Study of DNA replication in Drosophila using cell free in vitro system

Abstract

Using Drosophila early egg extracts we have developed an optimized cell free system to study DNA replication. The efficiency of replication depends on a cold treatment of Drosophila embryos before the extract preparation and a formation of nuclei facilitated by the addition of membrane fractions to the extracts. In vitro DNA replication is ORC and CDC6 dependent, as a removal of these proteins from the extracts abolishes DNA replication. The N-terminal part of Orc1 protein, which is important for non-replicative functions of ORC, is dispensable for the replication in vitro. We also show that the conserved ATPase motif of CDC6 is crucial for the replication. Our studies indicate that a Drosophila cell free system proves to be an extremely useful tool for a functional dissection of the processes and factors involved in DNA replication in metazoans.

Introduction

Eukaryotic cells duplicate their genomes with remarkable precision during the course of growth and division. This process depends on stringent regulatory molecular mechanisms that couple DNA replication and cell cycle progression. In the last two decades a model has emerged that explains the coupling of initiation of DNA replication to the cell cycle.^1–4 During the G₁ phase of the cell cycle, the replication initiation factors including ORC (Origin Recognition Complex), Cdc6, Cdt1, MCMs and others form a multi-protein pre-replication complex (pre-RC). Pre-RC formation is restricted to G₁ phase and marks potential origins of replication. Origin activation occurs after cells enter the S phase and requires the action of kinases, which modify the pre-RC components and other replication factors resulting in recruitment of the DNA synthesis machinery. During this transformation the pre-RC is disassembled to prevent a new round of initiation within the same cell cycle.⁴ The mechanisms by which the pre-RC is transformed into an active replication fork and the specific biochemical roles of initiation factors are the areas of intense interest.

The best approaches for studying DNA replication in eukaryotes have been genetics and the analysis in cell free in vitro systems. To this date the cell free systems have been described for Xenopus,⁵ Drosophila⁶ and human cells.⁷ Xenopus egg extracts represent a powerful biochemical system to study DNA replication.⁵ These extracts assemble added sperm chromatin into nuclei that undergo a complete round of semiconservative DNA replication. The replication in egg extracts requires initiation factors such as ORC, Cdc6, Cdt1, MCMs, Cdc45, etc.^2,4 A variation of the egg extract system has also been developed in which nuclear assembly is not required for replication.⁸ A cell free system to study DNA replication has been described for Drosophila⁶ which mimics protocols described for Xenopus egg extracts. In our earlier work we used Drosophila egg extracts to study ORC dependent DNA replication.⁹ The DNA synthesis in these extracts was, in our hands, at least 10 times less efficient than that by synchronized Xenopus egg extracts. In the current work we present an optimized protocol for studying DNA replication in a Drosophila cell free system. DNA replication in Drosophila egg extracts depend on replication initiation factors and can be used for biochemical characterization of replication factors complementing the genetic approach in Drosophila.

Results and Discussion

The cell free in vitro system to study DNA replication has been described for Drosophila,⁶ however, it proved to be difficult to reproduce in our hands. In this study we optimized the published protocol and show that this optimized assay can be used to elucidate the functions of the proteins involved in the initiation of DNA replication. Our main problem with the published protocol was the inability for Xenopus sperm DNA to facilitate a nuclei formation in Drosophila early 0–2 hours egg extracts, which resulted in extremely low levels of replication. Young Drosophila embryos should have large stores of the components required to build nuclei, since they have to go through the multiple rounds of DNA replication during initial cycles of development. Centrifugation is required to clear the extract from debris and endogenous egg nuclei. However, spinning the extract with high RPM (24,000 g as indicated in a previously published protocol⁶) resulted in our hands in a very low extract replication efficiency (Fig. 1A). We found that the optimal speed for centrifugation is ~14,000 g which corresponds to 14,500 RPM in TLS 55 Beckman rotor. Centrifugation with a lower speed (less than 10,000 g) resulted in a residual presence of the Drosophila egg nuclei in the extract leading to inability to perform immunodepletion and rescue add back experiments (data not shown). Spinning extracts with higher speed (more than 20,000 g) significantly decreased replication efficiency of the extract (Fig. 1A). Xenopus sperm chromatin, incubated in these extracts, was not able to facilitate the formation of pseudonuclei important for DNA replication. Consistent with this conclusion, the microscopic analysis of Xenopus sperm after incubation in egg extract revealed only modest degree of chromatin decondensation (Fig. 1E).

Figure 1.

In vitro DNA replication in Drosophila egg extracts. The effects of centrifugation force (A), cold shock (B), freezing of extracts (C) and the effect of an addition of membranes (D) are shown. (E) Visualization of in vitro DNA replication in Drosophila egg extracts prepared after high and low RPM centrifugation. The replication in Xenopus sperm nuclei after addition of membrane fraction is also shown. Merged confocal images are presented. (F) DNA replication in Drosophila egg extracts is ORC dependent as shown by density substitution analysis of replicated HL (heavy/light) DNA. Xenopus sperm DNA in Drosophila egg extract (blue) and in ORC-depleted Drosophila extract (red) are presented on the density profiles. (G) The time course of DNA replication is shown as measured by TCA precipitation. DNA synthesis is shown in extracts after high (green) and low (red) speed centrifugation and in extracts supplemented by the addition of the membranes (blue).

Similarly to a published protocol⁶ we found that the incubation of eggs on ice necessary for embryo synchronization greatly increased the ability of the extract to replicate DNA (Fig. 1B). The cold treatment of Drosophila cells has been shown to disrupt the centrosome causing a metaphase block,¹⁰ which results in cell synchronization. The extracts prepared from these synchronized embryos facilitated nuclei formation and supported DNA replication. Up to 25–30% of template Xenopus sperm nuclei became decondensed rather than the 2–3% obtained from the incubation in the extracts prepared from untreated embryos. We conclude that the cold treatment results in synchronization of young Drosophila embryos and facilitates further decondensation of template DNA leading to the elevated levels of nuclear formation and DNA replication.

We also found that the addition of glycerol (10%), suggested by earlier studies⁶ for the prolonged storage of the extract, significantly reduced the replication ability of the extract. Extracts, frozen in the presence of glycerol and subsequently thawed, consistently displayed only 10–20% of the activity of the freshly prepared extracts which did not contain glycerol (Fig. 1C). Therefore in our studies we always used freshly prepared extracts. No more than 5% of glycerol was used for prolonged storage of the extracts in liquid nitrogen.

Finally, we found that the addition of Xenopus egg membranes further increases DNA replication ability of extract up to 10 fold (Fig. 1D). Microscopic analysis revealed that under these conditions Xenopus sperm nuclei undergo complete decondensation and form pseudonuclei with a high level of DNA replication (Fig. 1E). Figure 1F shows the analysis of replication products by density substitution experiments and proves that DNA replication observed in prepared Drosophila egg extracts is ORC dependent. In a parallel experiment DNA synthesis was measured by TCA precipitations in a time course manner (Fig. 1G). As for the gel analysis DNA was incubated in the extracts at the concentration ~20 ng/μl. After the incubation DNA was isolated, precipitated with TCA and analyzed for the incorporation of labeled nucleotide as described.⁵ A low rate of DNA synthesis observed in the extracts isolated after high speed centrifugation increase 2 to 3 fold when extracts were isolated with a low speed (Fig. 1A and G). The efficiency of DNA replication increased even further when membrane fractions were added to the replication extracts prepared with a low speed (Fig. 1D and G). The important role for the nuclear envelope in activating DNA replication was suggested by the experiments performed in Xenopus egg extracts.^5,11,12 It was also shown that any perturbation of the nuclear envelope disrupts DNA replication.^12,13 The role of the nuclear envelope is to create, through selective nuclear transport, an intranuclear environment that is permissive for DNA replication.^8,14 The nuclear envelope also plays a more direct role in potentiating DNA replication. Using the Xenopus cell free system, Lemaitre and colleagues¹⁵ have shown that at S phase entry in early development, nuclei are organized into short loops and replicons, allowing recruitment of a large amount of ORC protein. Loop size increases progressively during the S phase. Subsequent mitosis reprograms nuclei so that they again include short loops and small replicons, enabling the rapid DNA replication in the early embryo.¹⁵

The hexameric Origin Recognition Complex (ORC) is an important component for eukaryotic DNA replication. It was originally discovered in budding yeast S. cerevisiae and subsequent studies both in yeast and higher eukaryotes laid the foundation for understanding the functions of this important key initiation factor. ORC binds to origin sites in an ATP dependent manner and serves as a scaffold for the assembly of other initiation factors.¹⁶ ORC also directly participates in the loading of initiation factors.^17,18 ORC localization and origin selection involve many elaborate pathways with many regulators intervening upstream and downstream of ORC chromatin association.^16,19–23

The extensive studies in both Drosophila and mammalian systems indicate that Orc1 is more loosely associated with other subunits and is degraded during G₂ and M phases of the cell cycle.^24,25 This process is one of the mechanisms to prevent re-replication in metazoan cells.^22,25 The cellular levels of Orc1 in Drosophila tissues change dramatically throughout the development and are controlled by E2F.²⁶ In our earlier work we consistently observed two peaks of ORC activity during ORC purification from Drosophila embryos.²⁷ The highest apparent molecular weight peak contained all ORC subunits. The smaller complex was also detected that was apparently without Orc1 subunit. Both complexes can be reconstituted in vitro and purified. Figure 2A shows a silver stained gel containing both ORC(1-6) and ORC(2-6) recombinant complexes used in this study. The wild type baculovirus expressed recombinant ORC containing all six subunits can rescue DNA replication in ORC depleted egg extracts (Fig. 2C–E).⁹ In contrast, recombinant ORC(2-6) complex lacking the largest ORC subunit, Orc1, was not able to restore DNA replication in ORC-depleted extract (Fig. 2C–E). Orc1 in Drosophila consists of a highly conserved C-terminal domain (amino acids 555–927) which bears a homology with CDC6 protein and a variable N-terminal domain (amino acids 1–555) which does not display significant homology between Orc1 subunits derived from different species.^16,28 We asked if the deletion of the N-terminal domain of Orc1 would have an effect of DNA replication activity of ORC. Complete deletion of the N-terminal domain resulted in an inability of truncated Orc1 to form a complex with other subunits (not shown). The N-terminal domain of Orc1 in Drosophila contains a motif (amino acids 119–327) responsible for the interaction with Hp1 protein.²⁸ We found that Orc1Δn subunit missing amino acids 1–327 readily entered ORC complex (Fig. 2A). The resulting ORC(1Δn-6) complex was also able to rescue DNA replication in vitro in ORC depleted extracts (Fig. 2C–E). For the experiments described above, early (0–2 hr) egg extracts were immunodepleted of ORC using antibody raised against Drosophila Orc1, Orc2 and Orc6 subunits. The efficiency of immunodepletion was tested by western blotting (Fig. 2B). In add back experiments increasing amounts of recombinant, baculovirus produced wild type and mutant ORC proteins were added to ORC depleted extracts. Replication efficiency was analyzed by in vitro DNA replication assays (Fig. 2C), followed by a measurement of DNA synthesis by TCA precipitation (Fig. 2D) and microscopy analysis (Fig. 2E). We conclude that Drosophila ORC, like budding yeast ORC, can not function without its largest Orc1 subunit. The N-terminal domain of Orc1 is important for the interaction with other ORC subunits however, the deletion of the N-terminus responsible for the non-replicative functions of Orc1 had little or no effect on ORC-dependent DNA replication in vitro.

Figure 2.

In vitro DNA replication in Drosophila extracts is ORC dependent. (A) Silver stained gel of wild type ORC(1-6)—lane 2, ORC(1Δn-6) containing truncated at N-terminus Orc1 subunit (lane 4) and ORC(2-6) lacking Orc1 subunit (lane 5). Marker proteins are present in lanes 1 and 3. (B) Western immunoblotting analysis of Drosophila egg extract depleted of ORC using antibodies against Orc1, Orc2 and Orc6 subunits. (C) In vitro DNA replication in ORC depleted Drosophila extracts can be rescued by the addition of recombinant wild type ORC(1-6) and by the addition of ORC(1Δn-6) complex containing truncated at N-terminus Orc1 subunit, but not with ORC(2-6) lacking Orc1 subunit. In a parallel experiment (D) DNA replication was measured by TCA precipitation. (E) Visualization of in vitro DNA replication in Drosophila egg extracts after immunodepletion of ORC and add back rescue experiments with recombinant wild type ORC(1-6), ORC(1Δn-6) and ORC(2-6) complexes.

CDC6 protein is an important component for DNA replication.^3,4 One function of Cdc6 homologues is to load the MCMs onto chromatin, a critical step in licensing the DNA for replication. Similar to many proteins involved in DNA replication CDC6 contains motifs important for binding and hydrolyzing ATP. To date, some work has probed the requirement for these sequences in yeast, Xenopus and human Cdc6. Cdc6 proteins containing disrupted Walker A motif are nonfunctional in these organisms,^29–32 suggesting that Cdc6 requires ATP binding to form a productive and stable interaction with ORC and MCMs at replication origins. Microinjection of Walker A mutant human Cdc6 (HsCdc6) into HeLa cells results in a dominant negative phenotype characterized by a block in replication,³³ suggesting that the human Walker A mutant protein disrupts the ability of endogenous wild-type Cdc6 to load MCMs stably onto chromatin.

As expected, removal of CDC6 (Fig. 3B) from replication extract by immunodepletion resulted in the inability of the extracts to replicate DNA, as shown by gel electrophoresis of radioactively labeled replication products (Fig. 3A). This result was confirmed by TCA precipitation of replicated DNA (Fig. 3C), fluorescence microscopy (Fig. 3D) and density substitution experiments (Fig. 3E). Again, replication can be rescued by the addition of recombinant CDC6 to CDC6-depleted extracts, but not with a recombinant CDC6(KA) which carries a mutation in ATP binding domain of the protein (Fig. 3A, C and D). Interestingly, the peak corresponding to the position of heavy-heavy chains of DNA can be detected during rescue experiments in a density substitution assay suggesting that the excess of CDC6 may force re-replication of DNA in our in vitro assays.

Figure 3.

In vitro DNA replication in Drosophila extracts is CDC6 dependent. (A) In vitro DNA replication in CDC6 depleted Drosophila extracts can be rescued by the addition of 100 ng (+) or 200 ng (++) recombinant wild type CDC6 but not by 100 ng CDC6(KA) containing mutation that disrupts ATPase motif of CDC6. (B) Western immunoblotting analysis of Drosophila egg extract depleted of CDC6 (one (+) and two (++) rounds of depletion were used) using antibodies against CDC6. (C) CDC6 dependent DNA synthesis in the extracts was also measured by TCA precipitations. (D) Visualization of in vitro DNA replication in Drosophila egg extracts after immunodepletion using anti-CDC6 antibody and add back rescue experiment with 100 ng of either recombinant wild type CDC6 or mutant CDC6(KA) proteins. (E) DNA replication in Drosophila egg extracts is CDC6 dependent as shown by density substitution analysis of replicated DNA. The replication of Xenopus sperm DNA in Drosophila egg extract (green) and after addition to CDC6-depleted extract of 100 ng of recombinant purified wild type CDC6 (blue) or CDC6(KA) mutant (red) are presented on the density profiles. HL and HH show positions of heavy/light and heavy/heavy DNAs.

In conclusion, the experiments presented herein provide further evidence that Walker A is an essential motif of Cdc6 that is required for proper pre-RC assembly. Disruption of ATP binding in CDC6(KA) mutant results in an inability of the mutant protein to support DNA replication in vitro, most likely due to the assembly of a nonproductive pre-RC.

Overall, the described cell free system that uses Drosophila extracts is a very useful complement of the analogous in vitro system in Xenopus and will help in understanding the process of DNA replication. The efficiency of DNA replication observed in our experiments was comparable to the Xenopus in vitro system. Moreover, the amenability of Drosophila to genetic manipulations should open new approaches not possible with studies using Xenopus, such as the potential to use specific fly mutants and/or transgenic animals with a gene that is expected to have a clear phenotype in the in vitro replication assay. Our findings also provide a biochemical framework with which to dissect further the role of ORC, Cdc6 and other initiator proteins in initiating DNA replication.

Materials and Methods

Cloning and mutagenesis

cDNAs for Orc1 and CDC6 were cloned by PCR from a Drosophila embryonic library (Clontech MATCHMAKER library). N-terminal deletion of Orc1-Orc1(320-927), the addition of GST-tag and the His-tag were generated with standard PCR technique. The Lysine to Alanine substitution at the position of 303 within the ATPase domain (GKT) of CDC6-CDC6(KA) was introduced with Stratagene’s site-directed mutagenesis protocol (http://www.stratagene.com/manuals/200516.pdf). All constructs were analyzed by sequencing. cDNAs were subcloned into desired vectors with standard molecular biology techniques.

Purification of recombinant drosophila proteins

Recombinant baculoviruses were generated by using Bac-to-Bac expression system (GIBCO/BRL) as described.²⁷ For wild type ORC(1-6) complex containing all six ORC subunits, Orc1 gene was fused with 6x His N-terminal tag. Orc2 subunit was tagged to facilitate purification of ORC(2-6) complex which lacks Orc1 subunit. CDC6 gene was similarly fused with 6x His N-terminal tag to facilitate purification of the protein. Cell infections and protein purification was performed as described earlier.²⁷

In vitro replication in drosophila egg extracts

The preparation of egg extracts was based on a procedure described previously.⁶ Drosophila embryos (0–2 h) were washed with extraction buffer, cold treated, and homogenized. The homogenate was centrifuged for 20 min at 11,000–15,000 rpm in TLS 55 Beckmann rotor. The middle layer was collected and re-centrifuged. For long-term storage in liquid nitrogen the supernatant was adjusted to 5% with respect to glycerol and 1 mM to ATP. The extract was frozen in 20 μl beads in liquid nitrogen. Demembraned sperm chromatin was prepared essentially as described.⁵ The membrane fraction was prepared as described.³⁴

Before the experiment egg extract was supplemented with an ATP regeneration system (60 mM phosphocreatine and 150 μg/ml creatine phosphokinase) and membrane fraction (1 μl). Xenopus sperm DNA was added and incubated in extracts for 1 hr at a concentration of 2–5 ng/μl in the presence of [α³²-P] dCTP. The reactions were stopped with stop solution, DNA was extracted and ethanol precipitated, resuspended in TE buffer and submitted to electrophoresis in a 0.8% agarose gel. The gel was dried and autoradiographed.

Microscopy and indirect immunofluorescence experiments were performed as described previously.³⁵ For these experiments Xenopus sperm DNA (10 ng/μl) was incubated in Drosophila egg extracts in the presence of biotin-16-UTP. The extracts were applied to cover slips and subjected to low speed centrifugation, nuclei were fixed and stained for detection of the incorporated UTP analog with fluorescein-conjugated streptavidin. DNA was counterstained with propidium iodide. Merged confocal images are presented.

For immunodepletion experiments affinity purified polyclonal rabbit antibodies against Orc1, Orc2, Orc6 and Cdc6 were used as described.⁹ Completeness of immunodepletions was monitored by immunoblotting. To rescue DNA replication, add back experiments were performed by the addition of increasing amounts of baculovirus expressed reconstituted ORC or Cdc6.

Density substitution analysis of replicated DNA

Demembraned Xenopus sperm DNA was incubated for 1 hr in Drosophila egg extract at a concentration of 10 ng/μl in the presence of BrdUTP and [α-³²P]dCTP. DNA was extracted and subjected to centrifugation through a gradient of CsCl. For density substitution experiments,⁵ in addition to labeled dCTP, BrdUTP was added to a concentration of 1 mM. The reactions were stopped, and DNA was extracted and loaded onto a CsCl density gradient. The gradient was spun in a Beckman 50Ti rotor at 36,000 rpm at 2°C for 40 h. Fractions were collected and counted by Cerenkov radiation. Positions of LL and HL peaks were determined in parallel experiments using single-stranded M13 DNA as a template as described.⁹ TCA precipitations and measurements of the DNA synthesis were performed as described.⁵