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The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila.

Lancaster OM, Breuer M, Cullen CF, Ito T, Ohkura H - PLoS Genet. (2010)

Bottom Line: An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation.Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes.Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.

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Unrepaired DNA breaks suppress NHK-1 kinase activity.(A) H2A T119 phosphorylation in wild type, nhk-1E24/Df and spnD2. Ovaries at stage 5–7 were immunostained with anti-dH2ApT119 antibody and propidium iodide. Arrowheads indicate meiotic chromosomes in oocytes. Bar = 10 µm. (B) The H2ApT119 signal intensity on the chromosomes in oocytes was measured relative to that in follicle cells. The bars on the graph represent standard error of the mean (SEM). A minimum of eight oocytes from each genotype were quantified. NHK-1 activity measured by H2A T119 phosphorylation was significantly reduced in nhk-1 and spn mutant oocytes (p<0.01; marked with asterisks). H2A T119 phosphorylation in oocytes expressing wild-type BAF and non-phosphorylatable BAF (BAF-3A) was comparable to that in wild type, indicating the karyosome abnormality itself is not the cause of low dH2ApT119 signals in spn mutants.
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pgen-1001179-g002: Unrepaired DNA breaks suppress NHK-1 kinase activity.(A) H2A T119 phosphorylation in wild type, nhk-1E24/Df and spnD2. Ovaries at stage 5–7 were immunostained with anti-dH2ApT119 antibody and propidium iodide. Arrowheads indicate meiotic chromosomes in oocytes. Bar = 10 µm. (B) The H2ApT119 signal intensity on the chromosomes in oocytes was measured relative to that in follicle cells. The bars on the graph represent standard error of the mean (SEM). A minimum of eight oocytes from each genotype were quantified. NHK-1 activity measured by H2A T119 phosphorylation was significantly reduced in nhk-1 and spn mutant oocytes (p<0.01; marked with asterisks). H2A T119 phosphorylation in oocytes expressing wild-type BAF and non-phosphorylatable BAF (BAF-3A) was comparable to that in wild type, indicating the karyosome abnormality itself is not the cause of low dH2ApT119 signals in spn mutants.

Mentions: Ovaries from spn mutants (spnA, spnB, spnD and vasa) were dissected and immunostained with the anti-dH2ApT119 antibody. As a control, we also examined wild type and the nhk-1E24/Df mutant in parallel. Compared to wild type, we found that the H2ApT119 signal was greatly reduced on meiotic chromosomes in oocytes from spn mutants, as well as in oocytes from the nhk-1E24/Df mutant (Figure 2A and Figure S3A).


The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila.

Lancaster OM, Breuer M, Cullen CF, Ito T, Ohkura H - PLoS Genet. (2010)

Unrepaired DNA breaks suppress NHK-1 kinase activity.(A) H2A T119 phosphorylation in wild type, nhk-1E24/Df and spnD2. Ovaries at stage 5–7 were immunostained with anti-dH2ApT119 antibody and propidium iodide. Arrowheads indicate meiotic chromosomes in oocytes. Bar = 10 µm. (B) The H2ApT119 signal intensity on the chromosomes in oocytes was measured relative to that in follicle cells. The bars on the graph represent standard error of the mean (SEM). A minimum of eight oocytes from each genotype were quantified. NHK-1 activity measured by H2A T119 phosphorylation was significantly reduced in nhk-1 and spn mutant oocytes (p<0.01; marked with asterisks). H2A T119 phosphorylation in oocytes expressing wild-type BAF and non-phosphorylatable BAF (BAF-3A) was comparable to that in wild type, indicating the karyosome abnormality itself is not the cause of low dH2ApT119 signals in spn mutants.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2965759&req=5

pgen-1001179-g002: Unrepaired DNA breaks suppress NHK-1 kinase activity.(A) H2A T119 phosphorylation in wild type, nhk-1E24/Df and spnD2. Ovaries at stage 5–7 were immunostained with anti-dH2ApT119 antibody and propidium iodide. Arrowheads indicate meiotic chromosomes in oocytes. Bar = 10 µm. (B) The H2ApT119 signal intensity on the chromosomes in oocytes was measured relative to that in follicle cells. The bars on the graph represent standard error of the mean (SEM). A minimum of eight oocytes from each genotype were quantified. NHK-1 activity measured by H2A T119 phosphorylation was significantly reduced in nhk-1 and spn mutant oocytes (p<0.01; marked with asterisks). H2A T119 phosphorylation in oocytes expressing wild-type BAF and non-phosphorylatable BAF (BAF-3A) was comparable to that in wild type, indicating the karyosome abnormality itself is not the cause of low dH2ApT119 signals in spn mutants.
Mentions: Ovaries from spn mutants (spnA, spnB, spnD and vasa) were dissected and immunostained with the anti-dH2ApT119 antibody. As a control, we also examined wild type and the nhk-1E24/Df mutant in parallel. Compared to wild type, we found that the H2ApT119 signal was greatly reduced on meiotic chromosomes in oocytes from spn mutants, as well as in oocytes from the nhk-1E24/Df mutant (Figure 2A and Figure S3A).

Bottom Line: An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation.Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes.Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.

Show MeSH
Related in: MedlinePlus