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Differences in DNA synthesis in vitro using isolated nuclei from regenerating livers of young and aged rats
Mechanisms of Ageing and Development 122 (2001) 141 – 155 www.elsevier.com/locate/mechagedev
Differences in DNA synthesis in vitro using isolated nuclei from regenerating livers of young and aged rats Takahiko Taguchi *, Mitsugu Fukuda, Mochihiko Ohashi Department of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakae-cho, Itabashi-ku, Tokyo 173 -0015, Japan Received 26 January 2000; received in revised form 4 June 2000; accepted 5 October 2000
Abstract To detect changes in DNA synthesis during ageing, we compare DNA synthesis in the livers of young and aged rats. As an intermediate between an in vivo system using intact cells and an in vitro system using purified DNA polymerases, isolated nuclei were prepared and used as the machinery for DNA synthesis. The DNA synthesizing capacity of nuclei from regenerating liver was higher than that of nuclei from normal liver and these capacities from liver and regenerating liver were lower in nuclear preparations from aged rats. DNA synthesis using isolated nuclei was stimulated by ATP and the cytoplasmic preparation. The cytoplasmic preparation from regenerating rat liver was found to stimulate DNA synthesis more than the preparation from normal liver. The activity in regenerating liver from young rats was also greater than in that from aged rats. It is well known that DNA replication is inhibited by aphidicolin and DNA repair by ddTTP. We examined the effects of aphidicolin and ddTTP on DNA synthesis using the nuclear system. Surprisingly, the inhibition by aphidicolin was 30% of total DNA synthesis using the nuclear system from young rats. On the other hand, the inhibition by ddTTP was : 80%. We measured the sizes of the DNA synthesized in the presence of both inhibitors. DNA synthesis was allowed to proceed for 10 min using isolated nuclei from regenerating liver of young rats and the size of the DNA was determined by sucrose density gradient centrifugation analysis. DNA products appeared in two fractions. Following a chase of 50 min in the presence or absence of aphidicolin, the short DNA product grew larger in both cases, although the amount of DNA in the presence of aphidicolin was :90% that in its absence. In the same experiment using nuclei from aged
* Corresponding author. Tel.: +81-3-39643241, ext. 3060; fax: +81-3-35794776. E-mail address: [email protected] (T. Taguchi). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 2 6 - 8
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rats, the amount in the presence of aphidicolin was : 60% that in its absence. These results suggest that DNA polymerase b is closely related to abnormal replication when DNA polymerases a and d are inhibited and that the effect of cytosol on DNA synthesis, as well as the DNA synthetic capacity of isolated nuclei, becomes lower in regenerating rat liver during ageing. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Regenerating liver; DNA polymerases; Replication and repair; Aphidicolin; ddTTP; Ageing
1. Introduction Eukaryotic DNA polymerases comprise a, b, g, d and o types. We have reported that aphidicolin is a specific inhibitor of DNA polymerase a (Ohashi et al., 1978) and it has been shown that this inhibitor is also effective against DNA polymerases d and o (Wang, 1991). Three enzymes — DNA polymerase a, d and o — have been shown to be involved in DNA replication (Weissbach, 1975; Focher et al., 1988; Wang, 1991; Linn, 1991). DNA polymerases b and g are inhibited by dideoxynucleoside 5%-triphosphate (ddNTP) (Waqar et al., 1978). DNA polymerase g is present only in mitochondria, where it is involved in mitochondrial DNA synthesis (Weissbach, 1975). DNA polymerase b is involved in base excision repair by filling the gaps formed by excision (Mosbaugh and Linn, 1983 Wang, 1991). Various systems have been used to study DNA replication in eukaryotes (Morioka et al., 1973 Seki and Oda, 1977 Ikeda et al., 1980 Wobbe et al., 1985). Isolated nuclei form one good system in which to clarify DNA replication and repair. Within cells, the nuclei contain the most machinery for DNA replication and repair. However, DNA synthesis using isolated hepatic nuclei (Kaufmann et al., 1982) and bleomycin-pretreated permeable cells (Seki et al., 1987) is stimulated by ATP. Moreover, the presence of a cytoplasmic factor that stimulates nuclear DNA synthesis has been reported (Jazwinski et al., 1976 Wong et al., 1987). Thus, some factors that are not contained in nuclei are necessary for DNA replication. On the other hand, it has been reported that the rate of DNA synthesis declines in aged animals. As the causes for this decrease, lower DNA polymerase a activity (Fry et al., 1984), telomere shortening (Harley, 1990), higher levels of expression of senescent cell-derived inhibitors of DNA synthesis (Noda et al., 1994) and a decreased level of proliferating cell nuclear antigen (Tanno et al., 1996) have been proposed. However, the details of the age dependent changes in DNA replication and repair are not well known. Nuclei contain DNA polymerases a, b, d and o. As described above, DNA polymerases a, d and o are almost completely inhibited by aphidicolin, whereas DNA polymerases b and g are unaffected by aphidicolin, but are inhibited by ddNTP. Mitochondria are removed by the preparation procedure. Therefore, the amounts of DNA replication or repair may be clarified by studying the effect of aphidicolin or ddNTP on the amount and size of the DNA synthesized using a nuclear system.
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2. Materials and methods
2.1. Animals Wistar strain male rats, 4- and 24-months-old, were supplied by the animal facility of the Tokyo Metropolitan Institute of Gerontology. Partial hepatectomy was carried out by the method of Higgins and Anderson (1931). Regenerating livers were removed from the rats 48 h after partial hepatectomy.
2.2. Chemicals Chemicals used were purchased as follows: ribonucleoside triphosphate and deoxyribonucleoside triphosphates from Boehringer Mannheim-Yamanouchi, Tokyo; [3H]deoxyribonucleoside triphosphates from Dupont/New England Nuclear, Boston, MA; ddTTP from Pharmacia P-L Biochemicals Inc., Milwaukee, WI. Aphidicolin was kindly provided by Dr S. Urano (Tokyo Metropolitan Institute of Gerontology).
2.3. Preparation of cells and isolated nuclei The cell suspension was prepared by the following method. Rat liver or regenerating liver was gently forced through No. 40 stainless steel wire gauze with a special syringe and 0.5 g of cells were suspended in 1 ml of 10 mM Tris–HCl, pH 7.4 containing 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2 (buffer A). To prevent nucleoplasm leakage, nuclei were collected using a modification of the method of Lynch et al. (1975). The removed liver or regenerating liver was homogenized (30 strokes) in 9 vol of 0.3 M sucrose containing 3 mM MgCl2 with a loose-fitting rubber pestle. The suspension was centrifuged at : 1000× g for 10 min, and the supernatant was removed and kept on ice for the preparation of cytoplasmic extract. The pellet was resuspended in : 9 vol of 2.2 M sucrose containing 3 mM MgCl2 and centrifuged at 40 000 ×g for 60 min. Pure nuclei were isolated as the pellet following this centrifugation. The pellet was resuspended in buffer A (1/10 the buffer volume used in the first homogenization). The supernatant from the earlier centrifugation at 1000×g was centrifuged at 100 000× g for 120 min; the supernatants were used as cytoplasmic extracts.
2.4. DNA synthesis using nuclei DNA polymerase activity was measured by the procedure of Taguchi and Ono (1972). The standard reaction mixture contained 2.5 mCi per 0.625 nmol of [3H]dTTP, 187.5 nmol each of dATP, dCTP and dGTP, 200 nmol each of ATP, CTP, GTP and UTP for primer synthesis, 10 mmol of MgCl2, 15 mmol of dithiothreitol, 25 mmol of Tris–HCl buffer, pH 8.3 and 0.25 ml of nuclear suspension in a final volume of 1.25 ml. As necessary, 0.25 ml of cytoplasmic extract, 3.75 mmol of ATP, 0.125 mmol of TTP, 125 mg of aphidicolin or 25 nmol
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of ddTTP was added. The reaction mixture was incubated for 60 min at 37°C. To measure the amount of DNA synthesis, a 50 ml aliquot from each reaction mixture after incubation was applied to a Whatman 3MM paper disc, 2.4 cm in diameter, previously soaked in 0.1 M Na4P2O7 and dried. The discs were then quickly immersed in a large volume of cold 5% trichloroacetic acid (TCA) solution. After 15 min, the TCA solution was discarded by decantation. This washing was repeated three times and the discs were finally placed in 95% ethanol. The discs were dried and put in counting vials containing 10 ml of toluene scintillator composed of 5 g of 2,5-diphenyloxazole (PPO) and 0.1 g of 1,4-bis-2(5-phenyloxazole)-benzene (POPOP) per liter of toluene. The radioactivity was counted in an Aloka LSC-650 liquid scintillation spectrometer (Aloka Co. Ltd., Tokyo, Japan).
2.5. DNA analysis by sucrose density gradient centrifugation To measure the size of the synthesized DNA, the 1.2 ml mixture just after reaction was applied to the 2% SDS layer of a 5–20% alkaline sucrose density gradient containing 0.1 N NaOH, 0.1 M NaCl and 1 mM EDTA in a centrifugation tube. Centrifugation was carried out at 25 000 rpm at 20°C for 15 h using a SW 27 rotor (Beckman Instruments Inc., Palo Alto, CA). After centrifugation, the sucrose gradient containing the labelled DNA was fractionated by dripping from the bottom of the centrifugation tube. Fractions of : 0.9 ml were collected yielding 40 fractions from of each tube. A 50-ml aliquot of each fraction was applied to a Whatman 3MM paper disc and the discs were washed and counted as described above.
2.6. Other methods Protein content was estimated by a modification of the Lowry method using a BCA Protein Assay Reagent (Pierce Chemical Co., IL) with bovine serum albumin as the standard.
3. Results Chromosomal DNA replication and repair must be accomplished in nuclei. All of the machinery required for complete DNA replication and repair is not yet known, however, it is possible that much of the machinery remains in isolated nuclei. Therefore, we used isolated nuclei to study differences in DNA synthesis involved in DNA replication and DNA repair in normal and regenerating livers from young and aged rats. DNA polymerase a is found in the cytoplasmic fraction following the usual extraction procedure. This is due to leakage of the enzyme into the cytoplasmic fraction during the preparation of isolated nuclei. DNA polymerase a is necessary for complete DNA replication. However, the nuclear preparation obtained by the method of Lynch et al. (1975) still retains a type DNA polymerase. We prepared nuclei by this method and used them as the key material in the DNA
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synthesis system. The four nucleoside 5%-triphosphates needed for RNA primer synthesis were also added to the nuclear system as substrates. The amount of DNA synthesis by isolated nuclei prepared from normal rat liver is lower than that by isolated nuclei from regenerating liver (Table 1). As described by Kaufmann et al. (1982) and Seki et al. (1987), the amount of DNA synthesis using nuclear preparations from normal and regenerating livers is increased by the addition of 3 mM ATP. Furthermore, as described previously, DNA synthesis using nuclei is stimulated by the addition of the cytoplasmic preparation (Jazwinski et al., 1976 Wong et al., 1987), which may contain certain factor(s) essential for DNA replication or repair. We also examined the effect of the cytosol fraction on DNA synthesis using nuclei and found a stimulatory effect. Significantly, the cytoplasm from regenerating liver produced a greater stimulation than that from normal rat liver. The stimulation of DNA synthesis by ATP and cytoplasm together was greater than the effect of ATP alone. To compare the amounts of nuclear and cytoplasmic protein, we measured their amounts. The results are summarized in Table 1. We were interested in the size of the newly synthesized DNA. Liver cells, separated under mild conditions to protect against scission of the DNA, were placed in the 2% SDS layer of a 5–20% alkaline sucrose density gradient. DNA from regenerating liver cells of young rat passed through the SDS layer to sediment at the bottom or near the bottom of the tubes after centrifugation. However, DNA from nuclear preparations of the same tissues was found as a peak with a maximum in fraction 23 (Fig. 1A). A similar result was obtained using regenerating liver from aged rats (data not shown). Centrifugation following in vitro synthesis for 1 min Table 1 Effect of ATP and the cytoplasmic fraction on DNA synthesis in nuclei from normal and regenerating liver of 4-month-old ratsa Conditions
Incorporated dTMP (CPM) NRL
Nuclei Nuclei+ATP Nuclei+ATP+cyto (NRL) Nuclei+ATP+cyto (RRL)
402 (0.375)b 993 1191 (55)d 1959 (52)e
RRL 1552 (0.315)c 2557 3328 6283
a The experimental procedures are the same as described in Section 2. NRL; normal rat liver; RRL; regenerating rat liver; cyto; Cytoplasmic fraction. b Number in parentheses represents miligrams protein amount in 0.25 ml nuclei extract from normal rat liver. c Number in parentheses represents miligram protein amount in 0.25 ml nuclei extract from regenerating rat liver. d Number in parentheses represents miligram protein amount in 0.25 ml cytoplasmic extract from normal rat liver. e Number in parentheses represents miligram protein amount in 0.25 ml cytoplasmic extract from regenerating rat liver.
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Fig. 1. DNA sizes were analysed by alkaline sucrose density gradient centrifugation as described in Section 2. Amounts of non-labeled DNA were measured by absorbance at 260 nm and amounts of labeled DNA were determined by measuring the cpm of the incorporated [3H]TMP. DNA sizes in cell preparations from regenerating rat liver and nuclear preparations from regenerating rat liver are indicated as ( — — ) and ( — — ), respectively, in (A). DNA synthesis using 0.25 ml of nuclear preparation from regenerating rat liver in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract was performed for 1 min ( — — ), 5 min ( — —) and 10 min ( — — ) as indicated in (B).
using the same nuclei gave a peak of newly synthesized DNA in fraction 33. DNA synthesis for 5 min resulted in two peaks with maxima in fractions 30 and 33. Reaction for 10 min produced increases in the amounts of the same two peaks (Fig. 1B). However, only the peak centered at fraction 33 was found following DNA synthesis for 10 min using nuclei from normal rat liver (data not shown). It is well known that DNA polymerases are inhibited by specific inhibitors. DNA polymerases a, d and o, which are involved in DNA replication, are inhibited by aphydicolin; DNA polymerase b, which is related to DNA repair, is inhibited by ddNTP. When aphidicolin (25 mg/0.25 ml) was added to the DNA synthesizing system, using nuclei activated by ATP and the cytoplasmic fraction, the amount of DNA synthesis was inhibited by 20–30%. This inhibition was found using nuclear preparations from both normal and regenerating rat livers (Tables 2 and 3). The same concentration of aphidicolin inhibits DNA polymerases a and d by 80% or more (Ohashi, et al., 1978 Aoyagi et al., 1994), while the inhibition by ddTTP is 60% or more (Tables 2 and 3). Although the amount of DNA synthesis using isolated nuclei from aged animals was lower, similar inhibitory effects were ob-
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Table 2 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from the livers of 4-month-old ratsa Inhibitor
Conditions Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 402 324 166 77
Nuclei+ATP+cyto
1191 878 195 163
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the liver of a 4-month-old rat.
served (Tables 4 and 5). The amounts of protein in the nuclear extracts from normal and regenerating liver of young rats are similar when prepared by the same methods (data not shown), and similar results were obtained using both cytoplasmic extracts. On the other hand, the amounts of protein in the nuclear extract from normal liver, the nuclear extract from regenerating liver and the cytoplasmic extracts from normal liver and regenerating liver of aged rats were 0.345 mg protein/0.25 ml, 0.290 mg protein/0.25 ml, 52 mg protein/0.25 ml and 46 mg protein/0.25 ml, respectively. The inhibitory effect of aphidicolin on DNA synthesis was unexpectedly small, while that by ddTTP was intense. The kinetics of DNA synthesis with and without each inhibitor was studied (Fig. 2). DNA synthesis was performed using nuclei from regenerating rat liver stimulated by ATP and the cytoplasmic fraction. When aphidicolin was added at the start of DNA synthesis, the DNA synthesis proceeded at a rate : 60% that of the control. When aphidicolin was added after 20 min, the rate of DNA synthesis was similar to that when aphidicolin was added at the start. The same results were obtained using ddTTP except that DNA synthesis proceeded at a rate of :20% that of the control level (Fig. 2.). Table 3 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from regenerating livers of 4-month-old ratsa Inhibitor
Conditions Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 1552 1098 449 407
Nuclei+ATP+cyto
6997 5209 2230 929
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the regenerating liver of a 4-month-old rat.
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Table 4 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from the livers of 24-month-old ratsa Inhibitor
Condition Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 288 206 130 52
Nuclei+ATP+cyto
820 593 154 88
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the liver of a 24-month-old rat.
Following DNA synthesis for 10 min, two distinct DNA peaks with maxima in fractions 30 and 33 were found, as described above (Fig. 3). At this time, 2000-fold concentrated cold dTTP was added and the labeled DNA was chased for 50 min. By this chase, the peak at fraction 33 shifted to fraction 30, however, no new peak at a heavier position was found (Fig. 3). Finally, the effects of aphidicolin and ddTTP on the size of the DNA after chase using nuclear preparations from regenerating livers of young and aged rats were studied. DNA synthesis was carried out for 10 min using the nuclear system from the regenerating liver of young rats in the presence of the cytoplasmic fraction and ATP and synthesis was continued for 50 min after the addition of cold TTP. This time, when the inhibitor — aphidicolin or ddTTP — was added after 10 min, cold TTP was also added and the chase reaction was continued for another 50 min. In the case of no inhibitor, a peak at fraction 30 was observed after chase for 50 min (Fig. 4). The same finding was observed in the sedimentation profile after DNA synthesis using the preparation from aged rats (Fig. 5). However, the addition of aphidicolin caused the amount of DNA synthesis using nuclear preparations from young rats to decrease to : 10– 20% the level in the absence of inhibitor (Fig. 4). The decrease using nuclear Table 5 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from the regenerating livers of 24-month-old ratsa Inhibitor
Conditions Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 1231 611 430 395
Nuclei+ATP+cyto
4956 3338 1735 783
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the regenerating liver of a 24-month-old rat.
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Fig. 2. Effects of aphydicolin and ddTTP on DNA synthesis. DNA synthesis was performed using the nuclear system from regenerating rat liver in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract ( — — ). Aphydicolin (112.5 mg) was added from the start of the reaction ( — — ) or 20 min after the start of the reaction ( — —); ddTTP (20 mM) was added from the start of the reaction ( — — ) or 20 min after the start of the reaction (— — ).
preparations from aged animals was 30% or more (Fig. 5). On the other hand, the addition of ddTTP did not result in a transfer of the smaller DNA to the larger after the chase (Fig. 4). The sedimentation profile of the DNA after chase was similar to that with no chase (Figs. 3 and 5). The amount of [3H] labeled DNA using the nuclear and cytosol fractions from 4-month-old rats was : 1.5-fold higher than for the same fractions from 24-month-old rats (Figs. 4 and 5) and a DNA peak was observed only in fraction 30, as in the chase experiments. Thus, the sizes of the synthesized DNA in nuclear preparations from young and aged rats were similar except for quantitative differences and were not changed by the addition of aphidicolin. However, using nuclei from young rats, two peaks with maxima in fractions 30 and 33, were found by the addition of ddTTP and the amounts of these two peaks were about half those in the absence of inhibitor (Fig. 4). DNA synthesis using the nuclei from aged rats, resulted in only one peak at fraction 33 upon the addition of ddTTP; the size of this peak was : 20% that in the absence of inhibitor (Fig. 5).
4. Discussion DNA synthesis using nuclear preparations closely approximates DNA replication in vivo. The DNA synthesizing capacity of nuclei from the regenerating liver of
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young rats is higher than that of normal liver. This supports the notion that regenerating liver is highly proliferative. The DNA synthetic capacity of nuclei from aged rats is lower than that from young rats. This suggests that cell proliferation may decrease in aged rat liver. Furthermore, DNA synthesis using isolated nuclei was stimulated by ATP and the cytoplasmic preparation (Table 2). This indicates that the machinery for DNA synthesis in isolated nuclei is insufficient for maximal activity and that additional factor(s) are required for DNA replication or repair. A requirement for certain cytoplasmic factor(s) for DNA synthesis is likely. Cytoplasmic preparations from regenerating rat liver stimulate DNA synthesis more than cytoplasmic preparations from normal liver (Table 1). This result seems reasonable. When the nuclear and cytosolic fractions were prepared from the regenerating livers of young and aged rats and DNA synthesis using the two subcellular fractions from the same aged rat were examined, the stimulation ratios of DNA synthesis by ATP and cytosolic factor(s) were similar (Tables 3 and 5). Similar results were obtained using the two fractions from the livers of young and aged rats (Tables 2 and 4). However, the stimulation of DNA synthesis using nuclei from the regenerating liver of a young rat and the cytosol fraction from an aged rat was less than when both preparations were from the young rat (data not shown). The DNA synthesizing capacity of nuclei and the DNA synthesis stimulating capacity of cytosol appear to decline similarly during ageing, suggesting the machinery shares specific relationships.
Fig. 3. DNA synthesis was performed using the nuclear system from regenerating rat liver in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract. The standard reaction mixture is described in Section 2. After 10 min, the reaction was stopped and the DNA synthesized in vitro was analyzed by sucrose density gradient centrifugation as described in Section 2 (— —) or 1.25 mmol of cold TTP was added and the reaction was continued for 50 min. DNA chased with cold TTP was analyzed by the same method ( — — ).
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Fig. 4. DNA synthesis was performed using the nuclear system from the regenerating liver of a 4-month-old rat in a standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract. The standard reaction mixture is described in Section 2. After 10 min, 1.25 mmol of cold TTP only ( — — ), cold TTP and 125 mg of aphidicolin (— —), or cold TTP and 25 mM of ddTTP ( — — ) was added and the reactions were continued for 50 min. The DNA synthesized in vitro was analyzed by sucrose density gradient centrifugation as described in Section 2.
It is well known that DNA polymerases a and d are inhibited by aphidicolin (Ohashi et al., 1978 Hammond et al., 1987) and DNA polymerase b by ddTTP (Waqar et al., 1978). It is accepted that the decline in the amount of DNA synthesis caused by the inhibition of DNA replication is large, but that caused by the inhibition of DNA repair, especially base excision repair, is small. However, our data indicate that the inhibition by aphidicolin is 30–40% of total DNA synthesis using the nuclear system from young rats, whereas, the inhibition by ddTTP is :80%. It has been reported that ddTTP inhibits the incorporation of ddNTP by DNA polymerases b and g (Waqar et al., 1978). The mode of inhibition by ddNTP is well known. If ddNMP is incorporated into DNA by DNA polymerase b, DNA strands cannot incorporate the next deoxynucleotide for 2% 3% dideoxyribose. Similarly, the connection of DNA strands by DNA ligase does not occur. Therefore, DNA strands that incorporate ddNMPs do not extend. Thus, it is reasonable that small DNA strands do not enlarge (Fig. 4). On the other hand, when DNA synthesis was inhibited by the addition of aphidicolin after 10 min, DNA synthesis was inhibited by 30% at the most and the small DNA strands that peak in fraction 33 enlarge to those with a maximum in fraction 30 by chase (Fig. 4). According to our previous report (Ohashi et al., 1978), the aphidicolin concentration used inhibits DNA polymerase a by 90% or more and DNA polymerase d by : 80% (Hammond et al., 1987). When permeable cells are used, the inhibition of DNA synthesis by this concentration of aphidicolin is \ 90% (Hammond et al., 1987). As
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described above, the inhibition of DNA synthesis by the addition of aphidicolin at the start of the reaction is no more than 40% (Tables 2–5 and Fig. 3). As explanations for these results, the following hypotheses were considered. In animals, oxygen radicals are produced through normal metabolism (Chance et al., 1979) and oxidative DNA damage increases during the ageing process (Kaneko et al., 1966 Fraga et al., 1990 Higami et al., 1994) with misincorporations due to oxidative damage having been reported (Kuchino et al., 1987). It is well known that transcription-coupled repair is needed for the accurate transfer of genetic information to mRNA (Bohr, 1988). However, the accurate transfer of genetic information is the most important in DNA replication. Therefore, it is not surprising that an increase in repair capacity occurs before replication. There may be a replicationcoupled repair system to defend against the appearance of mismatched base pairs. If this hypothesis is correct, DNA synthesis is controlled by DNA polymerase b, which manages DNA repair. Namely, DNA synthesis cannot proceed if there is no correction of DNA damage or if ddTTP is incorporated. Therefore, the inhibition of DNA synthesis by ddTTP is extensive (Tables 2–5, Figs. 2, 4 and 5) and these results support the above hypothesis. On the other hand, chase with and without aphidicolin after DNA synthesis for 10 min resulted in the DNA products sedimenting in the same fraction and the amount of the large size DNA after chase with aphidicolin was :90% that in the case of no inhibitor (Fig. 4). From these results, it appears that DNA polymerase b is strongly related to DNA elongation.
Fig. 5. DNA synthesis was performed using the nuclear system from the regenerating liver of a 24-month-old rat in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract. The standard reaction mixture is described in Section 2. After 10 min, 1.25 mmol of cold TTP only ( — — ), cold TTP and 125 mg of aphidicolin ( — —) or cold TTP and 25 mM of ddTTP ( — — ) were added and the reactions were continued for 50 min. The DNA synthesized in vitro was analyzed by sucrose density gradient centrifugation as described in Section 2.
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Similar evidence has been reported from experiments using an in vitro replication system of adenovirus DNA (Ikeda et al., 1980). However, it has been reported that the most likely model for the multi-protein complex at the eukaryotic replication fork does not contain DNA polymerase b (Tsurimoto and Stillman, 1989) and that this enzyme is involved in gap filling of short patches produced by base excision repair (Mosbaugh and Linn, 1983). Therefore, DNA polymerase b may play a role in abnormal replication when DNA polymerases d and a are inhibited. DNA synthesis using an isolated nuclear system from the regenerating livers of aged rats is :80% of that using the isolated nuclei from the regenerating livers of young rats (Figs. 4 and 5). The newly synthesized DNA found by chase after the addition of aphidicolin is the large size only, as found also for young rats. However, the amount of DNA synthesis using nuclei from regenerating livers of aged rat in the presence of aphidicolin is : 60% that in the case of no inhibitor (Fig. 5). As described above, the value for young rats is : 90% (Fig. 4). Thus, DNA synthesis after the addition of aphidicolin is decreased in isolated nuclei from aged rats. On the other hand, early DNA synthesis had to be estimated by the addition of ddTTP, because the size of the DNA remains almost unchanged for the above-mentioned reason. Although a DNA peak after synthesis for 10 min was found only in fraction 33, the counts of [3H]TMP by DNA synthesis shifted to fraction 30. This shift was found even though DNA synthesis was inhibited by aphidicolin after 10 min. DNA polymerase b may also play a role in the elongation of DNA under emergency conditions. On the other hand, the large size DNA was produced slowly during DNA synthesis using isolated nuclei from aged rats. These results suggest that the amount of complete DNA is small and the velocity of DNA synthesis is slow in isolated nuclei from the regenerating liver of aged rats. The reasons for this decrease and the slow-down in DNA synthesis in aged animals may include damage to the protein machinery and template DNA for DNA synthesis. The ratios of DNA polymerase a activities extracts from the nucleoplasmic and cytoplasmic fractions of regenerating liver of young rats prepared by the method of Lynch et al. were :97 and 3%, respectively (data was not shown), and similar results were obtained from aged rats (data not shown). The DNA sizes in the nuclei prepared from regenerating livers of young and aged rats are approximately the same. Moreover, it has been reported that nuclear size distributions from rapidly growing cells are smaller than from slow or non-growing cells (Mitsui and Schneider, 1976). Therefore, it is not likely that the nuclei from aged rats are more broken and susceptible to permeability than nuclei from young rats. Lambda phage DNA (48 kb) as a marker was sedimented in fraction 31. On the other hand, replication units (replicons) are heterogeneous in size, varying from 50 to 300 kb (Hand, 1978). The newly synthesized DNA produced in the isolated nuclear system appeared in fraction 30 and this value is included in the above variation. DNA from liver cells sediments near the bottom of the tube and DNA from nuclei sediments to fraction 23 (Fig. 1A). The DNA from pure nuclei may be broken due to the physical force of centrifugation. In all cases, the size of the DNA synthesized in rat liver is considerably longer than that synthesized in vitro. The DNA synthesized early appeared in fraction 33, but changed over time to two
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peaks in fractions 30 and 33 (Fig. 1B). This result suggests that the DNA in fraction 30 may represent the average size of the complete replicon. We can clarify some factors related to the decline in DNA synthesis in normal and regenerating liver from aged rats. Decreases in DNA replication and repair with ageing may be caused by the factors described above as well as by machinery for DNA synthesis in nuclei.
Acknowledgements We would like to thank Dr Shiro Urano for the generous gift of aphidicolin and Dr Margaret Dooley-Ohto for assistance with the manuscript.
References Aoyagi, N., Matsuoka, S., Furunobu, A., Matsukage, A., Sakaguchi, K., 1994. Drosophila DNA polymerase d: purification and characterization. J. Biol. Chem. 169, 6045 – 6050. Bohr, V.A., 1988. DNA repair and transcriptional activity in genes. J. Cell Sci. 90, 175 – 178. Chance, B., Sies, H., Boveris, A., 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527–605. Focher, F., Ferrari, E., Spadari, S., Hubsher, U., 1988. Do DNA polymerases d and a act coordinately as leading and lagging strand replicates? FEBS Lett. 229, 6 – 10. Fraga, C.G., Shigenaga, M.K., Park, J.-W., Degan, P., Ames, B.N., 1990. Oxidative damage to DNA during aging: 8-hydroxy-2%-deoxyguanosine in rat organ DNA and urine. Proc. Natl. Acad. Sci. USA 87, 4533–4537. Fry, M., Silber, J., Loeb, L.A., Martin, G.M., 1984. Delayed and reduced cell replication and diminishing levels of DNA polymerase-a in regenerating liver of aging mice. J. Cell. Physiol. 118, 225–232. Hammond, R.A., Byrnes, J.J., Miller, M.R., 1987. Identification of DNA polymerase d in CV-1 cells: studies implicating both DNA polymerase d and DNA polymerase a in DNA replication. Biochemistry 26, 6817–6824. Hand, R., 1978. Eucaryotic DNA: organization of the genome for replication. Cell 15, 317 – 325. Harley, C.B., Futcher, B., Greider, C.W., 1990. Teromeres shorten during ageing of human fibroblasts. Nature 345, 458–460. Higami, Y., Shimokawa, I., Okimoto, T., Ikeda, T., 1994. An age-related increase in the basal level of DNA damage and vulnerability to oxygen radicals in the individual hepatocytes of male F344 rats. Mutat. Res. 316, 59–67. Higgins, G.M., Anderson, R.M., 1931. Experimental pathology of the liver in the white rat following partial surgical removal. Arch. Pathol. 12, 186 – 202. Ikeda, J.-E., Longiaru, M., Horwitz, M.S., Hurwitz, J., 1980. Elongation of primed DNA templates by eukaryotic DNA polymerases. Proc. Natl. Acad. Sci. USA 77, 5827 – 5831. Jazwinski, S.M., Wang, J.L., Edelman, G.M., 1976. Initiation of replication in chromosomal DNA induced by extracts from proliferating cells. Proc. Natl. Acad. Sci. USA 73, 2231 – 2235. Kaneko, T., Tahara, S., Matsuo, M., 1966. Non-linear accumulation of 8-hydroxy-2%-deoxyguanosine, a marker of oxidized DNA damage during aging. Mutat. Res. 316, 277 – 285. Kaufmann, W.K., Kaufmann, D.G., Stenstrom, M., Grisham, J.W., 1982. Requirements for adenosine triphosphate in DNA repair in isolated hepatic nuclei. Biochem. Biophys. Res. Commun. 108, 1040–1047.
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Kuchino, Y., Mori, F., Kasai, H., Inoue, H., Iwai, S., Miura, K., Ohtsuka, E., Nishimura, S., 1987. Misreading of DNA templates containing 8-hydroxydeoxyguanosine at the modified base and at adjacent residues. Nature 327, 77–79. Linn, S., 1991. How many pols does it take to replicate nuclear DNA? Cell 66, 185 – 187. Lynch, W.E., Surrey, S., Lieberman, I., 1975. Nuclear deoxyribonucleic acid polymerases of liver. J. Biol. Chem. 1975, 8179–8183. Mitsui, Y., Schneider, L., 1976. Increased nuclear sizes in senescent human diploid fibroblast cultures. Exp. Cell. Res. 100, 147–152. Morioka, K., Shimada, H., Terayama, H., 1973. Serum factors stimulating DNA synthesis in the isolated nuclear system from rat liver. Biochem. Biophys. Res. Commun. 51, 451 – 460. Mosbaugh, D.W., Linn, S., 1983. Excision repair and DNA synthesis with a combination of HeLa DNA polymerase b and DNase V. J. Biol. Chem. 258, 108 – 118. Noda, A., Ning, Y., Venable, S.F., Pereira-Smith, O.M., Smith, J.R., 1994. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell. Res. 211, 90 – 98. Ohashi, M., Taguchi, T., Ikegami, S., 1978. Aphidicolin: a specific inhibitor of DNA polymerases in the cytosol of rat liver. Biochem. Biophys. Res. Commun. 82, 1084 – 1090. Seki, S., Oda, T., 1977. DNA synthesis in detergent-treated mouse ascites sarcoma cells. Biochim. Biophys. Acta 476, 24–31. Seki, S., Mori, S., Nakashima, A., Oda, T., 1987. Effects of ATP and other nucleotides on DNA repair synthesis in bleomycin-pretreated permeable mouse sarcoma cells. Carcinogenesis 8, 1391 – 1394. Taguchi, T., Ono, T., 1972. Changes in enzymes in the mouse spleen after Friend virus infection. GANN Monograph 12, 191–202. Tanno, M., Ogihara, M., Taguchi, T., 1996. Age-related changes in proliferating cell nuclear antigen levels. Mech. Ageing Dev. 92, 53–66. Tsurimoto, T., Stillman, B., 1989. Multiple replication factors augment DNA synthesis by the two eukaryotic DNA polymerases, a and d. EMBO J. 8, 3883 – 3889. Wang, T.S.-F., 1991. Eukaryotic DNA polymerases. Annu. Rev. Biochem. 60, 513 – 552. Waqar, M.A., Evans, M.J., Huberman, J.A., 1978. Effect of 2%,3%-dideoxythymidine-5%-triphosphate on HeLa cell in vitro DNA synthesis: evidence that DNA polymerase a is the only polymerase required for cellular DNA replication. Nucleic Acids Res. 5, 1933 – 1946. Weissbach, A., 1975. Vertebrate DNA polymerases. Cell 5, 101 – 108. Wobbe, C.R., Dean, F., Weissbach, L., Hurwitz, J., 1985. In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc. Natl. Acad. Sci. USA 82, 5710 – 5714. Wong, R.L., Gutowski, J.K., Katz, M., Goldfarb, R.H., Cohen, S., 1987. Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors. Proc. Natl. Acad. Sci. USA 84, 241–245.
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Differences in DNA synthesis in vitro using isolated nuclei from regenerating livers of young and aged rats Takahiko Taguchi *, Mitsugu Fukuda, Mochihiko Ohashi Department of Gene Regulation and Protein Function, Tokyo Metropolitan Institute of Gerontology, 35 -2 Sakae-cho, Itabashi-ku, Tokyo 173 -0015, Japan Received 26 January 2000; received in revised form 4 June 2000; accepted 5 October 2000
Abstract To detect changes in DNA synthesis during ageing, we compare DNA synthesis in the livers of young and aged rats. As an intermediate between an in vivo system using intact cells and an in vitro system using purified DNA polymerases, isolated nuclei were prepared and used as the machinery for DNA synthesis. The DNA synthesizing capacity of nuclei from regenerating liver was higher than that of nuclei from normal liver and these capacities from liver and regenerating liver were lower in nuclear preparations from aged rats. DNA synthesis using isolated nuclei was stimulated by ATP and the cytoplasmic preparation. The cytoplasmic preparation from regenerating rat liver was found to stimulate DNA synthesis more than the preparation from normal liver. The activity in regenerating liver from young rats was also greater than in that from aged rats. It is well known that DNA replication is inhibited by aphidicolin and DNA repair by ddTTP. We examined the effects of aphidicolin and ddTTP on DNA synthesis using the nuclear system. Surprisingly, the inhibition by aphidicolin was 30% of total DNA synthesis using the nuclear system from young rats. On the other hand, the inhibition by ddTTP was : 80%. We measured the sizes of the DNA synthesized in the presence of both inhibitors. DNA synthesis was allowed to proceed for 10 min using isolated nuclei from regenerating liver of young rats and the size of the DNA was determined by sucrose density gradient centrifugation analysis. DNA products appeared in two fractions. Following a chase of 50 min in the presence or absence of aphidicolin, the short DNA product grew larger in both cases, although the amount of DNA in the presence of aphidicolin was :90% that in its absence. In the same experiment using nuclei from aged
* Corresponding author. Tel.: +81-3-39643241, ext. 3060; fax: +81-3-35794776. E-mail address: [email protected] (T. Taguchi). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 2 2 6 - 8
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rats, the amount in the presence of aphidicolin was : 60% that in its absence. These results suggest that DNA polymerase b is closely related to abnormal replication when DNA polymerases a and d are inhibited and that the effect of cytosol on DNA synthesis, as well as the DNA synthetic capacity of isolated nuclei, becomes lower in regenerating rat liver during ageing. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Regenerating liver; DNA polymerases; Replication and repair; Aphidicolin; ddTTP; Ageing
1. Introduction Eukaryotic DNA polymerases comprise a, b, g, d and o types. We have reported that aphidicolin is a specific inhibitor of DNA polymerase a (Ohashi et al., 1978) and it has been shown that this inhibitor is also effective against DNA polymerases d and o (Wang, 1991). Three enzymes — DNA polymerase a, d and o — have been shown to be involved in DNA replication (Weissbach, 1975; Focher et al., 1988; Wang, 1991; Linn, 1991). DNA polymerases b and g are inhibited by dideoxynucleoside 5%-triphosphate (ddNTP) (Waqar et al., 1978). DNA polymerase g is present only in mitochondria, where it is involved in mitochondrial DNA synthesis (Weissbach, 1975). DNA polymerase b is involved in base excision repair by filling the gaps formed by excision (Mosbaugh and Linn, 1983 Wang, 1991). Various systems have been used to study DNA replication in eukaryotes (Morioka et al., 1973 Seki and Oda, 1977 Ikeda et al., 1980 Wobbe et al., 1985). Isolated nuclei form one good system in which to clarify DNA replication and repair. Within cells, the nuclei contain the most machinery for DNA replication and repair. However, DNA synthesis using isolated hepatic nuclei (Kaufmann et al., 1982) and bleomycin-pretreated permeable cells (Seki et al., 1987) is stimulated by ATP. Moreover, the presence of a cytoplasmic factor that stimulates nuclear DNA synthesis has been reported (Jazwinski et al., 1976 Wong et al., 1987). Thus, some factors that are not contained in nuclei are necessary for DNA replication. On the other hand, it has been reported that the rate of DNA synthesis declines in aged animals. As the causes for this decrease, lower DNA polymerase a activity (Fry et al., 1984), telomere shortening (Harley, 1990), higher levels of expression of senescent cell-derived inhibitors of DNA synthesis (Noda et al., 1994) and a decreased level of proliferating cell nuclear antigen (Tanno et al., 1996) have been proposed. However, the details of the age dependent changes in DNA replication and repair are not well known. Nuclei contain DNA polymerases a, b, d and o. As described above, DNA polymerases a, d and o are almost completely inhibited by aphidicolin, whereas DNA polymerases b and g are unaffected by aphidicolin, but are inhibited by ddNTP. Mitochondria are removed by the preparation procedure. Therefore, the amounts of DNA replication or repair may be clarified by studying the effect of aphidicolin or ddNTP on the amount and size of the DNA synthesized using a nuclear system.
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2. Materials and methods
2.1. Animals Wistar strain male rats, 4- and 24-months-old, were supplied by the animal facility of the Tokyo Metropolitan Institute of Gerontology. Partial hepatectomy was carried out by the method of Higgins and Anderson (1931). Regenerating livers were removed from the rats 48 h after partial hepatectomy.
2.2. Chemicals Chemicals used were purchased as follows: ribonucleoside triphosphate and deoxyribonucleoside triphosphates from Boehringer Mannheim-Yamanouchi, Tokyo; [3H]deoxyribonucleoside triphosphates from Dupont/New England Nuclear, Boston, MA; ddTTP from Pharmacia P-L Biochemicals Inc., Milwaukee, WI. Aphidicolin was kindly provided by Dr S. Urano (Tokyo Metropolitan Institute of Gerontology).
2.3. Preparation of cells and isolated nuclei The cell suspension was prepared by the following method. Rat liver or regenerating liver was gently forced through No. 40 stainless steel wire gauze with a special syringe and 0.5 g of cells were suspended in 1 ml of 10 mM Tris–HCl, pH 7.4 containing 0.25 M sucrose, 25 mM KCl, 5 mM MgCl2 (buffer A). To prevent nucleoplasm leakage, nuclei were collected using a modification of the method of Lynch et al. (1975). The removed liver or regenerating liver was homogenized (30 strokes) in 9 vol of 0.3 M sucrose containing 3 mM MgCl2 with a loose-fitting rubber pestle. The suspension was centrifuged at : 1000× g for 10 min, and the supernatant was removed and kept on ice for the preparation of cytoplasmic extract. The pellet was resuspended in : 9 vol of 2.2 M sucrose containing 3 mM MgCl2 and centrifuged at 40 000 ×g for 60 min. Pure nuclei were isolated as the pellet following this centrifugation. The pellet was resuspended in buffer A (1/10 the buffer volume used in the first homogenization). The supernatant from the earlier centrifugation at 1000×g was centrifuged at 100 000× g for 120 min; the supernatants were used as cytoplasmic extracts.
2.4. DNA synthesis using nuclei DNA polymerase activity was measured by the procedure of Taguchi and Ono (1972). The standard reaction mixture contained 2.5 mCi per 0.625 nmol of [3H]dTTP, 187.5 nmol each of dATP, dCTP and dGTP, 200 nmol each of ATP, CTP, GTP and UTP for primer synthesis, 10 mmol of MgCl2, 15 mmol of dithiothreitol, 25 mmol of Tris–HCl buffer, pH 8.3 and 0.25 ml of nuclear suspension in a final volume of 1.25 ml. As necessary, 0.25 ml of cytoplasmic extract, 3.75 mmol of ATP, 0.125 mmol of TTP, 125 mg of aphidicolin or 25 nmol
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of ddTTP was added. The reaction mixture was incubated for 60 min at 37°C. To measure the amount of DNA synthesis, a 50 ml aliquot from each reaction mixture after incubation was applied to a Whatman 3MM paper disc, 2.4 cm in diameter, previously soaked in 0.1 M Na4P2O7 and dried. The discs were then quickly immersed in a large volume of cold 5% trichloroacetic acid (TCA) solution. After 15 min, the TCA solution was discarded by decantation. This washing was repeated three times and the discs were finally placed in 95% ethanol. The discs were dried and put in counting vials containing 10 ml of toluene scintillator composed of 5 g of 2,5-diphenyloxazole (PPO) and 0.1 g of 1,4-bis-2(5-phenyloxazole)-benzene (POPOP) per liter of toluene. The radioactivity was counted in an Aloka LSC-650 liquid scintillation spectrometer (Aloka Co. Ltd., Tokyo, Japan).
2.5. DNA analysis by sucrose density gradient centrifugation To measure the size of the synthesized DNA, the 1.2 ml mixture just after reaction was applied to the 2% SDS layer of a 5–20% alkaline sucrose density gradient containing 0.1 N NaOH, 0.1 M NaCl and 1 mM EDTA in a centrifugation tube. Centrifugation was carried out at 25 000 rpm at 20°C for 15 h using a SW 27 rotor (Beckman Instruments Inc., Palo Alto, CA). After centrifugation, the sucrose gradient containing the labelled DNA was fractionated by dripping from the bottom of the centrifugation tube. Fractions of : 0.9 ml were collected yielding 40 fractions from of each tube. A 50-ml aliquot of each fraction was applied to a Whatman 3MM paper disc and the discs were washed and counted as described above.
2.6. Other methods Protein content was estimated by a modification of the Lowry method using a BCA Protein Assay Reagent (Pierce Chemical Co., IL) with bovine serum albumin as the standard.
3. Results Chromosomal DNA replication and repair must be accomplished in nuclei. All of the machinery required for complete DNA replication and repair is not yet known, however, it is possible that much of the machinery remains in isolated nuclei. Therefore, we used isolated nuclei to study differences in DNA synthesis involved in DNA replication and DNA repair in normal and regenerating livers from young and aged rats. DNA polymerase a is found in the cytoplasmic fraction following the usual extraction procedure. This is due to leakage of the enzyme into the cytoplasmic fraction during the preparation of isolated nuclei. DNA polymerase a is necessary for complete DNA replication. However, the nuclear preparation obtained by the method of Lynch et al. (1975) still retains a type DNA polymerase. We prepared nuclei by this method and used them as the key material in the DNA
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synthesis system. The four nucleoside 5%-triphosphates needed for RNA primer synthesis were also added to the nuclear system as substrates. The amount of DNA synthesis by isolated nuclei prepared from normal rat liver is lower than that by isolated nuclei from regenerating liver (Table 1). As described by Kaufmann et al. (1982) and Seki et al. (1987), the amount of DNA synthesis using nuclear preparations from normal and regenerating livers is increased by the addition of 3 mM ATP. Furthermore, as described previously, DNA synthesis using nuclei is stimulated by the addition of the cytoplasmic preparation (Jazwinski et al., 1976 Wong et al., 1987), which may contain certain factor(s) essential for DNA replication or repair. We also examined the effect of the cytosol fraction on DNA synthesis using nuclei and found a stimulatory effect. Significantly, the cytoplasm from regenerating liver produced a greater stimulation than that from normal rat liver. The stimulation of DNA synthesis by ATP and cytoplasm together was greater than the effect of ATP alone. To compare the amounts of nuclear and cytoplasmic protein, we measured their amounts. The results are summarized in Table 1. We were interested in the size of the newly synthesized DNA. Liver cells, separated under mild conditions to protect against scission of the DNA, were placed in the 2% SDS layer of a 5–20% alkaline sucrose density gradient. DNA from regenerating liver cells of young rat passed through the SDS layer to sediment at the bottom or near the bottom of the tubes after centrifugation. However, DNA from nuclear preparations of the same tissues was found as a peak with a maximum in fraction 23 (Fig. 1A). A similar result was obtained using regenerating liver from aged rats (data not shown). Centrifugation following in vitro synthesis for 1 min Table 1 Effect of ATP and the cytoplasmic fraction on DNA synthesis in nuclei from normal and regenerating liver of 4-month-old ratsa Conditions
Incorporated dTMP (CPM) NRL
Nuclei Nuclei+ATP Nuclei+ATP+cyto (NRL) Nuclei+ATP+cyto (RRL)
402 (0.375)b 993 1191 (55)d 1959 (52)e
RRL 1552 (0.315)c 2557 3328 6283
a The experimental procedures are the same as described in Section 2. NRL; normal rat liver; RRL; regenerating rat liver; cyto; Cytoplasmic fraction. b Number in parentheses represents miligrams protein amount in 0.25 ml nuclei extract from normal rat liver. c Number in parentheses represents miligram protein amount in 0.25 ml nuclei extract from regenerating rat liver. d Number in parentheses represents miligram protein amount in 0.25 ml cytoplasmic extract from normal rat liver. e Number in parentheses represents miligram protein amount in 0.25 ml cytoplasmic extract from regenerating rat liver.
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Fig. 1. DNA sizes were analysed by alkaline sucrose density gradient centrifugation as described in Section 2. Amounts of non-labeled DNA were measured by absorbance at 260 nm and amounts of labeled DNA were determined by measuring the cpm of the incorporated [3H]TMP. DNA sizes in cell preparations from regenerating rat liver and nuclear preparations from regenerating rat liver are indicated as ( — — ) and ( — — ), respectively, in (A). DNA synthesis using 0.25 ml of nuclear preparation from regenerating rat liver in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract was performed for 1 min ( — — ), 5 min ( — —) and 10 min ( — — ) as indicated in (B).
using the same nuclei gave a peak of newly synthesized DNA in fraction 33. DNA synthesis for 5 min resulted in two peaks with maxima in fractions 30 and 33. Reaction for 10 min produced increases in the amounts of the same two peaks (Fig. 1B). However, only the peak centered at fraction 33 was found following DNA synthesis for 10 min using nuclei from normal rat liver (data not shown). It is well known that DNA polymerases are inhibited by specific inhibitors. DNA polymerases a, d and o, which are involved in DNA replication, are inhibited by aphydicolin; DNA polymerase b, which is related to DNA repair, is inhibited by ddNTP. When aphidicolin (25 mg/0.25 ml) was added to the DNA synthesizing system, using nuclei activated by ATP and the cytoplasmic fraction, the amount of DNA synthesis was inhibited by 20–30%. This inhibition was found using nuclear preparations from both normal and regenerating rat livers (Tables 2 and 3). The same concentration of aphidicolin inhibits DNA polymerases a and d by 80% or more (Ohashi, et al., 1978 Aoyagi et al., 1994), while the inhibition by ddTTP is 60% or more (Tables 2 and 3). Although the amount of DNA synthesis using isolated nuclei from aged animals was lower, similar inhibitory effects were ob-
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Table 2 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from the livers of 4-month-old ratsa Inhibitor
Conditions Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 402 324 166 77
Nuclei+ATP+cyto
1191 878 195 163
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the liver of a 4-month-old rat.
served (Tables 4 and 5). The amounts of protein in the nuclear extracts from normal and regenerating liver of young rats are similar when prepared by the same methods (data not shown), and similar results were obtained using both cytoplasmic extracts. On the other hand, the amounts of protein in the nuclear extract from normal liver, the nuclear extract from regenerating liver and the cytoplasmic extracts from normal liver and regenerating liver of aged rats were 0.345 mg protein/0.25 ml, 0.290 mg protein/0.25 ml, 52 mg protein/0.25 ml and 46 mg protein/0.25 ml, respectively. The inhibitory effect of aphidicolin on DNA synthesis was unexpectedly small, while that by ddTTP was intense. The kinetics of DNA synthesis with and without each inhibitor was studied (Fig. 2). DNA synthesis was performed using nuclei from regenerating rat liver stimulated by ATP and the cytoplasmic fraction. When aphidicolin was added at the start of DNA synthesis, the DNA synthesis proceeded at a rate : 60% that of the control. When aphidicolin was added after 20 min, the rate of DNA synthesis was similar to that when aphidicolin was added at the start. The same results were obtained using ddTTP except that DNA synthesis proceeded at a rate of :20% that of the control level (Fig. 2.). Table 3 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from regenerating livers of 4-month-old ratsa Inhibitor
Conditions Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 1552 1098 449 407
Nuclei+ATP+cyto
6997 5209 2230 929
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the regenerating liver of a 4-month-old rat.
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Table 4 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from the livers of 24-month-old ratsa Inhibitor
Condition Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 288 206 130 52
Nuclei+ATP+cyto
820 593 154 88
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the liver of a 24-month-old rat.
Following DNA synthesis for 10 min, two distinct DNA peaks with maxima in fractions 30 and 33 were found, as described above (Fig. 3). At this time, 2000-fold concentrated cold dTTP was added and the labeled DNA was chased for 50 min. By this chase, the peak at fraction 33 shifted to fraction 30, however, no new peak at a heavier position was found (Fig. 3). Finally, the effects of aphidicolin and ddTTP on the size of the DNA after chase using nuclear preparations from regenerating livers of young and aged rats were studied. DNA synthesis was carried out for 10 min using the nuclear system from the regenerating liver of young rats in the presence of the cytoplasmic fraction and ATP and synthesis was continued for 50 min after the addition of cold TTP. This time, when the inhibitor — aphidicolin or ddTTP — was added after 10 min, cold TTP was also added and the chase reaction was continued for another 50 min. In the case of no inhibitor, a peak at fraction 30 was observed after chase for 50 min (Fig. 4). The same finding was observed in the sedimentation profile after DNA synthesis using the preparation from aged rats (Fig. 5). However, the addition of aphidicolin caused the amount of DNA synthesis using nuclear preparations from young rats to decrease to : 10– 20% the level in the absence of inhibitor (Fig. 4). The decrease using nuclear Table 5 Effects of aphidicolin and ddTTP on DNA synthesis in nuclei from the regenerating livers of 24-month-old ratsa Inhibitor
Conditions Nuclei
– Aphidicolin ddTTP Aphidicolin+ddTTP
Incorporated [3H]dTMP (cpm) 1231 611 430 395
Nuclei+ATP+cyto
4956 3338 1735 783
a The experimental procedures are the same as described in Section 2. cyto; Cytoplasmic fraction from the regenerating liver of a 24-month-old rat.
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Fig. 2. Effects of aphydicolin and ddTTP on DNA synthesis. DNA synthesis was performed using the nuclear system from regenerating rat liver in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract ( — — ). Aphydicolin (112.5 mg) was added from the start of the reaction ( — — ) or 20 min after the start of the reaction ( — —); ddTTP (20 mM) was added from the start of the reaction ( — — ) or 20 min after the start of the reaction (— — ).
preparations from aged animals was 30% or more (Fig. 5). On the other hand, the addition of ddTTP did not result in a transfer of the smaller DNA to the larger after the chase (Fig. 4). The sedimentation profile of the DNA after chase was similar to that with no chase (Figs. 3 and 5). The amount of [3H] labeled DNA using the nuclear and cytosol fractions from 4-month-old rats was : 1.5-fold higher than for the same fractions from 24-month-old rats (Figs. 4 and 5) and a DNA peak was observed only in fraction 30, as in the chase experiments. Thus, the sizes of the synthesized DNA in nuclear preparations from young and aged rats were similar except for quantitative differences and were not changed by the addition of aphidicolin. However, using nuclei from young rats, two peaks with maxima in fractions 30 and 33, were found by the addition of ddTTP and the amounts of these two peaks were about half those in the absence of inhibitor (Fig. 4). DNA synthesis using the nuclei from aged rats, resulted in only one peak at fraction 33 upon the addition of ddTTP; the size of this peak was : 20% that in the absence of inhibitor (Fig. 5).
4. Discussion DNA synthesis using nuclear preparations closely approximates DNA replication in vivo. The DNA synthesizing capacity of nuclei from the regenerating liver of
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young rats is higher than that of normal liver. This supports the notion that regenerating liver is highly proliferative. The DNA synthetic capacity of nuclei from aged rats is lower than that from young rats. This suggests that cell proliferation may decrease in aged rat liver. Furthermore, DNA synthesis using isolated nuclei was stimulated by ATP and the cytoplasmic preparation (Table 2). This indicates that the machinery for DNA synthesis in isolated nuclei is insufficient for maximal activity and that additional factor(s) are required for DNA replication or repair. A requirement for certain cytoplasmic factor(s) for DNA synthesis is likely. Cytoplasmic preparations from regenerating rat liver stimulate DNA synthesis more than cytoplasmic preparations from normal liver (Table 1). This result seems reasonable. When the nuclear and cytosolic fractions were prepared from the regenerating livers of young and aged rats and DNA synthesis using the two subcellular fractions from the same aged rat were examined, the stimulation ratios of DNA synthesis by ATP and cytosolic factor(s) were similar (Tables 3 and 5). Similar results were obtained using the two fractions from the livers of young and aged rats (Tables 2 and 4). However, the stimulation of DNA synthesis using nuclei from the regenerating liver of a young rat and the cytosol fraction from an aged rat was less than when both preparations were from the young rat (data not shown). The DNA synthesizing capacity of nuclei and the DNA synthesis stimulating capacity of cytosol appear to decline similarly during ageing, suggesting the machinery shares specific relationships.
Fig. 3. DNA synthesis was performed using the nuclear system from regenerating rat liver in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract. The standard reaction mixture is described in Section 2. After 10 min, the reaction was stopped and the DNA synthesized in vitro was analyzed by sucrose density gradient centrifugation as described in Section 2 (— —) or 1.25 mmol of cold TTP was added and the reaction was continued for 50 min. DNA chased with cold TTP was analyzed by the same method ( — — ).
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Fig. 4. DNA synthesis was performed using the nuclear system from the regenerating liver of a 4-month-old rat in a standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract. The standard reaction mixture is described in Section 2. After 10 min, 1.25 mmol of cold TTP only ( — — ), cold TTP and 125 mg of aphidicolin (— —), or cold TTP and 25 mM of ddTTP ( — — ) was added and the reactions were continued for 50 min. The DNA synthesized in vitro was analyzed by sucrose density gradient centrifugation as described in Section 2.
It is well known that DNA polymerases a and d are inhibited by aphidicolin (Ohashi et al., 1978 Hammond et al., 1987) and DNA polymerase b by ddTTP (Waqar et al., 1978). It is accepted that the decline in the amount of DNA synthesis caused by the inhibition of DNA replication is large, but that caused by the inhibition of DNA repair, especially base excision repair, is small. However, our data indicate that the inhibition by aphidicolin is 30–40% of total DNA synthesis using the nuclear system from young rats, whereas, the inhibition by ddTTP is :80%. It has been reported that ddTTP inhibits the incorporation of ddNTP by DNA polymerases b and g (Waqar et al., 1978). The mode of inhibition by ddNTP is well known. If ddNMP is incorporated into DNA by DNA polymerase b, DNA strands cannot incorporate the next deoxynucleotide for 2% 3% dideoxyribose. Similarly, the connection of DNA strands by DNA ligase does not occur. Therefore, DNA strands that incorporate ddNMPs do not extend. Thus, it is reasonable that small DNA strands do not enlarge (Fig. 4). On the other hand, when DNA synthesis was inhibited by the addition of aphidicolin after 10 min, DNA synthesis was inhibited by 30% at the most and the small DNA strands that peak in fraction 33 enlarge to those with a maximum in fraction 30 by chase (Fig. 4). According to our previous report (Ohashi et al., 1978), the aphidicolin concentration used inhibits DNA polymerase a by 90% or more and DNA polymerase d by : 80% (Hammond et al., 1987). When permeable cells are used, the inhibition of DNA synthesis by this concentration of aphidicolin is \ 90% (Hammond et al., 1987). As
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described above, the inhibition of DNA synthesis by the addition of aphidicolin at the start of the reaction is no more than 40% (Tables 2–5 and Fig. 3). As explanations for these results, the following hypotheses were considered. In animals, oxygen radicals are produced through normal metabolism (Chance et al., 1979) and oxidative DNA damage increases during the ageing process (Kaneko et al., 1966 Fraga et al., 1990 Higami et al., 1994) with misincorporations due to oxidative damage having been reported (Kuchino et al., 1987). It is well known that transcription-coupled repair is needed for the accurate transfer of genetic information to mRNA (Bohr, 1988). However, the accurate transfer of genetic information is the most important in DNA replication. Therefore, it is not surprising that an increase in repair capacity occurs before replication. There may be a replicationcoupled repair system to defend against the appearance of mismatched base pairs. If this hypothesis is correct, DNA synthesis is controlled by DNA polymerase b, which manages DNA repair. Namely, DNA synthesis cannot proceed if there is no correction of DNA damage or if ddTTP is incorporated. Therefore, the inhibition of DNA synthesis by ddTTP is extensive (Tables 2–5, Figs. 2, 4 and 5) and these results support the above hypothesis. On the other hand, chase with and without aphidicolin after DNA synthesis for 10 min resulted in the DNA products sedimenting in the same fraction and the amount of the large size DNA after chase with aphidicolin was :90% that in the case of no inhibitor (Fig. 4). From these results, it appears that DNA polymerase b is strongly related to DNA elongation.
Fig. 5. DNA synthesis was performed using the nuclear system from the regenerating liver of a 24-month-old rat in standard reaction mixture containing 3.0 mM ATP and 0.25 ml of cytoplasmic extract. The standard reaction mixture is described in Section 2. After 10 min, 1.25 mmol of cold TTP only ( — — ), cold TTP and 125 mg of aphidicolin ( — —) or cold TTP and 25 mM of ddTTP ( — — ) were added and the reactions were continued for 50 min. The DNA synthesized in vitro was analyzed by sucrose density gradient centrifugation as described in Section 2.
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Similar evidence has been reported from experiments using an in vitro replication system of adenovirus DNA (Ikeda et al., 1980). However, it has been reported that the most likely model for the multi-protein complex at the eukaryotic replication fork does not contain DNA polymerase b (Tsurimoto and Stillman, 1989) and that this enzyme is involved in gap filling of short patches produced by base excision repair (Mosbaugh and Linn, 1983). Therefore, DNA polymerase b may play a role in abnormal replication when DNA polymerases d and a are inhibited. DNA synthesis using an isolated nuclear system from the regenerating livers of aged rats is :80% of that using the isolated nuclei from the regenerating livers of young rats (Figs. 4 and 5). The newly synthesized DNA found by chase after the addition of aphidicolin is the large size only, as found also for young rats. However, the amount of DNA synthesis using nuclei from regenerating livers of aged rat in the presence of aphidicolin is : 60% that in the case of no inhibitor (Fig. 5). As described above, the value for young rats is : 90% (Fig. 4). Thus, DNA synthesis after the addition of aphidicolin is decreased in isolated nuclei from aged rats. On the other hand, early DNA synthesis had to be estimated by the addition of ddTTP, because the size of the DNA remains almost unchanged for the above-mentioned reason. Although a DNA peak after synthesis for 10 min was found only in fraction 33, the counts of [3H]TMP by DNA synthesis shifted to fraction 30. This shift was found even though DNA synthesis was inhibited by aphidicolin after 10 min. DNA polymerase b may also play a role in the elongation of DNA under emergency conditions. On the other hand, the large size DNA was produced slowly during DNA synthesis using isolated nuclei from aged rats. These results suggest that the amount of complete DNA is small and the velocity of DNA synthesis is slow in isolated nuclei from the regenerating liver of aged rats. The reasons for this decrease and the slow-down in DNA synthesis in aged animals may include damage to the protein machinery and template DNA for DNA synthesis. The ratios of DNA polymerase a activities extracts from the nucleoplasmic and cytoplasmic fractions of regenerating liver of young rats prepared by the method of Lynch et al. were :97 and 3%, respectively (data was not shown), and similar results were obtained from aged rats (data not shown). The DNA sizes in the nuclei prepared from regenerating livers of young and aged rats are approximately the same. Moreover, it has been reported that nuclear size distributions from rapidly growing cells are smaller than from slow or non-growing cells (Mitsui and Schneider, 1976). Therefore, it is not likely that the nuclei from aged rats are more broken and susceptible to permeability than nuclei from young rats. Lambda phage DNA (48 kb) as a marker was sedimented in fraction 31. On the other hand, replication units (replicons) are heterogeneous in size, varying from 50 to 300 kb (Hand, 1978). The newly synthesized DNA produced in the isolated nuclear system appeared in fraction 30 and this value is included in the above variation. DNA from liver cells sediments near the bottom of the tube and DNA from nuclei sediments to fraction 23 (Fig. 1A). The DNA from pure nuclei may be broken due to the physical force of centrifugation. In all cases, the size of the DNA synthesized in rat liver is considerably longer than that synthesized in vitro. The DNA synthesized early appeared in fraction 33, but changed over time to two
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peaks in fractions 30 and 33 (Fig. 1B). This result suggests that the DNA in fraction 30 may represent the average size of the complete replicon. We can clarify some factors related to the decline in DNA synthesis in normal and regenerating liver from aged rats. Decreases in DNA replication and repair with ageing may be caused by the factors described above as well as by machinery for DNA synthesis in nuclei.
Acknowledgements We would like to thank Dr Shiro Urano for the generous gift of aphidicolin and Dr Margaret Dooley-Ohto for assistance with the manuscript.
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