomyces cerevisiae led to the identification of the RAD52 epistasis group of genes (which includes RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2) as components of the recombinational repair pathway. These genes are conserved from yeast to humans. A central protein in this pathway is Rad51, which is related to the bacterial RecA protein. Both Rad51 and RecA are able to mediate strand invasion and annealing to yield a D loop, which is a key step in the recombination process. In this reaction, Rad51 (or RecA) forms a nucleoprotein filament on single-stranded DNA in the presence of ATP, and this filament is used for homologous pairing with a double-stranded DNA molecule. The efficiency of strand pairing by Rad51 (between single-stranded DNA and homologous duplex DNA) has been shown to be stimulated by the presence of additional factors such as RP-A, the Rad55-Rad57 heterodimer, Rad52, and Rad54 (Alexiadis, 2002 and references therein).

To study homologous recombination in the context of chromatin, focus was placed on the ability of purified recombinant Rad51 and Rad54 to catalyze D-loop formation between single-stranded DNA and homologous double-stranded DNA that is packaged into chromatin. The function of Rad54 in chromatin is of particular interest because it is a member of the Snf2-like family of ATPases. The Snf2-like family includes proteins such as Swi2/Snf2, Sth1, ISWI, Ino80, and Mi-2/CHD3/CHD4, which are the ATPase subunits of chromatin-remodeling factors that catalyze the mobilization of nucleosomes. It thus seemed possible that Rad54 would be important for homologous recombination in chromatin. Therefore, whether purified Rad51 and Rad54 can mediate D-loop formation with chromatin was investigated (Alexiadis, 2002).

To study the biochemical properties of Rad51 and Rad54, focus was placed Drosophila Rad51 and Rad54 (with C-terminal Flag tags) in Sf9 cells by using a baculovirus expression system, and then the proteins were purified to near homogeneity by FLAG immunoaffinity chromatography. The ability of these factors to mediate D-loop formation between a radiolabeled, single-stranded oligonucleotide (termed DL2; 135 nt) and a homologous, double-stranded plasmid (pU6LNS; 3291 bp) was tested. In this reaction, Rad51 assembles onto the single-stranded oligonucleotide in the presence of ATP to give a nucleoprotein filament, and then Rad54 interacts with the Rad51-oligonucleotide complex and facilitates the strand-pairing reaction. These experiments reveal that purified recombinant Drosophila Rad51 and Rad54 can catalyze the formation of D loops in a manner that is dependent on Rad51, Rad54, ATP, and homologous plasmid DNA (Alexiadis, 2002).

Next, the ability of Rad51 and Rad54 to catalyze D-loop formation in chromatin was tested. In these experiments, chromatin was reconstituted by salt dialysis techniques. The salt dialysis chromatin (SD chromatin) was prepared by gradually decreasing the salt concentration in a mixture of plasmid DNA and purified core histones from Drosophila embryos, and fully reconstituted chromatin was separated from partially reconstituted chromatin by sucrose gradient sedimentation. Micrococcal nuclease digestion analysis of the chromatin samples revealed that the salt dialysis chromatin consisted of closely packed arrays of nucleosomes. D-loop reactions were performed with the SD chromatin. These experiments revealed that Rad51 and Rad54 are able to form D loops with SD chromatin at an efficiency that is slightly higher than that obtained with naked DNA. Moreover, the rate of D-loop formation by Rad51 and Rad54 with chromatin is similar to that seen with naked DNA. In contrast, the Escherichia coli RecA protein is able to mediate D-loop formation with naked DNA, but not with chromatin. Thus, these experiments, which were performed with completely purified components, show that Rad51 in cooperation with Rad54 can mediate D-loop formation with chromatin with comparable efficiency and kinetics as with DNA, whereas the bacterial recombinase RecA is unable to mediate strand pairing with chromatin. The inability of RecA to function with chromatin is consistent with previous studies carried out with mononucleosomes, and further suggests that RecA is lacking a chromatin-specific function that is present in Rad51 and/or Rad54. In this regard, whether Rad54 could stimulate D-loop formation in chromatin by RecA was tested, but no activity was demonstrated (Alexiadis, 2002).

The bulk of the eukaryotic genome appears to possess little superhelical tension, and therefore the effect of torsional stress upon D-loop formation by Rad51 and Rad54 was investigated. To this end, the salt dialysis chromatin was relaxed with purified topoisomerase I. The salt dialysis chromatin was reconstituted by using supercoiled plasmid DNA in the absence of topoisomerases. Under these conditions, the DNA remains chemically unchanged, since no phosphodiester bonds are broken. Hence, in the absence of topoisomerase I, the numbers of supercoils in the naked DNA and chromatin (which was deproteinized prior to electrophoresis) are essentially identical. When topoisomerase I is added to the chromatin, the unconstrained supercoils are relaxed, but upon deproteinization, the resulting DNA exhibits supercoils that are caused by the wrapping of the DNA in nucleosomes, because the wrapping of the DNA around each histone octamer constrains approximately one negative supercoil (Alexiadis, 2002).

Strand-pairing reactions were performed with DNA and salt dialysis chromatin in the absence or presence of topoisomerase I. With naked DNA, a >100-fold reduction in the efficiency of D-loop formation was observed upon relaxation of the template with topoisomerase I. Notably, this >100-fold decrease in strand-pairing efficiency is much more pronounced than the twofold reduction seen with yeast Rad51, Rad54, and RPA. This difference could potentially be due to the use of yeast versus Drosophila factors, the presence or absence of RPA, the length of the single-stranded DNA (5386 nt, Van Komen, 2000; 135 nt, this study), and/or the concentration of Rad51 in the reaction medium (1500 nM, Van Komen, 2000; 200 nM, this study). Note, however, that stimulation of D-loop formation by purified RPA was not observed in reactions performed in this study. In contrast to the effects seen with naked DNA, relaxation of the chromatin by topoisomerase I has little effect on the efficiency of D-loop formation by Rad51 and Rad54. Thus, in the absence of superhelical tension, strand pairing by Rad51 and Rad54 occurs with higher efficiency in chromatin than in naked DNA (Alexiadis, 2002).

Because Rad54 is related to the ATPase subunit of chromatin-remodeling complexes, whether Rad54 possesses chromatin-remodeling activity was tested. The ability of Rad54 and/or Rad51 to facilitate the access of a restriction enzyme (HaeIII) to DNA packaged into nucleosome arrays was tested. ACF was used as a positive control. This type of restriction-enzyme accessibility assay has been used for the analysis of chromatin remodeling in vivo, the biochemical purification of the CHRAC chromatin-remodeling factor, the characterization of the INO80.com remodeling complex, and the comparative analysis of six chromatin-remodeling complexes (ySWI/SNF, yRSC, hSWI/SNF, xMi-2, dCHRAC, dNURF). Neither Rad54 alone nor Rad51 alone exhibits any detectable chromatin-remodeling activity in the absence or presence of the DL2 oligonucleotide. In sharp contrast, Rad54 and Rad51 function cooperatively in the ATP-dependent remodeling of chromatin. The ability of Rad54 and Rad51 to rearrange chromatin structure is consistent with their ability to catalyze strand pairing with chromatin. It is also notable that Rad54 requires the presence of Rad51 to function as a chromatin-remodeling factor (Alexiadis, 2002).

In conclusion, these studies have revealed that D-loop formation by Rad54 and Rad51 occurs with >100-fold higher efficiency with chromatin relative to naked DNA in the absence of superhelical torsion. In addition, Rad54 and Rad51 act cooperatively in the ATP-dependent remodeling of chromatin. This ability of Rad54 and Rad51 to alter chromatin structure is likely to be related to their chromatin-specific function in the strand-pairing reaction. These findings provide an example of optimized function of eukaryotic DNA-using proteins in chromatin. Moreover, it is possible that the use of chromatin templates, instead of naked DNA templates, might similarly increase the efficiency of targeted homologous recombination in vivo (Alexiadis, 2002).

okra: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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