BACKGROUND
reliable dna replication during s-phase is vital for the sustainability of a cell and, ultimately, an organism.
the proteins that assemble at licensed replication forks come together to form the replisome progression complex .
this complex must faithfully copy an organism's genome in the context of cell cycle regulation and chromatid segregation while at the same time safeguarding against dna damage.
in response to this highly integrated nature of the rpc, recent studies have generated models at the replication fork that link mcm helicase activity to primase activity and establishment of sister chromatid cohesion throughout s-phase  <cit> .
the ctf <dig> protein has recently become the subject of several investigations in eukaryotes due to its position as a central component of the rpc.
it was initially identified in yeast during a screen for mutants affecting chromosome transmission fidelity, after which it is named  <cit> .
the ctf <dig> gene in drosophila  encodes a protein that is conserved across lower and higher eukaryotes.
ctf <dig> has been proposed to be a member of the fork protection complex  shown to link the mcm helicase activity to lagging-strand polymerase α-primase activity in eukaryotes  <cit> .
while depletion studies in xenopus laevis  <cit>  have suggested its importance in the stabilization of this interaction and thus replication fidelity in general, ctf <dig> has also been implicated in proper chromatid segregation and cohesion from their initial establishment in s-phase to separation in anaphase  <cit> .
in fact, other members of the fpc have been shown to be essential for proper chromatid cohesion during s-phase, including tim1/tipin, which interact with cohesin in humans  <cit> .
this information suggests that replication fidelity is coordinated with chromatid cohesion.
this study is the first to undertake phenotypic analysis of the effects of ctf <dig> knockdown on development in a whole-organism, multi-cellular eukaryote.
using a gal4/uas-driven expression and heat-shock driven expression of rnai targeting ctf <dig>  we demonstrate that ctf <dig> is an essential gene in drosophila that is required for normal cell cycle progression, sister chromatid cohesion, endoreplication, and response to replication stress.
RESULTS
ctf <dig> is a conserved protein
ctf <dig> has been found, without exception, in a wide range of eukaryotes.
the human and1/ctf <dig> sequence was used to blast the drosophila genome and we identified cg <dig> as the drosophila homolog of ctf <dig>  multiple alignments of drosophila ctf <dig> against a range of homologs from other species reveals that protein contains a series of wd <dig> repeats at the n-terminus predicted to facilitate protein-protein interactions, a central conserved domain in all eukaryotes, and  an hmg  box domain at the c terminus that has dna-binding activity .
the wd <dig> repeats and the central domain are conserved across all studied species.
however, in vertebrates the c terminus has expanded to include an hmg box domain  <cit> .
drosophila ctf <dig> interacts with dna replication proteins
studies in yeast and xenopus have shown a conserved role for ctf <dig> in tying the activity of polymerase α primase to the mcm helicase complex  <cit> , and that mcm <dig> recruits ctf <dig> to this role  <cit> .
in order to test whether or not drosophila ctf <dig> interacted with similar replication proteins as in other eukaryotes investigated, ctf <dig> was cloned from drosophila and fused to the gal activation domain for yeast 2-hybrid analysis.
as expected, drosophila ctf <dig> was found to interact with polymerase α primase.
in addition, consistent interaction with psf <dig>  psf <dig> and mcm <dig> was also detected .
gad fusion proteins tested showed no one-hybrid activity .
ctf <dig> mrna is reduced via rnai
utilizing a gal <dig> driven rnai transgene specific to ctf <dig> we were able to significantly reduce levels of ctf <dig> transcript .
ctf <dig> transcript levels were measured by rtpcr in 3rd instar larvae and were found to be reduced by 87% in flies containing both the actin promoter driven gal <dig> and the uas driven ctf <dig> rnai transgene as compared to the sibling control that contained only the uas driven ctf <dig> rnai transgene .
ctf <dig> transcript levels were likewise reduced in females by utilizing a heat-shock promoter upstream of the ctf <dig> rnai construct.
ctf <dig> transcript levels were reduced by 81% in the females containing the rnai construct compared to wild-type females .
ctf <dig> is required for viability
using the actin promoter driven gal <dig> driving expression of ctf <dig> specific dsrna we investigated the impact of ctf <dig> depletion of the viability of drosophila through development.
we discovered that maternal loading in combination with low levels of zygotic transcription was sufficient for viability from the egg stage to pupation as the approximately 1: <dig>  mendelian ratio was observed in the progeny of uas:ctf <dig> dsrna/uas:ctf <dig> dsrna crossed to actin:gal <dig> cyo,gfp .
however, only  <dig> % of ctf <dig> knockdown flies succeeded in eclosing .
moreover, of those flies surviving this stage were all female and exhibited 100% lethality after < <dig> days and were severely emaciated compared to wild-type .
depletion of ctf <dig> results in cell cycle progression defects
reductions in the levels of dna replication proteins have been shown to cause cell cycle delays in drosophila wandering 3rd instar larval brain tissue  <cit> .
in addition the expression level of ctf <dig> has been shown to be relatively high in the larval brain   <cit> .
cell cycle progression was assayed by determining the relative mitotic indices for ctf <dig> depleted and control larvae.
ctf <dig> depletion resulted in a significant  reduction in the percent of cells in mitosis  compared to the sibling control  .
in addition to a lower mitotic index, mitotic figures were observed in the ctf <dig> depleted larvae that demonstrated premature sister chromatid separation .
no such sister chromatid separations were observed in the mitotic spreads in control larvae .
a reduction in the faction of cells in m phase is consistent with a possible increase in the fraction of cells in s phase in the ctf <dig> depleted larvae.
to directly test for a possible s-phase delay edu  incorporation and detection by fluorescence was used.
edu is a thymidine analog incorporated during dna replication whose reactive alkyne group is detected by forming a triazole ring  <cit> .
quantitation of edu incorporation in ctf <dig> knockdown larval brains showed a severe s-phase delay with  <dig> % ±  <dig> % of cells in s phase when ctf <dig> was depleted compared to  <dig> % ±  <dig> % in the control  .
ctf <dig> is required for endoreplication
while the larval brain tissue provides for the analysis of cells undergoing the canonical cell cycle , whereas the drosophila salivary glands undergo repeated rounds of replication without mitosis producing large polytene chromosomes.
some but not all dna replication proteins have been shown to be required for endoreplication of these tissues  <cit> .
to investigate the impact of ctf <dig> knockdown on endoreplication, drosophila salivary glands were dissected and spread.
a survey of polytene micrographs revealed a consistent thinning of polytene chromosomal arms , suggesting under-replication.
levels of replication were quantitated as the stained area of the chromosome/chromosome length.
overall, ctf <dig> depletion resulted in polytene chromosome that were reduced in thickness by  <dig> %  compared to the sibling controls   .
ctf <dig> is required for early embryo cell cycles
maternal loading of ctf <dig> prevents the analysis of the requirement of ctf <dig> in early embryo cell cycles using the gal <dig> uas:rnai system.
to circumvent maternal loading and probe the requirement for ctf <dig> in early embryos we took advantage of the heat-shock promoter upstream of the ctf <dig> dsrna construct and subjected transgene containing females and wild-type females to heat-shock  and examined the resulting embryos.
normally the syncytial nuclei divide rapidly and synchronously with no gap phase.
these nuclei can become asynchronous and become linked to one another via anaphase bridges if dna replication is compromised .
while the wild-type control embryos appeared normal, asynchrony and anaphase bridging were observed in 69% of the embryos  laid by ctf <dig> depleted females .
ctf <dig> knockdown results in early degeneration of egg chambers
the expression profile of ctf <dig> in adult drosophila indicates it is most actively transcribed in females within the ovaries  <cit> .
these ovaries are subdivided into individual ovarioles that themselves consist of multiple egg chambers.
egg chambers are progressively more mature in the posterior direction and contain nurse cells that provide materials which nurture the oocyte and ultimately are required for the development of the embryo prior to the activation of zygotic transcription  <cit> .
during mid-oogenisis, stages 7- <dig>  egg chambers will naturally degenerate if the egg chamber is defective and unable to support the subsequent growth of the oocyte and embryo  <cit> .
this pre-vitellogenic check-point is triggered in egg chambers where ctf <dig> has been depleted and results in degeneration of  <dig> % of the stage 7- <dig> egg chambers  as compared to no degeneration observed in the wild-type control  .
ctf <dig> is required for coping with replication stress
an important facet of the ctf <dig> protein is its role in the fork protection complex.
this complex is thought to assist in the stabilization of paused replication forks during low nucleotide availability that occurs during replication of repetitive sequences, as well as during repair events that lead to stalling  <cit> .
in order to assess whether or not ctf <dig> plays a role in protecting paused dna replication forks from damage we treated larvae with hydroxyurea  which depletes the nucleotide pool during dna replication leading to replication fork pausing  <cit> .
using the gal <dig> uas ctf <dig> rnai system  we scored surviving 3rd instar larvae.
just over half  of the ctf <dig> depleted larvae survived relative to controls .
these results indicate that normal levels of ctf <dig> are required for coping with the replication stress induced by fork pausing.
ctf <dig> knockdown larvae exhibit a decrease in relative survival compared to sibling control.
survival ratios represent the percentage of glowing to non-glowing larvae.
parentheses indicate total number of larvae scored.
relative survival is the ratio of  hydroxyurea survival to  hydroxyurea survival, and shows the effect of hydroxyurea treatment on ctf <dig> knockdown larvae.
hydroxyurea group is not significant).
discussion
ctf <dig> has been shown to stabilize helicase-polymerase association during s phase by interaction with the cmg complex.
the stabilization of this association is required at the replication fork to ensure the faithful duplication of genetic information and passage through s phase  <cit> .
surprisingly, though this a conserved function across the species examined, it is not likely mediated through the highly conserved wd <dig> domain found in the n terminus of the protein.
instead, the c terminal portion of the protein has been implicated in interaction with the cmg complex  <cit> .
we demonstrate that drosophila ctf <dig> interacts with mcm <dig>  polymerase alpha primase, and members of the gins complex.
these interactions are consistent with a central role for ctf <dig> in coordinating the helicase-polymerase association during s phase.
it has been reported in budding yeast that ctf <dig> interacts only with the gins complex via interaction with sld <dig>  <cit> .
drosophila ctf <dig> appears to be different in that it interacts with the psf <dig> and psf <dig> subunits of the gins complex.
given the structural similarities between the subunits of the gins complex  <cit>  it is not unreasonable to speculate that specific subunit association of ctf <dig> may be fluid over evolutionary time.
ctf <dig> has been repeatedly implicated in proper sister chromatid cohesion  <cit> .
we show here that when ctf <dig> is depleted in drosophila, premature sister chromatid separation is observed.
our observations are made in tissues that are normally progressing through the cell cycle and are not treated with mitosis inhibitors which can cause ectopic sister chromatid separation to occur.
research using unicellular eukaryotes, tissue culture, and xenopus egg extracts has clearly shown a role for ctf <dig> in dna replication  <cit> .
we demonstrate here, for the first time that within the context of a multicellular metazoan, ctf <dig> is required for normal s-phase progression and viability through development.
moreover, we show that ctf <dig> is required for endoreplication, ovary development, and early embryo cell cycles.
previous research has indicated that there is a link between ctf <dig> and mcm <dig>  <cit> .
indeed both mcm <dig> and ctf <dig> appear to share many of the same functions in that they both coordinate aspects of the cmg complex and polymerase alpha  <cit> .
using the yeast two-hybrid system we could not confirm a direct interaction between these two drosophila proteins .
however it is interesting to note that depletion of mcm <dig> by use of a hypomorphic allele results in similar s phase delays, early embryo anaphase bridges, and defects in ovary development  <cit> .
in addition mcm <dig> like ctf <dig> has been shown to interact with the gins complex in drosophila  <cit> .
curiously, the knockdown of mcm <dig> in drosophila only results in a partial lethality and fly stocks may be kept homozygous for the hypomorphic mcm <dig> allele  <cit> .
within this context it is also interesting to note that conserved domain analysis of the ctf <dig> consensus sequence reveals a cryptic mcm <dig> like domain in the c terminal   <cit> .
the literature, combined with our observations, suggest that mcm <dig> and ctf <dig> may share some redundant functions with respect to coordination at the replication fork.
if this is the case then the foundation laid by our work will inform double mutant analysis of these two genes leading to a better understanding of how replication fork progression complexes are coordinated and regulated.
CONCLUSIONS
recent investigations into ctf <dig> have placed it as an integral member of the dna replication machinery with respect to coordinating helicase and polymerase activity.
here with lay the groundwork for studying the role of this protein within the context of a multicellular metazoan.
we have found that this highly conserved protein interacts with a member of the mcm2- <dig> helicase, members of the gins complex, and dna pol alpha.
moreover, we demonstrate efficient rnai knockdown of ctf <dig> in drosophila and use these animals to show that ctf <dig> is required for viability, sister chromatid cohesion, s phase progression, endoreplication, ovariole development, early embryo cell cycles, and coping with replication stress.
taken altogether, ctf <dig> continues to be a key protein in our understanding of the underlying processes required for cell viability and organismal development.

 reliable dna replication during s-phase is vital for the sustainability of a cell and, ultimately, an organism. the proteins that assemble at licensed replication forks come together to form the replisome progression complex. this complex must faithfully copy an organism's genome in the context of cell cycle regulation and chromatid segregation while at the same time safeguarding against dna damage. in response to this highly integrated nature of the rpc, recent studies have generated models at the replication fork that link mcm helicase activity to primase activity and establishment of sister chromatid cohesion throughout s-phase. the ctf protein has recently become the subject of several investigations in eukaryotes due to its position as a central component of the rpc. it was initially identified in yeast during a screen for mutants affecting chromosome transmission fidelity, after which it is named. the ctf gene in drosophila encodes a protein that is conserved across lower and higher eukaryotes. ctf has been proposed to be a member of the fork protection complex shown to link the mcm helicase activity to lagging-strand polymerase α-primase activity in eukaryotes. while depletion studies in xenopus laevis have suggested its importance in the stabilization of this interaction and thus replication fidelity in general, ctf has also been implicated in proper chromatid segregation and cohesion from their initial establishment in s-phase to separation in anaphase. in fact, other members of the fpc have been shown to be essential for proper chromatid cohesion during s-phase, including tim1/tipin, which interact with cohesin in humans. this information suggests that replication fidelity is coordinated with chromatid cohesion. this study is the first to undertake phenotypic analysis of the effects of ctf knockdown on development in a whole-organism, multi-cellular eukaryote. using a gal4/uas-driven expression and heat-shock driven expression of rnai targeting ctf we demonstrate that ctf is an essential gene in drosophila that is required for normal cell cycle progression, sister chromatid cohesion, endoreplication, and response to replication stress.  ctf is a conserved protein ctf has been found, without exception, in a wide range of eukaryotes. the human and1/ctf sequence was used to blast the drosophila genome and we identified cg as the drosophila homolog of ctf multiple alignments of drosophila ctf against a range of homologs from other species reveals that protein contains a series of wd repeats at the n-terminus predicted to facilitate protein-protein interactions, a central conserved domain in all eukaryotes, and an hmg box domain at the c terminus that has dna-binding activity. the wd repeats and the central domain are conserved across all studied species. however, in vertebrates the c terminus has expanded to include an hmg box domain. drosophila ctf interacts with dna replication proteins studies in yeast and xenopus have shown a conserved role for ctf in tying the activity of polymerase α primase to the mcm helicase complex, and that mcm recruits ctf to this role. in order to test whether or not drosophila ctf interacted with similar replication proteins as in other eukaryotes investigated, ctf was cloned from drosophila and fused to the gal activation domain for yeast 2-hybrid analysis. as expected, drosophila ctf was found to interact with polymerase α primase. in addition, consistent interaction with psf psf and mcm was also detected. gad fusion proteins tested showed no one-hybrid activity. ctf mrna is reduced via rnai utilizing a gal driven rnai transgene specific to ctf we were able to significantly reduce levels of ctf transcript. ctf transcript levels were measured by rtpcr in 3rd instar larvae and were found to be reduced by 87% in flies containing both the actin promoter driven gal and the uas driven ctf rnai transgene as compared to the sibling control that contained only the uas driven ctf rnai transgene. ctf transcript levels were likewise reduced in females by utilizing a heat-shock promoter upstream of the ctf rnai construct. ctf transcript levels were reduced by 81% in the females containing the rnai construct compared to wild-type females. ctf is required for viability using the actin promoter driven gal driving expression of ctf specific dsrna we investigated the impact of ctf depletion of the viability of drosophila through development. we discovered that maternal loading in combination with low levels of zygotic transcription was sufficient for viability from the egg stage to pupation as the approximately 1: mendelian ratio was observed in the progeny of uas:ctf dsrna/uas:ctf dsrna crossed to actin:gal cyo,gfp. however, only % of ctf knockdown flies succeeded in eclosing. moreover, of those flies surviving this stage were all female and exhibited 100% lethality after < days and were severely emaciated compared to wild-type. depletion of ctf results in cell cycle progression defects reductions in the levels of dna replication proteins have been shown to cause cell cycle delays in drosophila wandering 3rd instar larval brain tissue. in addition the expression level of ctf has been shown to be relatively high in the larval brain. cell cycle progression was assayed by determining the relative mitotic indices for ctf depleted and control larvae. ctf depletion resulted in a significant reduction in the percent of cells in mitosis compared to the sibling control. in addition to a lower mitotic index, mitotic figures were observed in the ctf depleted larvae that demonstrated premature sister chromatid separation. no such sister chromatid separations were observed in the mitotic spreads in control larvae. a reduction in the faction of cells in m phase is consistent with a possible increase in the fraction of cells in s phase in the ctf depleted larvae. to directly test for a possible s-phase delay edu incorporation and detection by fluorescence was used. edu is a thymidine analog incorporated during dna replication whose reactive alkyne group is detected by forming a triazole ring. quantitation of edu incorporation in ctf knockdown larval brains showed a severe s-phase delay with % ± % of cells in s phase when ctf was depleted compared to % ± % in the control. ctf is required for endoreplication while the larval brain tissue provides for the analysis of cells undergoing the canonical cell cycle, whereas the drosophila salivary glands undergo repeated rounds of replication without mitosis producing large polytene chromosomes. some but not all dna replication proteins have been shown to be required for endoreplication of these tissues. to investigate the impact of ctf knockdown on endoreplication, drosophila salivary glands were dissected and spread. a survey of polytene micrographs revealed a consistent thinning of polytene chromosomal arms, suggesting under-replication. levels of replication were quantitated as the stained area of the chromosome/chromosome length. overall, ctf depletion resulted in polytene chromosome that were reduced in thickness by % compared to the sibling controls. ctf is required for early embryo cell cycles maternal loading of ctf prevents the analysis of the requirement of ctf in early embryo cell cycles using the gal uas:rnai system. to circumvent maternal loading and probe the requirement for ctf in early embryos we took advantage of the heat-shock promoter upstream of the ctf dsrna construct and subjected transgene containing females and wild-type females to heat-shock and examined the resulting embryos. normally the syncytial nuclei divide rapidly and synchronously with no gap phase. these nuclei can become asynchronous and become linked to one another via anaphase bridges if dna replication is compromised. while the wild-type control embryos appeared normal, asynchrony and anaphase bridging were observed in 69% of the embryos laid by ctf depleted females. ctf knockdown results in early degeneration of egg chambers the expression profile of ctf in adult drosophila indicates it is most actively transcribed in females within the ovaries. these ovaries are subdivided into individual ovarioles that themselves consist of multiple egg chambers. egg chambers are progressively more mature in the posterior direction and contain nurse cells that provide materials which nurture the oocyte and ultimately are required for the development of the embryo prior to the activation of zygotic transcription. during mid-oogenisis, stages 7- egg chambers will naturally degenerate if the egg chamber is defective and unable to support the subsequent growth of the oocyte and embryo. this pre-vitellogenic check-point is triggered in egg chambers where ctf has been depleted and results in degeneration of % of the stage 7- egg chambers as compared to no degeneration observed in the wild-type control. ctf is required for coping with replication stress an important facet of the ctf protein is its role in the fork protection complex. this complex is thought to assist in the stabilization of paused replication forks during low nucleotide availability that occurs during replication of repetitive sequences, as well as during repair events that lead to stalling. in order to assess whether or not ctf plays a role in protecting paused dna replication forks from damage we treated larvae with hydroxyurea which depletes the nucleotide pool during dna replication leading to replication fork pausing. using the gal uas ctf rnai system we scored surviving 3rd instar larvae. just over half of the ctf depleted larvae survived relative to controls. these results indicate that normal levels of ctf are required for coping with the replication stress induced by fork pausing. ctf knockdown larvae exhibit a decrease in relative survival compared to sibling control. survival ratios represent the percentage of glowing to non-glowing larvae. parentheses indicate total number of larvae scored. relative survival is the ratio of hydroxyurea survival to hydroxyurea survival, and shows the effect of hydroxyurea treatment on ctf knockdown larvae. hydroxyurea group is not significant). discussion ctf has been shown to stabilize helicase-polymerase association during s phase by interaction with the cmg complex. the stabilization of this association is required at the replication fork to ensure the faithful duplication of genetic information and passage through s phase. surprisingly, though this a conserved function across the species examined, it is not likely mediated through the highly conserved wd domain found in the n terminus of the protein. instead, the c terminal portion of the protein has been implicated in interaction with the cmg complex. we demonstrate that drosophila ctf interacts with mcm polymerase alpha primase, and members of the gins complex. these interactions are consistent with a central role for ctf in coordinating the helicase-polymerase association during s phase. it has been reported in budding yeast that ctf interacts only with the gins complex via interaction with sld. drosophila ctf appears to be different in that it interacts with the psf and psf subunits of the gins complex. given the structural similarities between the subunits of the gins complex it is not unreasonable to speculate that specific subunit association of ctf may be fluid over evolutionary time. ctf has been repeatedly implicated in proper sister chromatid cohesion. we show here that when ctf is depleted in drosophila, premature sister chromatid separation is observed. our observations are made in tissues that are normally progressing through the cell cycle and are not treated with mitosis inhibitors which can cause ectopic sister chromatid separation to occur. research using unicellular eukaryotes, tissue culture, and xenopus egg extracts has clearly shown a role for ctf in dna replication. we demonstrate here, for the first time that within the context of a multicellular metazoan, ctf is required for normal s-phase progression and viability through development. moreover, we show that ctf is required for endoreplication, ovary development, and early embryo cell cycles. previous research has indicated that there is a link between ctf and mcm. indeed both mcm and ctf appear to share many of the same functions in that they both coordinate aspects of the cmg complex and polymerase alpha. using the yeast two-hybrid system we could not confirm a direct interaction between these two drosophila proteins. however it is interesting to note that depletion of mcm by use of a hypomorphic allele results in similar s phase delays, early embryo anaphase bridges, and defects in ovary development. in addition mcm like ctf has been shown to interact with the gins complex in drosophila. curiously, the knockdown of mcm in drosophila only results in a partial lethality and fly stocks may be kept homozygous for the hypomorphic mcm allele. within this context it is also interesting to note that conserved domain analysis of the ctf consensus sequence reveals a cryptic mcm like domain in the c terminal. the literature, combined with our observations, suggest that mcm and ctf may share some redundant functions with respect to coordination at the replication fork. if this is the case then the foundation laid by our work will inform double mutant analysis of these two genes leading to a better understanding of how replication fork progression complexes are coordinated and regulated.  recent investigations into ctf have placed it as an integral member of the dna replication machinery with respect to coordinating helicase and polymerase activity. here with lay the groundwork for studying the role of this protein within the context of a multicellular metazoan. we have found that this highly conserved protein interacts with a member of the mcm2- helicase, members of the gins complex, and dna pol alpha. moreover, we demonstrate efficient rnai knockdown of ctf in drosophila and use these animals to show that ctf is required for viability, sister chromatid cohesion, s phase progression, endoreplication, ovariole development, early embryo cell cycles, and coping with replication stress. taken altogether, ctf continues to be a key protein in our understanding of the underlying processes required for cell viability and organismal development. 
using in vivo rnai knockdown of ctf in drosophila we show that ctf is required for viability, s phase progression, sister chromatid cohesion, endoreplication, and coping with replication stress. proper coordination of the functions at the dna replication fork is vital to the normal functioning of a cell. the ctf 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, and as being important for sister chromatid cohesion. we show that ctf function is conserved and that drosophila can be effectively used as a model to further probe the precise function of ctf as a member of the replication fork and possible roles in development. specifically the precise coordination of helicase and polymerase activity is crucial for efficient passage though s phase. we find that drosophila ctf is a conserved protein that interacts with members of the gins complex, mcm and polymerase α primase. ctf remains a central player in dna replication.
 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 ctf 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, and as being important for sister chromatid cohesion. as such, understanding the role of ctf 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 ctf is a conserved protein that interacts with members of the gins complex, mcm and polymerase α primase. using in vivo rnai knockdown of ctf in drosophila we show that ctf is required for viability, s phase progression, sister chromatid cohesion, endoreplication, and coping with replication stress.  ctf remains a central player in dna replication. our findings are consistent with what has been previously reported for ctf function in yeast, xenopus extracts, and human tissue culture. we show that ctf function is conserved and that drosophila can be effectively used as a model to further probe the precise function of ctf as a member of the replication fork and possible roles in development. 