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Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression.
Gosnell JA
,
Christensen TW
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Proper coordination of the functions at the DNA replication fork is vital to the normal functioning of a cell. Specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though S phase. The Ctf4 protein has been shown to be a central member of the replication fork and links the replicative MCM helicase and DNA polymerase α primase. In addition, it has been implicated as a member of a complex that promotes replication fork stability, the Fork Protection Complex (FPC), and as being important for sister chromatid cohesion. As such, understanding the role of Ctf4 within the context of a multicellular organism will be integral to our understanding of its potential role in developmental and disease processes. We find that Drosophila Ctf4 is a conserved protein that interacts with members of the GINS complex, Mcm2, and Polymerase α primase. Using in vivo RNAi knockdown of CTF4 in Drosophila we show that Ctf4 is required for viability, S phase progression, sister chromatid cohesion, endoreplication, and coping with replication stress. Ctf4 remains a central player in DNA replication. Our findings are consistent with what has been previously reported for CTF4 function in yeast, Xenopus extracts, and human tissue culture. We show that Ctf4 function is conserved and that Drosophila can be effectively used as a model to further probe the precise function of Ctf4 as a member of the replication fork and possible roles in development.
Figure 1. Ctf4 is conserved across species and interacts with DNA replication proteins. A. Alignment of Ctf4 from different organisms shows a conserved WD40 domain and central domain, as well as an HMG domain conserved only in vertebrates. B. Serial dilutions of PJ69α yeast two-hybrid reporter strain with the indicated fusion constructs. Drosophila Ctf4 fused to GAD interacts with Drosophila GBK fusions of Psf1, Psf2, Mcm2, and Pol alpha respectively as evidenced by growth on media lacking histidine, while no growth occurs in the control.
Figure 2. GAL4/UAS-driven RNAi system in Drosophila for knockdown of CTF4. A. Crossing scheme for the generation of CTF4 knockdown flies and rtPCR of resulting knockdown. GFP positive larvae are sibling controls for the non-glowing CTF4 knockdown larvae. Transcript levels are reduced by 87% in RNAi flies compared to sibling controls. Analysis was carried out on these groups as indicated. B. Alternate strategy for CTF4 knockdown using a heat-shock promoter upstream of the RNAi sequence. Analysis by rtPCR shows an 81% reduction in CTF4 transcript compared to wild-type controls.
Figure 3. Ctf4 is required for viability. A. Graph showing survival at the third-instar, pupation, and eclosion for CTF4 knockdown and sibling control showing that CTF4 is required for successful eclosion. Maternal loading is no longer sufficient to augment knockdown of Ctf4 at pupation. B. SEM images of aberrant phenotype in Ctf4 knockdown eclosion survivors.
Figure 4. Ctf4 is required for normal cell cycle progression. A. CTF4 knockdown results in reduced mitotic index in larval brain tissue as compared to sibling control. Boxplot of mitotic indices determined from 3rd-instar wandering larva brain squashes. Larvae with the genotype RNAi-Ctf4/Actin-GAL4 (Ctf4 knockdown) yielded a significantly lower ratio of cells progressing through M-phase compared to sibling control (P = 0.004). B-C. Micrographs of mitotic figures from control and RNAi knockdown larval brains. Ctf4 depletion results in premature sister chromatid separation as indicated by triangles (bars represent 10 μm). D. CTF4 knockdown results in S phase delay. Boxplot of S-phase indices of larval brains determined by EdU incorporation showing that significantly more cells are seen in S-phase within Ctf4 knockdown larval brains compared to sibling control (P = 0.009).
Figure 5. Ctf4 is required for normal endoreplication. A. Representative confocal micrographs of CTF4 knockdown polytene chromosome compared to sibling control showing thinning of chromosomes in CTF4 knockdown relative to control. B. Quantitation of polytene chromosomes in CTF4 knockdown compared to controls. Knockdown of CTF4 results in overall thinning of chromosome arms by an average of 43.4%.
Figure 6. Knockdown of CTF4 results in defects in ovary and early embryo development. Representative confocal micrographs of egg chamber ovarioles at stage 7 in CTF4 knockdown and wild-type controls showing apoptotic-like DNA condensation in CTF4 knockdown nurse cells (right panels). Representative confocal micrographs of DNA stained with DAPI in Drosophila early embryos from both CTF4 knockdown and wild-type controls (left panels). Mitotic bridging can be seen in the Ctf4 knockdown group but was absent from controls.
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