| dc.description.abstracteng | The early development of Drosophila melanogaster occurs in a syncytium, where nuclei undergo mitotic divisions without accompanying cytokinesis. Early cleavage cycles are very short and consist only of S and M phases. In cycle 14, the cell cycle is paused and a G2 phase is introduced. During this extended interphase the embryo undergoes cellularisation, a modified form of cytokinesis which transforms the syncytial into a cellular blastoderm. Despite extensive investigations, regulation of cellularisation and progression of early cell cycles is not fully understood. The mutant X238 was identified in a screen for morphological defects in early development and contains a mutation in the gene CG3011 encoding the enzyme Serine hydroxymethyltransferase (SHMT). SHMT is a metabolic enzyme converting serine and tetrahydrofolate (THF) into glycine and methylene-THF (mTHF), which is the major one-carbon source for a large number of anabolic reactions including thymidylate and S-adenosylmethionine (SAM) synthesis. X238 embryos precociously arrest the cell cycle in interphase 13 and fail to undergo cellularisation, although zygotic gene expression is only weakly disturbed. Furthermore, nuclear morphology becomes disturbed in late cycle 13 of SHMT mutant embryos. Although early cellularisation markers are expressed and localised as revealed by immunostaining against Slow as molasses (Slam), Diaphanous (Dia), and Discs large (Dlg), cellularisation is not initiated. Moreover, its depletion causes the activation of both checkpoint kinases Chk1 (Grapes in Drosophila) and Chk2 (Maternal nuclear kinase - Mnk) in cycle 13, which finally stops the cell cycle progression. This checkpoint activation indicates the ccurrence
of replication stress, an interference of transcription and replication throughout an extended S phase 13. These results were confirmed by life imaging of RpA70-eGFP expressing Drosophila embryos, which accumulated RpA70-eGFP in foci within the nuclei during S phase 13 of X238 mutant embryos indicating replication stress. Inhibition of transcription by -amanitin as well as injection of the heterochromatin binding protein HP1 is able to rescue both the cell cycle and lethality phenotypes. This reduction of transcriptional activity, thereby reducing replication stress and postponing the activation of checkpoint kinases, may allow the mutant embryos to finish DNA replication in S phase 13. Consistent with the current literature, in which it was shown that SHMT functions at the localised thymidylate supply at the nuclear periphery of mammalian cells and that its mutations causes mis-incorporation of nucleotides, the results of this thesis lead to the conclusion that the blastoderm defect of X238 could be caused by an interference with DNA replication due to a lack of localised thymidylate synthesis. This could induce stalling of replication forks at the nuclear periphery, which activates the DNA replication checkpoint and stops cell cycle progression. | de |