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

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.

Figure 1. Inactivation of the meiotic checkpoint did not suppress nhk-1 karyosome defects.

The karyosome morphology in an oocyte from spnA¹ (A), nhk-1^E24/Df (B), wild type (C), an mnk^p6 spnA¹ double mutant (D), and an mnk^p6 nhk-1^E24/Df double mutant (E). Bar = 10 µm. (F) The frequency of deformed karyosomes in oocytes with various genotypes. Inactivation of the meiotic checkpoint by the mnk^p6 mutation rescued the karyosome defect in spnA¹ (p<0.01), but not in nhk-1^E24/Df (G) The meiotic recombination checkpoint pathway in Drosophila oocytes (modified from 23).

Figure 2. Unrepaired DNA breaks suppress NHK-1 kinase activity.

(A) H2A T119 phosphorylation in wild type, nhk-1^E24/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.

Figure 3. The meiotic recombination checkpoint suppresses NHK-1 activity.

(A) H2A T119 phosphorylation in oocytes of wild type, spnD, and mnk spnD. Mnk (the Chk2 orthologue) is an essential kinase in the meiotic checkpoint. Ovaries at stage 5–7 were immunostained with anti-dH2ApT119 antibody and DAPI. (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). At least ten oocytes from each genotype were quantified. These samples were processed in parallel and compared only with each other, as exact values vary over time due to changes in factors including the conditions of the antibodies and fixative. NHK-1 activity measured by H2A T119 phosphorylation was significantly reduced in oocytes of a spnD mutant (p<0.01; marked with an asterisk), but not in those of an mnk spnD double mutant. Inactivation of the meiotic checkpoint rescued the suppression of NHK-1 activity in the presence of DSBs.

Figure 4. DSBs suppress NHK-1 activity in S2 cells.

(A) Kinase activity of NHK-1-GFP was reduced after X-ray irradiation. S2 cells stably expressing NHK-1-GFP or NHK-1(K77R)-GFP, together with untransfected S2 cells, were irradiated with X-rays. Cells were collected 15 minutes later and NHK-1-GFP was immunoprecipitated from cell extracts by a GFP antibody. For the kinase assays, ³²P-γATP was added and phosphorylation of co-immunoprecipitated BAF by NHK-1 was detected by autoradiograph. Cell extracts and immunoprecipitates used for kinase assays were immunoblotted with a GFP antibody. (B) DSBs were retained and the nuclear localisation of NHK-1-GFP was unaffected when cells were collected after X-ray treatment. DSBs and NHK-1-GFP were detected by immunostaining using antibodies against γH2Av and GFP, respectively. Bar = 10 µm.

Figure 5. Disassembly of the synaptonemal complex and loading of the condensin complex is delayed by meiotic checkpoint activation.

(A) Synaptonemal complex in wild-type and spn mutant oocytes. Ovaries were immunostained for the tranverse filament protein C(3)G and DNA. Bar = 10 µm. (B) The C(3)G staining pattern was classified as filamentous (arrowheads in A), fragmented or diffused (arrow in A). spn mutants significantly delayed disassembly of the synaptonemal complex (p<0.01; marked with asterisks). A minimum of thirteen oocytes were counted. (C) Condensin in wild-type and spn mutant oocytes. Ovaries were immunostained for the condensin subunit CAP-D2 and DNA. Bar = 10 µm. (D) Chromosome accumulation of CAP-D2 staining was classified into clear (arrow in C), weak or absent (arrowheads in C). spn mutants significantly delayed condensin loading (p<0.01; marked with asterisks). A minimum of nine oocytes were counted.

Figure 6. A central role for NHK-1 kinase in meiotic progression.

NHK-1 promotes nuclear reorganisation from a recombination phase to a post-recombination phase, including karyosome formation, synaptonemal complex disassembly and condensin loading. NHK-1 directly phosphorylates BAF that anchors meiotic chromosomes to the nuclear envelope. DSBs activate the meiotic recombination checkpoint that suppresses NHK-1 kinase to prevent nuclear reorganisation.