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1 Department of Biochemistry and Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan
2 Nara Institute of Science and Technology, Ikoma, Nara, Japan
3 Department of Biology, Graduate School of Science, Osaka University, Osaka, Japan
4 Laboratories for Biomolecular Network, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan
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Abstract |
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and 
(Pol and Pol) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication in Xenopus egg extracts in which Pol or Pol was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Pol depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and single-stranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Pol is essential for lagging strand synthesis and that this function of Pol cannot be substituted for by Pol. |
Introduction |
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, and (Pol, Pol and Pol) are thought to be the major replicative DNA polymerases (reviewed in Waga & Stillman 1998; Kawasaki & Sugino 2001; Hübscher et al. 2002). Pol is tightly associated with primase, and so it appears to function in the initiation of DNA synthesis of both the leading strand and Okazaki fragments. However, Pol/primase is only able to synthesize a short RNA-DNA primer, which then has to be extended by other processive DNA polymerases.
Previous studies of Simian Virus 40 (SV40) DNA replication have shown that Pol is responsible for the extension of both leading and lagging strands (reviewed in Waga & Stillman 1998; Hübscher et al. 2002). This mechanism involves polymerase switching from Pol to Pol, which is believed to take place during the synthesis of each Okazaki fragment. The proliferating cell nuclear antigen (PCNA) and replication factor C (RFC), which function as a sliding clamp and a clamp loader, respectively, are required to accomplish polymerase switching. RFC catalyses the loading of PCNA on to a primer-template junction in an ATP-dependent manner, and PCNA is required for processive DNA synthesis by Pol (reviewed in Waga & Stillman 1998; Hübscher et al. 2002).
Pol is another highly processive DNA polymerase (reviewed in Kawasaki & Sugino 2001). It has been suggested that yeast Pol is essential for cell viability and is required for DNA replication (Morrison et al. 1990; Araki et al. 1992). However, the polymerase activity of Pol in yeast cells is not required for cell viability (Kesti et al. 1999; Dua et al. 1999; Feng & D’Urso 2001), suggesting that, at least in yeast cells, the loss of Pol activity can be compensated for by another polymerase, which is likely to be Pol. On the other hand, recent analyses of yeast mutant cells lacking the polymerase activity of Pol have shown these cells to display a severe defect in S phase progression (Feng & D’Urso 2001; Ohya et al. 2002), suggesting that the polymerase activity of Pol is necessary for the proper control of S phase in yeast cells. Furthermore, our study of Xenopus Pol has shown that it is required for efficient DNA replication and that DNA replication is significantly impaired without Pol (Waga et al. 2001). Previous analyses also support the involvement of mammalian Pol in cellular DNA replication (Zlotkin et al. 1996). Notably, Pol and Pol, but not Pol, have been shown to be required for lagging strand synthesis coupled with de novo telomerase-dependent telomere addition in yeast cells (Diede & Gottschling 1999).
In addition, a previous analysis of human Pol has suggested a possible role of Pol in regions where specific chromatin structure is formed (Fuss & Linn 2002). Furthermore, previous mutational analyses of yeast suggest differential involvement of Pol and Pol in the synthesis of lagging and leading strands (for example, see Morrison & Sugino 1994; Shcherbakova & Pavlov 1996).
Pol has been shown to interact with the Dpb11 protein that is involved in the initiation of DNA replication and cell cycle control in yeast cells (Masumoto et al. 2000). Xenopus Cut5, a Xenopus homolog of Dpb11, is also required for the initiation of DNA replication in egg extracts (Hashimoto & Takisawa 2003). TopBP1, a human homolog of Dpb11, also interacts with Pol and is required for DNA replication (Mäkiniemi et al. 2001). Recently, Xenopus Gins, which is a homolog of yeast Gins that specifically interacts with Dpb11, has been shown to be required for the initiation of DNA replication, and it has been suggested that Gins may function along with Pol (Kubota et al. 2003; Takayama et al. 2003). Furthermore, a yeast Pol2–16p protein that lacks the catalytic domain of Pol binds to the origin region during initiation (Ohya et al. 2002), suggesting an essential role for Pol during initiation. Therefore, it is thought that Pol not only participates in replication of the whole chromosomal DNA region, but also in the initiation of DNA replication and cell cycle control.
Despite a number of lines of evidence that suggest both Pol and Pol are required for DNA replication, little is known about the precise roles of these polymerases in DNA synthesis. Here, we have examined the roles of Pol and Pol in DNA replication by comparative analyses of DNA replication in Xenopus egg extracts in which either Pol or Pol was immunodepleted. Both depletions caused severe defects in elongation of nascent DNA. More importantly, Pol depletion specifically led to various replication defects that probably resulted from deficient synthesis of lagging strands. Therefore, Pol and Pol participate in strand elongation during DNA replication in a distinct manner, and the probable roles of these polymerases in elongation are discussed.
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Results |
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-depleted egg extracts
DNA replication in Xenopus egg extracts was significantly impaired when Pol was immunodepleted (Waga et al. 2001). These results suggest that the remaining Pol did not substitute for the function of Pol. Here, we have conversely depleted Pol from the egg extracts to determine if Pol could substitute for the function of Pol.
Antibodies to the p125 and p66 subunits of Xenopus Pol were prepared and used for immunodepletion of Pol. Using this approach, more than 99% of Pol in the egg extracts was removed (data not shown, see also Fig. 2A). When a DNA replication assay was carried out using demembranated Xenopus sperm nuclei, the amount of DNA synthesis was markedly decreased in Pol-depleted extracts, compared to that in mock-depleted extracts (Fig. 1A). Importantly, the level of DNA synthesis in Pol-depleted extracts did not reach the level seen in mock-depleted extracts even after a 100-min incubation (Fig. 1A).
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-depleted extracts is due to the lack of Pol, we partially purified Pol from the egg extracts and added it back into the depleted extracts. DNA polymerase activity in the Pol fractions was barely detected without PCNA (Fig. 1B), implying little contamination of DNA polymerases other than Pol in these fractions. The p125, p66 and p55 subunits of Pol were apparent in these Pol fractions (Fig. 1C,D) (the fourth subunit of Xenopus Pol remains to be identified). DNA replication was restored to a level that was 80% that of the control by adding the purified Pol fractions (Fig. 1E,F). DNA replication was restored in a dose-dependent manner and about 80% restoration was attained by adding about 30% of the amount of Pol present in the extracts before immunodepletion (data not shown). More importantly, the extent of restoration was correlated with the amount of Pol added, except for a discrepancy seen when fraction 20 was tested (Fig. 1B–F). In this particular fraction, Pol was clearly detected by immunoblotting and a significant level of restoration of DNA replication was also observed when this fraction was added, but DNA polymerase activity was barely detected in the reaction using a synthetic primer-template (Fig. 1B, see Discussion). Although a number of additional polypeptides were seen in these Pol fractions, most of them were not immunoprecipitated with anti-Pol antibodies (Fig. 1D). Taken together, these results strongly argue that the defects seen in Pol-depleted extracts are indeed due to the lack of Pol. Therefore, Pol is also required for efficient DNA replication in Xenopus egg extracts.
DNA is minimally replicated without Pol and Pol
Although DNA replication was significantly impaired in the Pol- and Pol-depleted extracts, residual, but still significant, amounts of DNA synthesis were seen in both extracts (Fig. 1A, and Waga et al. 2001). To confirm that the residual DNA synthesis is carried out by other replicative DNA polymerases, we immunodepleted both Pol and Pol simultaneously in the same extracts.
DNA synthesis did indeed further decrease in the doubly depleted extracts, compared to that in each of the singly depleted extracts (Fig. 1G). This result is consistent with the possibility that the residual DNA synthesis occurring in either the Pol- or Pol-depleted extracts is carried out by other replicative DNA polymerases. While DNA synthesis decreased to an extremely low level in the doubly depleted extracts, MCM6, CDC45, Pol and RPA were loaded on to chromatin, and chromatin binding of RPA actually increased in the doubly depleted extracts (Fig. 1H), suggesting that both formation and activation of the prereplicative complex (pre-RC) (reviewed in Bell & Dutta, 2002) probably occurred at the replication origins in the doubly depleted extracts.
The size of replication products is different in Pol- and Pol-depleted extracts
To explore the difference, if any, in the roles of Pol and Pol in elongation, we compared the reactions in Pol- and Pol-depleted extracts. As far as we could determine, the amounts of replication proteins, other than the depleted polymerases, were not reduced in either of the depleted extracts (Fig. 2A), indicating that both depletions were highly specific. Furthermore, chromatin binding of either depleted polymerase was barely seen in the respective depleted extracts (data not shown), indicating that both depletions were indeed almost complete.
When the replication products were analysed by alkaline agarose gel electrophoresis, we found that short DNA products had accumulated in both depleted extracts. More importantly, the size distribution of the short products was remarkably different between the Pol- and Pol-depleted extracts (Fig. 2B). The short products in Pol-depleted extracts were heterogeneously distributed from about 0.2 kb to > 10 kb (Fig. 2B, lanes 4 and 5). In contrast, the short products seen in Pol-depleted extracts were mainly longer than about 2 kb (Fig. 2B, lanes 8–10).
Because radioactive nucleotides were continuously incorporated during the incubation, the intensity of products on the gel does not show a linear correlation with the number of product molecules. Thus, the estimated number of replication products made in each depleted extract was compared between the extracts (Fig. 2C). This further emphasizes the difference in size distribution of products between Pol- and Pol-depleted extracts; in particular, DNA products of < 0.7 kb significantly accumulated in Pol-depleted extracts.
Pol- or Pol-depletion causes the deficient elongation of nascent strands
The short DNA products described above may represent either accumulation of nascent DNA strands or accumulation of breakdown products of replicated genomic DNA, or both. To distinguish between these possibilities, we carried out the following experiments. Demembranated sperm nuclei were first incubated in Pol- or Pol-depleted extracts supplemented with [-32P]dATP. Each extract was then added to either a different type of depleted extracts, as a source of the missing DNA polymerase, or to the same type of depleted extract, as a control. The mixture was then incubated further with excess dATP, in order to chase the labelled products (Fig. 2D).
When depleted extracts of the same type were added to an already incubated extract, we observed no significant change in the sizes and amounts of DNA products (Fig. 2E, lanes 1–4 and 8–11). Importantly, further addition of the same type of depleted extracts did not lead to a further increase in the amount of short DNA molecules. In contrast, when depleted extracts of different types were combined, the short products made in the Pol- or Pol-depleted extracts were quickly converted to long products; the extension of short products were seen as quickly as 5 min after addition of the other type of depleted extract (lanes 5 and 12, compare to lanes 1 and 8). This indicates that the missing DNA polymerase that was supplied by addition of the second extract can cross the nuclear membrane and participate in elongation. Therefore, it is probable that the short products that accumulated in the Pol- or Pol-depleted extracts are nascent DNA strands that formed during elongation, rather than breakdown products.
Pol depletion causes a more severe elongation defect than Pol depletion
We next examined in more detail how nascent strands might be elongated in Pol- or Pol-depleted extracts. To do this, demembranated sperm nuclei were preincubated in each type of depleted extract in the presence of AraCTP, a dCTP analogue that inhibits elongation but not initiation (Walter & Newport 1997). Following this, excess dCTP was added to release the AraC arrest and start elongation simultaneously at all the fired origins. The nascent strands were analysed by alkaline agarose gel electrophoresis (Fig. 3A,B).
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-depleted extracts, but the elongation rate was significantly slower than in the control (80 and 260 bp/min in the Pol- and mock-depleted extracts, respectively), and the size of the nascent DNA reached only approximately 3 kb in a 30-min incubation (lane 12). This DNA size range is similar to the size of replication products that accumulated in the Pol-depleted extracts, as shown in Fig. 2B, indicating that Pol depletion causes slowing of the overall elongation rate.
In stark contrast, a more severe elongation defect was observed in Pol-depleted extracts (Fig. 3B, lanes 19–24); the majority of short nascent DNA molecules (approximately 0.6 kb) were barely elongated at least 10 min after release (lanes 19–22). While some of these nascent DNA products were elongated in the next 10–20 min (lanes 23 and 24), it was clear that short nascent DNA continued to appear during at least a 30-min incubation (lane 24), implying that short nascent DNA may be continuously synthesized during DNA replication in the Pol-depleted extracts. These results suggest that the remaining DNA polymerases participate in elongation in a manner that is different in the Pol- and Pol- depleted extracts.
It should be noted that both the amounts and sizes of nascent DNA products synthesized during the arrest phase were different between the depleted extracts. At least in part, this is probably due to different specificity of AraCTP inhibition of DNA polymerases; Xenopus Pol may be relatively resistant to AraCTP, compared to Pol, as previously suggested (Jiang et al. 2000).
Chromatin binding of replication proteins that frequently function in lagging strand synthesis is specifically markedly increased in the absence of Pol
To further understand the differential elongation defects in Pol- and Pol-depleted extracts, we analysed the chromatin binding of replication proteins. All the replication proteins examined here, except for ORC2, had gradually dissociated from chromatin at about the time of the completion of DNA synthesis in the mock-treated extracts (Fig. 4A,B, see Fig. 4E,F for ORC2). In contrast, such dissociation was not seen in either Pol- or Pol-depleted extracts, which is indicative of incomplete DNA synthesis (Fig. 4A,B).
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-depleted extracts, compared to those in mock-depleted extracts (Fig. 4A,C). Such accumulation was, however, not seen in Pol-depleted extracts; the levels of chromatin-bound RFC, PCNA and RPA were similar to those in mock-depleted extracts (Fig. 4B,D), and chromatin binding of FEN1 in the Pol- and mock-depleted extracts was barely detectable under the conditions used (Fig. 4C,D). Thus, elevated accumulation of RFC, PCNA, RPA and FEN1 on chromatin occurred specifically with Pol depletion.
Neither Pol nor Pol depletion significantly affected the levels of chromatin binding of the remaining DNA polymerase (Fig. 4A,4B), although chromatin binding of Pol was slightly increased in both depleted extracts (Fig. 4F). Thus, it seems unlikely that the increased chromatin binding of RFC and PCNA in Pol-depleted extracts reflects more vigorous action of the remaining Pol. Rather, RFC and PCNA are likely to remain bound to a primer-template junction. RFC, PCNA, RPA and FEN1 are all thought to function more frequently in lagging strand synthesis (reviewed in Waga & Stillman 1998). Therefore, these results suggest that Pol depletion causes a defect in certain specific processes during lagging strand synthesis.
Initiation seems to occur normally in Pol- or Pol-depleted extracts
As shown in Fig. 4, the levels of chromatin-bound ORC2, MCM3, MCM6 and CDC45 did not significantly change in the Pol- or Pol-depleted extracts, compared to the corresponding mock control. Moreover, the timing of CDC45 binding to chromatin did not seem to change in either depleted extract. These results suggest that neither formation nor activation of the pre-RC is significantly affected by the depletion of Pol or Pol. However, they do not exclude an effect of polymerase depletion on an unknown initiation process occurring after CDC45 binding, if any such process exists.
Single-stranded DNA gaps accumulate during DNA replication in Pol-depleted extracts
The accumulation of chromatin-bound RPA described above implies the accumulation of single-stranded DNA regions. To confirm this, we performed a primer-extension assay using T4 DNA polymerase and Xenopus genomic DNA that was purified after a replication reaction in the depleted extracts (Fig. 5A).
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-depleted extracts was subjected to a primer-extension reaction, a significant amount of nucleotide incorporation was detected, whereas much less incorporation was seen when DNA from Pol- or mock-depleted extracts was used (Fig. 5B), indicating that single-stranded DNA gaps accumulated during the incubation in Pol-depleted extracts to a much greater extent than in Pol- or mock-depleted extracts.
We also noticed that labelled products were heterogeneously distributed from about 0.6 kb to > 10 kb when DNA from Pol-depleted extracts was used (Fig. 5B, lanes 4–6). This suggests that the majority of the short nascent DNA products seen in Fig. 2B served as primers in a primer-extension reaction. Importantly, there seemed to be no significant change in the size distribution of labelled products after the incubation for 60–100 min (lanes 4–6), suggesting that single-stranded DNA gaps continue to accumulate throughout the incubation in Pol-depleted extracts.
We have found that Chk1, which is involved in replication checkpoint control, was slightly phosphorylated in each depleted extracts in an ATM (Ataxia telangiectasia mutated)/ATR (Ataxia- and Rad-rlated)-dependent manner (data not shown). However, the addition of caffeine, an inhibitor of ATM/ATR, to each depleted extracts diminished neither accumulation of short nascent DNA nor elevated chromatin binding of RFC and FEN1 (data not shown). Thus, it is unlikely that the ATM/ATR-dependent checkpoint mechanism is directly related to deficient replication in Pol- and Pol-depleted extracts.
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Discussion |
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and Pol for chromosomal DNA replication
In this study, it was shown that Pol depletion resulted in a marked inhibition of DNA replication, but that the addition of Xenopus Pol fractions purified from egg extracts could restore DNA replication. We observed a significant level of restoration in Pol-depleted extracts by adding a fraction, in which DNA polymerase activity was barely detected (fraction 20, Fig. 1B). This probably occurred because factors that inhibit the DNA polymerase reaction with a synthetic primer template might have been concentrated in this fraction, and these inhibitory factors may have been somehow suppressed in the crude cell extracts.
The data presented in this study, together with our previous study of Xenopus Pol (Waga et al. 2001), strongly argue that both Pol and Pol are required for efficient elongation during DNA replication in Xenopus egg extracts, and this is also likely to be true in somatic cells. Hence, these data provide the first direct evidence that Pol and Pol are the major DNA polymerases involved in chromosomal DNA replication in metazoans.
Pol depletion does not lead to a decrease in the amount of Pol in depleted extracts, and vice versa, as shown in Fig. 2A. Given that most replication proteins exist in large excess in egg extracts, the deficient elongation in both polymerase-deleted extracts suggests that the remaining polymerase cannot compensate for the lack of the depleted polymerase in either Pol- or Pol-depleted extracts. Therefore, Pol and Pol are likely to have distinct functions in elongation in the egg extracts.
The caveat in functional analyses performed by immunodepletion of a particular protein is that the consequences of the depletion might be indirect. In the case of depletion of DNA polymerases, it is possible that the depletion may result in the failure of proper structural assembly of the replication machinery at the replication fork, leading to deficient elongation. However, the relatively quick recovery observed upon adding the missing polymerase back into the extracts, as shown in Fig. 2E, suggests that the structure at the replication forks is not totally disrupted in the absence of Pol or Pol. Nevertheless, in order to confirm this conclusion it will be necessary to perform substitution experiments with mutant polymerases that specifically lack polymerase activity, but still form a replisome.
Roles of Pol in elongation during lagging strand synthesis
Various defects specifically seen in Pol-depleted extracts are consistent with the possibility that Pol is essential for lagging strand synthesis, for the following reasons. Both RFC and PCNA probably remain bound to a primer-template junction in the absence of Pol, as shown by the elevated chromatin binding of RFC and PCNA in Pol-depleted extracts. These interactions at a primer-template junction may result from inefficient polymerase switching from Pol/primase to Pol in the absence of Pol. Moreover, short nascent strands (about 0.6 kb) were continuously detected after release from AraC arrest during the incubation (Fig. 3B), suggesting that short nascent DNA molecules are continuously produced after release. Furthermore, single-stranded DNA gaps also accumulated in the absence of Pol, which is consistent with a marked increase of chromatin-bound RPA. The results of the primer extension assay indicate that each nascent DNA serves as a primer in this assay. Importantly, our data also suggest that single-stranded DNA gaps persist throughout the incubation in Pol-depleted extracts. Therefore, it is fairly reasonable to conclude that single-stranded DNA gaps are generated between adjacent short nascent strands. This means that the defects described above result predominantly from the deficient synthesis of lagging strands, during which short nascent strands might be continuously produced, but remain unligated. Therefore, it seems reasonable to conclude that Pol has an essential function in the completion of lagging strand synthesis during chromosomal DNA replication in metazoans.
We also observed an elevated level of chromatin-bound FEN1 specifically in the absence of Pol. FEN1 has roles in both processing of Okazaki fragments and DNA repair/recombination (reviewed in Lieber 1997; Waga & Stillman 1998), and so its elevated chromatin binding results from either deficient synthesis of lagging strands or increased assembly of the repair/recombination machinery, or both. In the former case, FEN1 might remain bound to either PCNA or to the 5'-end of an Okazaki fragment, as suggested previously (reviewed in Waga & Stillman 1998; also see Tom et al. 2000; Bae et al. 2001). Since the processing of Okazaki fragments is thought to be performed in a DNA synthesis-coupled manner (Hosfield et al. 1998; Bae et al. 2001; Maga et al. 2001; Jin et al. 2003), it is most likely that FEN1 cannot efficiently function in processing Okazaki fragments without efficient DNA synthesis, due to the lack of Pol.
The sustained presence of single-stranded gaps on the template might trigger the assembly of the repair/recombination machinery. Related to this, we observed an increased level of chromatin-bound WRN helicase (FFA-1 in Xenopus) (Chen et al. 2001) specifically in Pol-depleted extracts (Noriko Sasakawa et al. unpublished observation). Although FEN1 interacts with WRN helicase (Brosh et al. 2001), an increased level of chromatin-bound FEN1 was also observed in extracts depleted simultaneously of both Pol and FFA-1 (data not shown). Thus, the increase in chromatin binding of FEN1 is unlikely to be mediated by the FEN1–WRN interaction.
Mechanism of elongation of nascent strands by Pol and Pol
Previous analyses of SV40 DNA replication indicate that Pol along with Pol/primase is sufficient for DNA synthesis of both lagging and leading strands (reviewed in Waga & Stillman 1998). Thus, it is possible that Pol also synthesizes leading strands during cellular DNA replication. Furthermore, the existence of short nascent strands for a relatively long time (at least 5 min) without obvious elongation in the absence of Pol (Fig. 3) also implies inefficient elongation of the leading strand, as well as of the lagging strand.
Previous analyses of yeast Pol using the chromatin immunoprecipitation assay indicate that Pol binds to the origin region during initiation and remains associated with the replication fork during elongation (reviewed in Bell & Dutta 2002). The question, then, is what is the role of Pol in elongation of nascent strands? Our analysis has shown that the overall elongation rate in Pol-depleted extracts is clearly slower than that in the control. Although single-stranded DNA gaps appear to accumulate to a small extent in Pol-depleted extracts, the levels of chromatin-bound RPA in Pol-depleted extracts did not significantly change, compared to those in mock-depleted extracts. These results imply that both leading and lagging strands are somehow synthesized in the absence of Pol, albeit at a slower rate. Thus, we speculate that Pol may be involved in elongation by affecting the preceding DNA unwinding. Furthermore, the increase in the levels of chromatin-bound Pol in Pol-depleted extracts may suggest more frequent involvement of Pol in DNA synthesis without Pol. Thus, Pol might be required for proper control of Pol during initiation and/or elongation. In addition, it cannot be formally excluded that Xenopus Pol may also be directly involved in the initiation step.
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Xenopus EST clones (PBX0151F04, PBX0120A10) encoding the p66 subunit of Pol and the p37 subunit of RFC, respectively, were obtained, and both strands of the cDNA inserts were sequenced (accession numbers AB080267 and AB080268, respectively). The cDNA of Xenopus Pol p50 was obtained from the EST clone (PBX0151F04) and its amino acid sequence was identical to that reported previously (Reynolds & MacNeill 1999). The cDNA of Xenopus PCNA was cloned by PCR and found to encode the amino acid sequence in which one amino acid is different (245E to G) from that in the database (M34080).
Antibodies
The rabbit anti-Xenopus Pol p66, Pol p50, Pol p70, and RFC p37 antibodies were raised against bacterially expressed, 10 histidine-tagged recombinant proteins. The Pol p66-specific antibodies were affinity-purified using the antigen-immobilized Affi-Gel 10 (Bio-Rad). The rabbit anti-Xenopus Pol p180 antibody was raised against glutathione S-transferase-fused, amino-terminal polypeptide (from amino acid 1–547) (Mimura & Takisawa 1998). The rabbit anti-Xenopus Pol p125 antibody was raised against the synthetic peptide (SSQTKKLRGDWDDD) corresponding to the N-terminal region of Pol p125 (K. Yamauchi, M. Akiyama and H. Maki, unpublished observation). The rabbit anti-Xenopus Pol p58 and Pol p49 (primase subunits) antibodies were raised against bacterially expressed recombinant proteins (T. Fukui, A. sugino and S. Waga, unpublished). The rabbit anti-Xenopus Pol p260 antibody was raised against the polypeptide corresponding to the N-terminal region (amino acids 1–674) of Pol p260 (K. Shikata, S. Waga and A. Sugino, unpublished observation). The rabbit anti-Xenopus RFC p140 antibody was raised against the bacterially expressed polypeptide encoded by a Xenopus EST clone AW199747
[GenBank]
, which encodes the sequence homologous to the region from amino acid 362–484 of human RFC p140. The rabbit anti-Xenopus Pol p60 and CDC7 antibodies are described elsewhere (Waga et al. 2001; Furukohri et al. 2003). The rabbit anti-Xenopus MCM6, MCM3, CDC45 and RPA p70 antibodies were generous gifts from Dr H. Takisawa (Osaka University, Japan), the antibodies against PCNA and human FEN1 were from Dr B. Stillman (Cold Spring Harbour Laboratory), the rabbit anti-Xenopus CDC6 antibody was from Dr J. Walter (Harvard Medical School) and the rabbit anti-Xenopus ORC2 antibody was from Dr Y. Kawasaki (Osaka University, Japan).
Egg extracts and DNA replication assay
Preparation of Xenopus egg extracts and DNA replication with demembranated Xenopus sperm nuclei (2000 nuclei/ µl of extracts) were carried out as previously described (Waga et al. 2001). Replication products were analysed by neutral or alkaline agarose gel electrophoresis previously described (Waga et al. 2001). The elongation rate was calculated from the average size of nascent strands.
Analysis of chromatin-bound proteins
Demembranated Xenopus sperm nuclei were incubated in egg extracts (4000/µl of extracts) under the same conditions as DNA replication assay. Analysis of chromatin-bound proteins by immunoblotting were performed as described elsewhere (Furukohri et al. 2003).
Primer extension assay
DNA replication was carried out in egg extracts with demembranated sperm nuclei (4000 nuclei/µl of extracts). After incubation, an aliquot (10 µl) of the reaction mixture was withdrawn, chromatin was isolated as above, and the genomic DNA was purified. The purified DNA was then incubated with 12 units of T4 DNA polymerase (TAKARA) and [-32P]dATP in a buffer containing 33 mM Tris-HCl, pH 7.9, 10 mM MgCl2, 6.6 mM potassium acetate, 0.5 mM dithiothreitol (DTT), 0.01% BSA and 200 µM each of dNTP for 15 min at 30 °C. The labelled products were analysed by alkaline agarose gel electrophoresis.
Immunodepletion
The antibody to Pol p66, Pol p125 or Pol p60 and whole rabbit IgG (Pierce) as a control were bound to Dynabeads-Protein A (DYNAL) (0.25–1 µg of IgG per µl of bead suspension). For immunodepletion of Pol, egg extracts were incubated sequentially with the Pol p66 antibody-beads (2 times, 15 min each) and the Pol p125 antibody-beads (2 times, 15 min each). For immunodepletion of Pol, the extracts were treated 4 times with the Pol p60 antibody-beads. For immunodepletion of both Pol and Pol simultaneously, the extracts were treated 2 times with the mixture of the Pol p66 antibody- and the Pol p60 antibody-beads and then 2 times with the mixture of the Pol p125 antibody- and the Pol p60 antibody-beads.
Purification of Xenopus Pol
Xenopus Pol was purified as follows (Shikata et al. 2001). Purified recombinant Xenopus PCNA (T. Muroya, M. Akiyama and H. Maki, unpublished observation) was crosslinked to Affigel 15 (Bio-Rad) (7.5 mg PCNA/ml gel). Egg extracts were spun at 220 000 g for 90 min at 4 °C and the supernatant (30 ml) was applied on 5 ml of BSA (4 mg)-crosslinked Affigel 15 that had been equilibrated with 0.1 M NaCl, 1 mM EDTA, 0.01% NP40, buffer A (25 mM Tris-HCl [pH 7.7], 1 mM DTT, 1 mM PMSF, 10% glycerol). The flow through fractions were applied on 10 ml of the PCNA-affinity column, and the column was washed sequentially with 20 ml of 0.1 M NaCl, 1 mM EDTA, 0.01% NP40, buffer A and 3 ml of 0.1 M NaCl, buffer A. Pol was then eluted with 0.3 M NaCl, buffer A. The Pol fraction was diluted 3-fold with 1 mM EDTA, 0.005% NP40, buffer A and loaded on a MonoQ column (1 ml) (Amersham Pharmacia), and Pol was eluted with a linear gradient of 0.1–0.7 M NaCl in 1 mM EDTA, 0.005% NP40, buffer A.
The polymerase activity of Pol was assayed using oligo dT/poly dA as a primer-template for 15 min at 30 °C in a buffer containing 50 mM bisTris-HCl, pH 7.9, 10 mM KCl, 6 mM MgCl2, 50 µM TTP, 1 mM DTT, and 0.4 mg/ml BSA in the presence or absence of 10 µg/ml Xenopus PCNA.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: swaga{at}biken.osaka-u.ac.jp
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References |
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Received: 6 November 2003
Accepted: 16 December 2003
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