Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Drosophila Ctf4 is essential for efficient DNA replication and normal cell cycle progression.
Gosnell JA
,
Christensen TW
.
???displayArticle.abstract???
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.
Apger,
Multiple functions for Drosophila Mcm10 suggested through analysis of two Mcm10 mutant alleles.
2010, Pubmed
Apger,
Multiple functions for Drosophila Mcm10 suggested through analysis of two Mcm10 mutant alleles.
2010,
Pubmed
Asano,
Endoreplication: the advantage to initiating DNA replication without the ORC?
2009,
Pubmed
Banga,
Hypersensitivity of Drosophila mei-41 mutants to hydroxyurea is associated with reduced mitotic chromosome stability.
1986,
Pubmed
Bermudez,
Influence of the human cohesion establishment factor Ctf4/AND-1 on DNA replication.
2010,
Pubmed
Bianchi,
Changes of deoxyribonucleoside triphosphate pools induced by hydroxyurea and their relation to DNA synthesis.
1986,
Pubmed
Buszczak,
Eggs to die for: cell death during Drosophila oogenesis.
2000,
Pubmed
Chintapalli,
Using FlyAtlas to identify better Drosophila melanogaster models of human disease.
2007,
Pubmed
Christensen,
Drosophila MCM10 interacts with members of the prereplication complex and is required for proper chromosome condensation.
2003,
Pubmed
Das-Bradoo,
Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast.
2006,
Pubmed
Dej,
The endocycle controls nurse cell polytene chromosome structure during Drosophila oogenesis.
1999,
Pubmed
Errico,
Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts.
2007,
Pubmed
,
Xenbase
Errico,
Tipin/Tim1/And1 protein complex promotes Pol alpha chromatin binding and sister chromatid cohesion.
2009,
Pubmed
,
Xenbase
Gambus,
A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome.
2009,
Pubmed
Gouge,
Drosophila Sld5 is essential for normal cell cycle progression and maintenance of genomic integrity.
2010,
Pubmed
Gregan,
Fission yeast Cdc23/Mcm10 functions after pre-replicative complex formation to promote Cdc45 chromatin binding.
2003,
Pubmed
,
Xenbase
Gygax,
SepBCTF4 is required for the formation of DNA-damage-induced UvsCRAD51 foci in Aspergillus nidulans.
2005,
Pubmed
Hanna,
Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion.
2001,
Pubmed
Im,
Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins.
2009,
Pubmed
Kouprina,
CTF4 (CHL15) mutants exhibit defective DNA metabolism in the yeast Saccharomyces cerevisiae.
1992,
Pubmed
Labib,
Replication fork barriers: pausing for a break or stalling for time?
2007,
Pubmed
Labib,
Making connections at DNA replication forks: Mrc1 takes the lead.
2008,
Pubmed
Leman,
Human Timeless and Tipin stabilize replication forks and facilitate sister-chromatid cohesion.
2010,
Pubmed
Lengronne,
Establishment of sister chromatid cohesion at the S. cerevisiae replication fork.
2006,
Pubmed
MacNeill,
Structure and function of the GINS complex, a key component of the eukaryotic replisome.
2010,
Pubmed
,
Xenbase
Marchler-Bauer,
CDD: specific functional annotation with the Conserved Domain Database.
2009,
Pubmed
Park,
The origin recognition complex is dispensable for endoreplication in Drosophila.
2008,
Pubmed
Ricke,
Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha.
2004,
Pubmed
Ricke,
A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-alpha in budding yeast.
2006,
Pubmed
Salic,
A chemical method for fast and sensitive detection of DNA synthesis in vivo.
2008,
Pubmed
Su,
Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.
1998,
Pubmed
Tanaka,
Ctf4 coordinates the progression of helicase and DNA polymerase alpha.
2009,
Pubmed
Tweedie,
FlyBase: enhancing Drosophila Gene Ontology annotations.
2009,
Pubmed
Van Hatten,
The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication.
2002,
Pubmed
,
Xenbase
Wang,
Ctf4p facilitates Mcm10p to promote DNA replication in budding yeast.
2010,
Pubmed
Wawrousek,
Xenopus DNA2 is a helicase/nuclease that is found in complexes with replication proteins And-1/Ctf4 and Mcm10 and DSB response proteins Nbs1 and ATM.
2010,
Pubmed
,
Xenbase
Xu,
Genetic dissection of parallel sister-chromatid cohesion pathways.
2007,
Pubmed
Yang,
Nuclear distribution and chromatin association of DNA polymerase alpha-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast.
2005,
Pubmed
Zhu,
Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication.
2007,
Pubmed
,
Xenbase
