DNA Replication in NPE

Sperm chromatin or plasmid DNA can be used as a template for efficient DNA replication when incubated sequentially in HSE and NPE (Fig. 1A). Incubation of the DNA template with HSE leads to pre-RC formation. Subsequent addition of NPE

stimulates replication initiation by providing high levels of several replication factors (see Subheading 1.), while also limiting DNA replication to a single round because of the presence of inhibitors that block the formation of pre-RCs (6). Complete DNA replication of a 3-kb plasmid usually takes 20 to 30 min, whereas replication of sperm chromatin takes 45 to 60 min.

1. Rapidly thaw 1 aliquot of HSE (50 ||L) and demembranated sperm (5 ||L; 100,000 sperm/|L), then transfer immediately to ice.

2. To 30 |L HSE, add 1 |L of ATP regeneration mix, 0.2 |L of 0.5 mg/mL nocodozole, 0.1 to 0.5 |L [a32P]dATP (3000 Ci/mmol), and either 3 |L of 100,000 sperm/|L or 20 ng/|L final concentration of a purified, supercoiled plasmid DNA (3-12 kb). Mix thoroughly by repeated pipeting (10 times).

3. Immediately subdivide the reaction into smaller aliquots (usually 3-5 ||L), which will later be stopped at different time-points, and incubate for 30 min at 22 to 23°C to allow the assembly of pre-RCs (see Note 2).

4. After pre-RC assembly has been initiated, thaw an appropriate amount of NPE and supplement it with ATP regeneration mix (1/30 vol) and DTT to 20 mM final concentration. The NPE is incubated at room temperature for 10 min before 2 vol are added to each aliquot of HS/DNA template. Mixing is carried out by flicking the tube or by gentle pipeting (see Notes 6 and 7).

5. The reaction is transferred to room temperature (22-23°C) and immediately subdivided into several 2.5-|L aliquots, each of which is stopped at different time-points. To stop the reactions, add 5 |L replication stop buffer in 20- to 30-min intervals and vortex briefly to mix.

6. Once all reactions have been stopped, add 1 |L 20 mg/mL proteinase K to each reaction and vortex briefly to mix. Incubate for 30 min at 37°C.

7. If sperm DNA is the template, vortex each reaction vigorously for 30 s to reduce the viscosity of the DNA. Then, load each reaction on a 0.8% agarose gel in 1X TBE. Electrophoresis is carried out at 25 V/cm, until the bromophenol dye migrates at least 2 cm. If the DNA template is plasmid, vortexing is not necessary, and electrophoresis is performed for 6 cm.

8. Cut the gel immediately above the dye front and discard the bottom part of the gel. Reduce the water content of the gel by placing, on both sides of the gel, a single sheet of Whatman paper and a stack of approx 10 paper towels (in the case of plasmid DNA, place a piece of diethylaminoethyl [DEAE] paper between the gel and the Whatman paper). Place a weight on top of the stack for approx 30 min. Remove the paper towels and Whatman paper (but not the DEAE paper) and place the gel on a fresh piece of Whatman paper, transfer to a preheated gel dryer (with the gel facing up), and cover with Saran Wrap. Dry at 80°C under vacuum.

9. The amount of input DNA replicated is calculated by determining the fraction of radioactive dATP that was consumed (1). To this end, spot 1 |L of the final replication reaction on the filter paper supporting the dried gel and air dry. Expose the gel to a PhosphorImager. Comparing the amount of radioactivity in the replicated DNA with the amount of radioactivity in the replication reaction and assuming 50 |M endogenous dATP concentrations for HSE and NPE, the picomoles of dATP consumed in the reaction can be calculated. An Excel spreadsheet that calculates replication efficiency is available on request.

3.7. Using DNA Structure to Monitor Replication Initiation Events: Origin Unwinding

An important advantage of the soluble DNA replication system is that it supports extremely efficient DNA replication of small circular plasmids, which have a structure that can be used to monitor different steps of DNA replication. For example, origin unwinding can be detected as a substantial increase in negative supercoiling of plasmid DNA (6). Although the assembly of nucleosomes onto plasmids in HSE causes the plasmids to become underwound and therefore negatively supercoiled on protein extraction (14), origin unwinding on addition of NPE leads to further underwinding of the template.

This underwinding is dramatically enhanced in the presence of aphidicolin, an inhibitor of replicative DNA polymerases. Aphidicolin appears to uncouple the activity of the helicase that unwinds the DNA from the replication fork, leading to extensive negative supercoiling. Because plasmid incubated in HSE is already underwound because of the presence of nucleosomes, the further increase in underwinding that results from initiation of DNA replication can only be detected when the extracted DNA is separated on a gel containing an appropriate concentration of chloroquine, an intercalating agent.

The structure of the plasmid can be used to observe other events in DNA replication. Most notably, when DNA replication is allowed to proceed in the absence of aphidicolin, several low-mobility forms of the plasmid are observed that persist after DNA replication has ceased. These likely represent catenated daughter molecules that are only later resolved, presumably by topoisomerase II. In this subheading, we describe the origin unwinding assay.

1. Follow steps 1-4 in the DNA replication protocol in Subheading 3.6., except that a 3- to 4-kb circular plasmid should be used as the DNA template at a concentration of 40 ng/| L in the HSE. Also, the NPE is supplemented with 50 |lg/mL aphidicolin.

2. Immediately before, and at different times after, NPE addition, aliquots containing 40 ng DNA (1 |L before NPE addition, 3 |L after NPE addition) are mixed with 100 |L unwinding stop solution. A potent preparation of NPE will completely unwind all added plasmid within 10 min.

3. After all time-points are collected, add 2 | L of 20 mg/mL proteinase K to each time-point and incubate for 30 min at 37°C.

4. Extract each sample with 100 |L of 1/1 phenol/chloroform. Do not vortex the samples. Mix gently by repeated inversion. Centrifuge at top speed in a microcentrifuge for 1 min.

5. Remove 80 |L of the aqueous layer to a new 1.5-mL tube. Precipitate the samples by adding 1 |L 20 mg/mL glycogen, 9 |L 3 M NaOAc, and 230 |L 100% EtOH. Mix well by inversion.

6. Chill on ice for 15 min. Spin at top speed in a microcentrifuge for 15 min. Aspirate the supernatant and allow the pellet to air dry for at least 5 min at room temperature.

7. Resuspend the pellet in 20 |L 1X TBE loading dye supplemented with 100 ng/mL ribo-nuclease A and incubate for at least 10 min at room temperature.

8. Load one-half of the sample on a freshly poured 0.8% TBE agarose gel supplemented with 1.8 |M chloroquine. Also, add fresh chloroquine to the TBE running buffer to a final concentration of 1.8 |M. Load 25 ng of a 1-kb DNA ladder as a marker.

9. Perform electrophoresis at 10 V/cm until the dye has migrated 9 to 10 cm. Stain the gel for 60 min in 100 mL Sybergold nucleic acid stain diluted 1/10,000 in water, keeping protected from light. Photograph using a yellow filter if available.

3.8. Chromatin Loading of Replication Factors in NPE

Most of the key events in DNA replication result in the loading or unloading of DNA replication proteins onto and off of chromatin. To monitor these events, chroma-tin that has been incubated in HSE or HSE followed by NPE can be isolated by cen-trifugation through a sucrose cushion and analyzed by immunoblotting. In HSE, ORC loading occurs within seconds, Cdc6 and Cdt1 loading occurs within 3 to 5 min, and maximal MCM2-7 loading requires 15 to 20 min. On NPE addition, Cdc7 and MCM10 load onto chromatin within 1 min, and Cdc45, RPA, and DNA polymerase-a load within 5 to 15 min. Some proteins, such as RPA, are sometimes isolated non-specifically during the chromatin isolation step after NPE addition. It is therefore important to include control reactions that lack sperm chromatin.

1. Repeat steps 1 to 4 in the DNA replication protocol.

2. Transfer the reaction to room temperature and subdivide into several 6-| L aliquots.

3. Prepare as many sucrose cushions as the number of aliquots. To do this, add 180 |L 1X ELB salts containing 0.5 M sucrose to 5 x 44 mm microfuge tubes (cat. no. 342867; Beckman) and place on ice.

4. At the desired time, supplement an aliquot with 60 |L cold ELB containing 0.2% Triton X-100 (but lacking cycloheximide or DTT), mix by pipeting up and down three times, and place on ice. Carefully overlay the entire volume (~70 |L) onto a sucrose cushion. Centrifuge for 25 s at 12,000 rpm (16,000g) in a Prima 18R horizontal centrifuge at 4°C.

5. Aspirate the supernatant with a narrow gel-loading tip, leaving behind about 3 | L. Do not touch the bottom of the tube with the gel-loading tip.

6. Add 200 | L cold ELB (lacking cycloheximide or DTT), but do not mix, and centrifuge as in step 4.

7. Aspirate the supernatant, this time leaving behind about 1 | L. Again, do not to touch the bottom of the tube with the gel-loading tip.

8. Add 12 |L 1X SDS sample buffer, vortex gently, boil for 2 min, collect the condensate with brief centrifugation, vortex gently again, and load the entire sample onto SDS-PAGE (polyacrylamide gel electrophoresis).

9. Perform Western blotting using antibodies against factors of interest.

3.9. Immunodepletion From HSE or NPE

An important tool in the study of DNA replication in Xenopus egg extracts is the removal of specific proteins using immunodepletion so that their function in DNA replication can be assessed. This subheading describes immunodepletion of HSE and NPE using crude antisera or affinity-purified antibody. For some factors, a single round of immunodepletion is sufficient to remove the protein, whereas in many cases, two to three rounds are needed. The number of rounds required must be determined empirically. Because of the high concentration of replication factors present in NPE, sometimes greater than 99% of a protein must be removed to observe a defect in DNA

replication. Unlike HSE, NPE is very sensitive to dilution, and it can become inactivated over time. Therefore, it is important first to determine the minimum number of depletions required to remove the factor of interest effectively. Typically, NPE is not depleted for longer than a total of 8 h, although some extract preparations are robust enough to survive 16 h of immunodepletion.

The protocol described next is for depletion from 40 ||L HSE or NPE, but all vol in the protocol can be scaled up or down proportionally. The smallest volume of extract that can be practically depleted using this protocol is 20 |L. It is important to perform a "mock" depletion using preimmune serum or an unrelated affinity-purified antibody. In addition, any defects in DNA replication should be reversible by adding back the depleted protein from a heterologous source.

1. Transfer 8 |L packed bed volume of Protein A Sepharose Fast Flow beads (cat. no. 17-127901; Amersham) to a siliconized 0.65-mL microfuge tube (cat. no. 3206; Costar). This is the volume of beads required for a single round of depletion of 40 |L of extract. If multiple rounds of immunodepletion are required, multiply 8 |L of beads by the number of rounds to be performed (see step 4). Wash the beads three times with 10 vol of ELB (lacking cycloheximide and DTT). For each wash, centrifuge for 40 s at 5000 rpm (2800g) in a horizontal centrifuge at 4°C to pellet the beads. Aspirate the supernatant with a 27-gage needle attached to a vacuum source.

2. Add 3 vol of crude antiserum to the beads and mix well. If using affinity-purified antibody, add roughly 3 to 6 |g of antibody in 3 to 5 vol of buffer. Incubate for 30 min at 4°C. Mix on a rotating wheel to prevent the beads from settling.

3. Wash the beads five times as in step 1. After the last wash, aspirate as much of the supernatant and void volume as possible by sticking the 27-gage needle directly into the settled Sepharose. The Sepharose will become opaque. If multiple rounds of immunodepletion will be performed, subdivide the antibody-coupled beads into multiple 8-|L aliquots before the final aspiration. Keep on ice until use.

4. Rapidly thaw HSE or NPE and transfer to ice. Supplement HSE with nocodozole (3.3 |g/mL final concentration; this obviates the need to add nocodozole during replication). Add 40 |L extract to the tube containing 8 |L of aspirated Sepharose and mix well. Incubate at 4°C on a rotating wheel. Care should be taken to ensure that the extract remains at the bottom of the tube, rather than coating the entire inside of the tube, because this will lead to loss and inactivation of extract. A single round of depletion is performed for 1 to 2 h. If the factor of interest is not completely depleted after a single round, transfer the supernatant from the first aliquot of Sepharose to a new aliquot of aspirated Sepharose (step 1) using the spin filter technique described in Note 8 and incubate at 4°C for an additional 2 h. Repeat if necessary.

5. After the final round of incubation, collect the supernatant in a new tube using the spin filter technique.

6. NPE is frequently inactivated because of oxidation during the immunodepletion procedure. Therefore, after immunodepletion, NPE is supplemented with 20 mM DTT.

7. The degree to which the factor was immunodepleted should be assessed for every experiment. This is done by analyzing 1 |L of immunodepleted extract alongside a dilution series of mock-depleted extract using Western blotting with antibodies against the factor of interest.

4. Notes

1. If the interface of the sperm and the 2.3 M sucrose layer are not thoroughly stirred prior to centrifugation, a significant amount of sperm may not sediment to the bottom. If after the first centrifugation you see a cloudy layer above the red blood cell layer, remix the cloudy layer and centrifuge for another 20 min.

2. Sperm settles quickly. Thoroughly resuspend sperm by pipeting 10 times before and about once every minute when aliquoting to maintain accurate concentration.

3. Healthy eggs have a uniformly light-to-dark-brown animal hemisphere with a germinal vesicle centered in the middle, appearing as a white spot. Batches of eggs in which the majority of eggs lack a germinal vesicle or with animal hemispheres that are extensively variegated or mottled in pigment should be discarded. It is normal for some eggs to lyse, resulting in an empty sphere, mostly white in appearance. Any batch of eggs in which more than 10% of the eggs have lysed should be discarded.

4. The first needle is used to poke a hole in the side of the tube. This needle cannot be used to withdraw the cytoplasm because it becomes clogged with the plug of polypropylene removed from the tube. Therefore, the second needle, attached to a syringe, is inserted in the hole to withdraw the cytoplasm. To minimize the amount of cytoplasm that flows out of the hole on removal of the first needle, seal the top of the tube with parafilm, which will prevent significant loss of cytoplasm by vacuum. Before withdrawing the cytoplas-mic fraction from the crushed eggs, be sure to remove the parafilm or the vacuum created by the syringe will perturb the layers. Avoid withdrawing from the mitochondrial layer immediately above the cytoplasmic layer and from the pigment layer below. Keep the beveled side of the needle tip facing up and centered in the tube. Withdraw slowly to minimize the amount of turbulence generated while withdrawing. Continue withdrawing until the cytoplasmic layer is less than 2 mm thick.

5. If the nuclear layer is too thin to harvest, check the following: first, verify that the average nucleus really grew to the desired size. Second, double check the concentration of the sperm preparation used. Third, examine the nuclei that are in the layer using Hoechst dye. Extracts sometimes undergo apoptosis, which can cause complete, irreversible destruction of nuclei within minutes.

6. Although HSE has never been observed to support DNA replication of duplex DNA in our hands, a control in which the NPE is supplemented with 50 |g/mL aphidicolin, an inhibitor of DNA polymerase-a can be included to verify that any DNA replication observed is caused by replicative DNA polymerases and not to repair synthesis.

7. Depending on the relative concentration of factors between preparations of NPE, NPE may not need to be used at full concentration to achieve efficient DNA replication. For each preparation of NPE, a titration experiment should be performed. Test NPE at 100, 80, and 60% concentrations (diluted with ELB) but always add 2 vol of NPE to 1 vol of HSE. Using NPE at less than 100% concentration effectively increases the usable size of a given preparation. Further, having a preparation of NPE that can support efficient replication even when diluted will facilitate extensive manipulation of the NPE. For example, if you are performing immunodepletion studies with NPE, the procedure (Subheading 3.9.) usually causes about 10 to 20% dilution of the NPE. Last, having a dilutable NPE preparation will also facilitate efficient replication if the NPE becomes diluted in experiments by addition of recombinant proteins when performing depletion/add-back studies.

8. To remove extract from a mixture with antibody beads with minimum volume loss, we use a homemade spin filter using a 0.65-mL microfuge tube, a P200 pipet tip, and a Nitex mesh membrane (cat. no. 03-20/14; Sefar). To fabricate the filter, cut the bottom 2 cm off of a P200 pipet tip with a clean razor blade and discard the bottom portion. Cut the top portion precisely where the protruding ridge along the side ends (this is where the pipet tip is "seated" in the pipet tip box). You now have a top segment of the pipet tip with the ridges and the narrower tapered segment. Cut a 1.5 x 1.5 cm square of Nitex mesh. Direct the mesh square into the upper segment through the top by using the narrow end of the lower segment. The lower segment will fit snugly into the upper segment and, because of its tapered shape, will fit more tightly the farther in it is pushed. The snug fit between the two segments holds the mesh in place. The whole filter "assembly" can hold up to 100 |L above the mesh membrane and fits inside a 0.65-mL microfuge tube. To recover extract from the Sepharose, pipet the mixture into the filter assembly and centrifuge for 40 s at 5000 rpm (2800g) in a Prima 18R horizontal centrifuge at 4°C. When another round of depletion follows, place the filter inside a new 0.65-mL tube containing a fresh aliquot of aspirated Sepharose. After the final round of depletion, place the filter assembly in a fresh empty tube. Using this approach, approx 5% of the starting volume of extract will be lost for every round of immunodepletion.

References

1 Blow, J. J. and Laskey, R. A. (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47, 577-587.

2 Lohka, M. J. and Masui, Y. (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719-721.

3 Munshi, R. and Leno, G. H. (1998) Replication of nuclei from cycling and quiescent mammalian cells in 6-DMAP-treated Xenopus egg extract. Exp. Cell Res. 240, 321-332.

4 Newport, J. (1987) Nuclear reconstitution in vitro: stages of assembly around protein-free DNA. Cell 48, 205-217.

5 Arias, E. E. and Walter, J. C. (2004) Initiation of DNA replication in Xenopus egg extracts. Front. Biosci. 9, 3025-3049.

6 Walter, J., Sun, L., and Newport, J. (1998) Regulated chromosomal DNA replication in the absence of a nucleus. Mol. Cell 1, 519-529.

7 Prokhorova, T. A., Mowrer, K., Gilbert, C. H., and Walter, J. C. (2003) DNA replication of mitotic chromatin in Xenopus egg extracts. Proc. Natl. Acad. Sci. USA 100, 13,241-13,246.

8. Walter, J. C. (2000) Evidence for sequential action of cdc7 and cdk2 protein kinases during initiation of DNA replication in Xenopus egg extracts. J. Biol. Chem. 275, 39,773-39,778.

9. Wohlschlegel, J. A., Dhar, S. K., Prokhorova, T. A., Dutta, A., and Walter, J. C. (2002) Xenopus mcm10 binds to origins of DNA replication after mcm2-7 and stimulates origin binding of cdc45. Mol. Cell 9, 233-240.

10 Hodgson, B., Li, A., Tada, S., and Blow, J. J. (2002) Geminin becomes activated as an inhibitor of Cdt1/RLF-B following nuclear import. Curr. Biol. 12, 678-683.

11 Lin, X. H., Walter, J., Scheidtmann, K., Ohst, K., Newport, J. and Walter, G. (1998) Protein phosphatase 2A is required for the initiation of chromosomal DNA replication. Proc. Natl. Acad. Sci. USA 95, 14,693-14,698.

12 Stokes, M. P., Van Hatten, R., Lindsay, H. D., and Michael, W. M. (2002) DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts. J. Cell Biol. 158, 863-872.

13 Walter, J. and Newport, J. (2000) Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase a. Mol. Cell 5, 617-627.

14 Almouzni, G. and Wolffe, A. P. (1993) Nuclear assembly, structure, and function: the use of Xenopus in vitro systems. Exp. Cell Res. 205, 1-15.

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