9 by Springer-Verlag 1978
Correlation between Deoxyribonuclease Activity and DNA Replication in the Embryonic Axes of Germinating Peas (Pisum sativum L.) Sheila M. Jenns and John A. Bryant* Department of Botany, University College, P.O. Box 78, Cardiff CF1 1XL, U.K.
Abstract. Crude chromatin preparations from pea seedlings contain calcium-dependent deoxyribonuclease activity, at least part of which is endonucleolytic. During germination, there is a dramatic increase in chromatin-bound deoxyribonuclease activity in the embryonic axis immediately prior to the onset of DNA replication. Evidence has been obtained for the presence of an inhibitor of deoxyribonuclease activity in chromatin preparations from embryonic axes not undergoing DNA replication, Key words: Chromatin - DNA Pisum - Replication (DNA).
Germination -
Introduction Chromosomes of eukaryotic organisms contain very long DNA molecules. Fibre autoradiography of these DNA molecules indicates that during replication, DNA synthesis is initiated at several hundred points along each molecule (Cairns, 1966; Huberman and Riggs, 1968; Callan, 1973; Huberman etal., 1973). The process of semiconservative replication requires strand separation prior to synthesis of new daughter strands. The difficulty of complete strand separation being initiated at several hundred points in one double helix strongly suggests that an endonucleolytic nicking of at least one strand occurs at each initiation point (Lewin, 1974; Bryant, 1976). This suggestion is supported by a growing body of evidence that nuclear or chromatin-bound endodeoxyribonuclease activity is associated with periods of DNA synthesis in animal cells (O'Connor, 1969, 1971; Ove etal. 1969 ; De Petrocellis and Parisi, 1972, 1973). In several instances, the endodeoxyribonuclease has been shown *
To whom reprint requests should be sent
to be calcium-dependent (Burzio and Koide, 1973; Hewish and Burgoyne, 1973; Junowicz and Spencer, 1973 a, b) and calcium-dependent "priming" of DNA for replication has been observed in isolated nuclei from rat liver and rat thymus (Burgoyne etal., 1970a,
b). There is also some evidence from plant cells that deoxyribonucleases may be associated with DNA replication. In the green algae Chlorella and Euglena, peaks of deoxyribonuclease activity are associated with periods of DNA synthesis, but there has been no characterization of the enzymes in terms of mode of action or cellular location (Sch6nherr et al., 1970; Walther and Edmunds, 1970). The nuclei of Tradescantia pollen grains contain an actinomycin-D-sensitire endodeoxyribonuclease (Kligman and Takats, 1975). Actinomycin-D inhibits DNA synthesis in these nuclei without affecting either the DNA template or DNA polymerase. This strongly suggests that the activity of the actinomycin-sensitive endonuclease is necessary for DNA synthesis. In this laboratory we are studying the regulation of DNA synthesis in the embryonic axis of germinating pea (Pisum sativum L.) seeds. During germination of whole seeds at 22~ C, there is a lag of 29 to 30 h before the onset of DNA replication (Robinson and Bryant, 1975a). Several hours before the onset of DNA replication, both soluble DNA polymerase (polymerase-~) and chromatin-bound DNA polymerase (polymerase-/3) show three- to four-fold increases in activity (Robinson and Bryant, 1975b). The timing of these increases in DNA polymerase activity suggests that polymerase activity on its own does not regulate the rate of DNA synthesis. We have therefore investigated a number of other possible regulatory mechanisms, and in this paper we show that there is a close correlation between increased deoxyribonuclease activity and the onset of DNA replication.
0032-0935/78/0138/0099/$01.00
100
S.M. Jenns and J.A. Bryant: Deoxyribonuclease Activity in Germinating Peas
Materials and Methods Germination and growth of plants was carried out as described previously (Robinson and Bryant, 1975a). Extraction and Assay of Chromatin-bound Deoxyribonuclease Crude chromatin pellets (prepared as described by Robinson and Bryant, 1975b) were resuspended in cold extraction buffer (2 ml for each original gram fresh weight of tissue). Deoxyribonuclease in the chromatin suspension was assayed in a total volume of 0.3 ml, containing 0.1 ml chromatin suspension, 100 lag native calfthymus DNA, 50 mM Tris-HC1 (buffered at pH 6.6 at 37~ C), 0.33 mM Na2EDTA, 8.3 mM sucrose, 3.3 mM mercaptoethanol and 8 mM CaC12. Blanks were stopped at time zero by the addition of 2.0 ml ethanol containing 0.7 M HC104. Assay tubes were incubated at 37~ for 30 min (during which time the reaction was linear), before the addition of ethanol-HC104. After stopping the reaction, the tubes were incubated at - 1 8 ~ for 17 to 20 h, and then centrifuged at 6000 xg for 20 rain at 20~ C. The supernatants were assayed for absorbance at 260 nm.
Ion-exchange Paper Chromatography For characterisation of the reaction products of deoxyribonucIease activity, [3H]thymidine-labelledpea DNA was t~sed in the assay mixture. The supernatants obtained from the assay (see above) were neutralised with KOH, evaporated to ca. 0.1 ml and aliquots of 50 gl were applied to Whatman DEAE-cellulose paper. Native DNA, sonicated DNA, dTMP and thymidine were used as stan9 dards. The papers were developed for ten hours, using 0.25 M NH4HCO 3 as solvent in a descending chromatography system. After drying the papers, spots were located by exposure to a U.V. light source (254 nm). Radioactivity on the chromatograms was estimated essentially as described by Bryant et al. (1974).
Detection of Endonuclease Activity Endonuelease activity was detected as described by McLennan and Keir (1975). The enzyme preparation was diluted five-fold and 0.1 ml of dilute preparation was added to 0.1 ml of assay mixture to give final concentrations of 50 mM Tris-HC1 (pH 6.6 at 37~ 0.33 mM NagEDTA, 8.3 mM Sucrose, 3 mM mercaptoethanol and 8 mM CaC12. Two to 50 gg 3H-labelled supercoiled, circular SV 40 DNA was present as substrate. Unlabelled supercoiled, circular DNA (SV 40 or Col El) or 3H-labelled linear DNA was added as a carrier if necessary. The reaction was stopped after 15 rain by rapid chilling to 0~ C, foliowed by addition of I0 txi of 2 M NaOH-0.2 M Na2EDTA. The reaction mixture was layered onto an alkaline sucrose gradient (pH 11, 5-20%, w/v, sucrose), containing 0.3% (w/v) Sarcosyl (Sodium lauroyl sarcosinate). Gradients were centrifuged at 39,000rpm for 2h at 15~ in the SW50 rotor of a Beckman L5-65 ultra-centrifuge. After centrifugation, tube contents were removed by upward displacement and fractionated into scintillation vials. The fractions were nuetralised with 3 M HC1 and then counted in Toluene-; Triton X100-, PPO-, POPOP-scintillation cocktail.
Results Optimal Assay Conditions In a series of p r e l i m i n a r y experiments we established the assay c o n d i t i o n s shown in Table 1. U n d e r these
Table l. Optimum assay conditions for chromatin-bound deoxyribonuclease DNA conc'n Caz+ conc'n Mgz+ conc'n pH
333 gg/ml 8 mM 0 mM 6.6
conditions, the reaction is linear for at least 30 m i n at 37 ~ C. The p H o p t i m u m is exhibited in b o t h citratep h o s p h a t e buffer a n d Tris-HC1 buffer. However, the c i t r a t e - p h o s p h a t e buffer causes r e m o v a l of c a l c i u m by precipitation, a n d so gives less consistent results t h a n the Tris-HC1 buffer. Tris-HC1 is therefore used routinely, a l t h o u g h p H 6,6 is m a r g i n a l l y outside its efficient buffering range, even a t 37 ~ C.
Characterization o f the Mode o f Action After i o n - e x c h a n g e p a p e r c h r o m a t o g r a p h y o f the hydrolysis p r o d u c t s p r o d u c e d f r o m labelled D N A by c h r o m a t i n - b o u n d deoxyribonuclease, we are able to detect radioactivity in regions of the c h r o m a t o g r a m c o r r e s p o n d i n g to oligonucleotides a n d to m o n o - n u cleotides (Fig. 1). The d i s t r i b u t i o n of radioactivity is n o t altered by the o m i s s i o n of c a l c i u m f r o m the assay, a l t h o u g h the reaction proceeds m o r e slowly. The presence of b o t h oligo- a n d m o n o - n u c l e o t i d e s suggests that the c h r o m a t i n extracts c o n t a i n b o t h e n d o - a n d exo-nuclease. The presence of radioactivity r u n n i n g faster t h a n m o n o n u c l e o t i d e s suggests that the crude extracts also c o n t a i n p h o s p h a t a s e activity. I n order to show conclusively w h e t h e r or n o t endonuclease is present in the c h r o m a t i n extracts, supercoiled, closed-circular SV40 D N A has been used as a substrate. Figure 2 clearly indicates that the chrom a t i n - b o u n d d e o x y r i b o n u c l e a s e is able to degrade closed-circular D N A , since there is n o rapidly-sedim e n t i n g radioactive m a t e r i a l (A, in Fig. 2a) after enzyme action. This m e a n s t h a t at least p a r t of the activity is end0nucleolytic. The crude e n z y m e prepar a t i o n can also reduce the size of linear D N A , since the p r o p o r t i o n of m a t e r i a l s e d i m e n t i n g n e a r the top of the g r a d i e n t at p o s i t i o n B (Fig. 2a) is c o n s i d e r a b l y reduced by enzyme action, whereas the p r o p o r t i o n o f m a t e r i a l floating o n the g r a d i e n t (C).is increased. This is c o n s i s t e n t with either e n d o - or exonuclease activity. O m i s s i o n o f c a l c i u m does n o t alter the enzyme's ability to degrade circular D N A , a l t h o u g h the rate of hydrolysis is reduced. C h a r a c t e r i s a t i o n of the m o d e of action thus shows that c h r o m a t i n preparations possess e n d o d e o x y r i b o n u c l e a s e activity, a n d also possibly e x o d e o x y r i b o n u c l e a s e activity.
S.M. Jenns and J.A. Bryant: Deoxyribonuclease Activity in Germinating Peas
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Correlation between Deoxyribonuclease Activity and DNA Replication in the Embryonic Axes of Germinating Peas (Pisum sativum L.) Sheila M. Jenns and John A. Bryant* Department of Botany, University College, P.O. Box 78, Cardiff CF1 1XL, U.K.
Abstract. Crude chromatin preparations from pea seedlings contain calcium-dependent deoxyribonuclease activity, at least part of which is endonucleolytic. During germination, there is a dramatic increase in chromatin-bound deoxyribonuclease activity in the embryonic axis immediately prior to the onset of DNA replication. Evidence has been obtained for the presence of an inhibitor of deoxyribonuclease activity in chromatin preparations from embryonic axes not undergoing DNA replication, Key words: Chromatin - DNA Pisum - Replication (DNA).
Germination -
Introduction Chromosomes of eukaryotic organisms contain very long DNA molecules. Fibre autoradiography of these DNA molecules indicates that during replication, DNA synthesis is initiated at several hundred points along each molecule (Cairns, 1966; Huberman and Riggs, 1968; Callan, 1973; Huberman etal., 1973). The process of semiconservative replication requires strand separation prior to synthesis of new daughter strands. The difficulty of complete strand separation being initiated at several hundred points in one double helix strongly suggests that an endonucleolytic nicking of at least one strand occurs at each initiation point (Lewin, 1974; Bryant, 1976). This suggestion is supported by a growing body of evidence that nuclear or chromatin-bound endodeoxyribonuclease activity is associated with periods of DNA synthesis in animal cells (O'Connor, 1969, 1971; Ove etal. 1969 ; De Petrocellis and Parisi, 1972, 1973). In several instances, the endodeoxyribonuclease has been shown *
To whom reprint requests should be sent
to be calcium-dependent (Burzio and Koide, 1973; Hewish and Burgoyne, 1973; Junowicz and Spencer, 1973 a, b) and calcium-dependent "priming" of DNA for replication has been observed in isolated nuclei from rat liver and rat thymus (Burgoyne etal., 1970a,
b). There is also some evidence from plant cells that deoxyribonucleases may be associated with DNA replication. In the green algae Chlorella and Euglena, peaks of deoxyribonuclease activity are associated with periods of DNA synthesis, but there has been no characterization of the enzymes in terms of mode of action or cellular location (Sch6nherr et al., 1970; Walther and Edmunds, 1970). The nuclei of Tradescantia pollen grains contain an actinomycin-D-sensitire endodeoxyribonuclease (Kligman and Takats, 1975). Actinomycin-D inhibits DNA synthesis in these nuclei without affecting either the DNA template or DNA polymerase. This strongly suggests that the activity of the actinomycin-sensitive endonuclease is necessary for DNA synthesis. In this laboratory we are studying the regulation of DNA synthesis in the embryonic axis of germinating pea (Pisum sativum L.) seeds. During germination of whole seeds at 22~ C, there is a lag of 29 to 30 h before the onset of DNA replication (Robinson and Bryant, 1975a). Several hours before the onset of DNA replication, both soluble DNA polymerase (polymerase-~) and chromatin-bound DNA polymerase (polymerase-/3) show three- to four-fold increases in activity (Robinson and Bryant, 1975b). The timing of these increases in DNA polymerase activity suggests that polymerase activity on its own does not regulate the rate of DNA synthesis. We have therefore investigated a number of other possible regulatory mechanisms, and in this paper we show that there is a close correlation between increased deoxyribonuclease activity and the onset of DNA replication.
0032-0935/78/0138/0099/$01.00
100
S.M. Jenns and J.A. Bryant: Deoxyribonuclease Activity in Germinating Peas
Materials and Methods Germination and growth of plants was carried out as described previously (Robinson and Bryant, 1975a). Extraction and Assay of Chromatin-bound Deoxyribonuclease Crude chromatin pellets (prepared as described by Robinson and Bryant, 1975b) were resuspended in cold extraction buffer (2 ml for each original gram fresh weight of tissue). Deoxyribonuclease in the chromatin suspension was assayed in a total volume of 0.3 ml, containing 0.1 ml chromatin suspension, 100 lag native calfthymus DNA, 50 mM Tris-HC1 (buffered at pH 6.6 at 37~ C), 0.33 mM Na2EDTA, 8.3 mM sucrose, 3.3 mM mercaptoethanol and 8 mM CaC12. Blanks were stopped at time zero by the addition of 2.0 ml ethanol containing 0.7 M HC104. Assay tubes were incubated at 37~ for 30 min (during which time the reaction was linear), before the addition of ethanol-HC104. After stopping the reaction, the tubes were incubated at - 1 8 ~ for 17 to 20 h, and then centrifuged at 6000 xg for 20 rain at 20~ C. The supernatants were assayed for absorbance at 260 nm.
Ion-exchange Paper Chromatography For characterisation of the reaction products of deoxyribonucIease activity, [3H]thymidine-labelledpea DNA was t~sed in the assay mixture. The supernatants obtained from the assay (see above) were neutralised with KOH, evaporated to ca. 0.1 ml and aliquots of 50 gl were applied to Whatman DEAE-cellulose paper. Native DNA, sonicated DNA, dTMP and thymidine were used as stan9 dards. The papers were developed for ten hours, using 0.25 M NH4HCO 3 as solvent in a descending chromatography system. After drying the papers, spots were located by exposure to a U.V. light source (254 nm). Radioactivity on the chromatograms was estimated essentially as described by Bryant et al. (1974).
Detection of Endonuclease Activity Endonuelease activity was detected as described by McLennan and Keir (1975). The enzyme preparation was diluted five-fold and 0.1 ml of dilute preparation was added to 0.1 ml of assay mixture to give final concentrations of 50 mM Tris-HC1 (pH 6.6 at 37~ 0.33 mM NagEDTA, 8.3 mM Sucrose, 3 mM mercaptoethanol and 8 mM CaC12. Two to 50 gg 3H-labelled supercoiled, circular SV 40 DNA was present as substrate. Unlabelled supercoiled, circular DNA (SV 40 or Col El) or 3H-labelled linear DNA was added as a carrier if necessary. The reaction was stopped after 15 rain by rapid chilling to 0~ C, foliowed by addition of I0 txi of 2 M NaOH-0.2 M Na2EDTA. The reaction mixture was layered onto an alkaline sucrose gradient (pH 11, 5-20%, w/v, sucrose), containing 0.3% (w/v) Sarcosyl (Sodium lauroyl sarcosinate). Gradients were centrifuged at 39,000rpm for 2h at 15~ in the SW50 rotor of a Beckman L5-65 ultra-centrifuge. After centrifugation, tube contents were removed by upward displacement and fractionated into scintillation vials. The fractions were nuetralised with 3 M HC1 and then counted in Toluene-; Triton X100-, PPO-, POPOP-scintillation cocktail.
Results Optimal Assay Conditions In a series of p r e l i m i n a r y experiments we established the assay c o n d i t i o n s shown in Table 1. U n d e r these
Table l. Optimum assay conditions for chromatin-bound deoxyribonuclease DNA conc'n Caz+ conc'n Mgz+ conc'n pH
333 gg/ml 8 mM 0 mM 6.6
conditions, the reaction is linear for at least 30 m i n at 37 ~ C. The p H o p t i m u m is exhibited in b o t h citratep h o s p h a t e buffer a n d Tris-HC1 buffer. However, the c i t r a t e - p h o s p h a t e buffer causes r e m o v a l of c a l c i u m by precipitation, a n d so gives less consistent results t h a n the Tris-HC1 buffer. Tris-HC1 is therefore used routinely, a l t h o u g h p H 6,6 is m a r g i n a l l y outside its efficient buffering range, even a t 37 ~ C.
Characterization o f the Mode o f Action After i o n - e x c h a n g e p a p e r c h r o m a t o g r a p h y o f the hydrolysis p r o d u c t s p r o d u c e d f r o m labelled D N A by c h r o m a t i n - b o u n d deoxyribonuclease, we are able to detect radioactivity in regions of the c h r o m a t o g r a m c o r r e s p o n d i n g to oligonucleotides a n d to m o n o - n u cleotides (Fig. 1). The d i s t r i b u t i o n of radioactivity is n o t altered by the o m i s s i o n of c a l c i u m f r o m the assay, a l t h o u g h the reaction proceeds m o r e slowly. The presence of b o t h oligo- a n d m o n o - n u c l e o t i d e s suggests that the c h r o m a t i n extracts c o n t a i n b o t h e n d o - a n d exo-nuclease. The presence of radioactivity r u n n i n g faster t h a n m o n o n u c l e o t i d e s suggests that the crude extracts also c o n t a i n p h o s p h a t a s e activity. I n order to show conclusively w h e t h e r or n o t endonuclease is present in the c h r o m a t i n extracts, supercoiled, closed-circular SV40 D N A has been used as a substrate. Figure 2 clearly indicates that the chrom a t i n - b o u n d d e o x y r i b o n u c l e a s e is able to degrade closed-circular D N A , since there is n o rapidly-sedim e n t i n g radioactive m a t e r i a l (A, in Fig. 2a) after enzyme action. This m e a n s t h a t at least p a r t of the activity is end0nucleolytic. The crude e n z y m e prepar a t i o n can also reduce the size of linear D N A , since the p r o p o r t i o n of m a t e r i a l s e d i m e n t i n g n e a r the top of the g r a d i e n t at p o s i t i o n B (Fig. 2a) is c o n s i d e r a b l y reduced by enzyme action, whereas the p r o p o r t i o n o f m a t e r i a l floating o n the g r a d i e n t (C).is increased. This is c o n s i s t e n t with either e n d o - or exonuclease activity. O m i s s i o n o f c a l c i u m does n o t alter the enzyme's ability to degrade circular D N A , a l t h o u g h the rate of hydrolysis is reduced. C h a r a c t e r i s a t i o n of the m o d e of action thus shows that c h r o m a t i n preparations possess e n d o d e o x y r i b o n u c l e a s e activity, a n d also possibly e x o d e o x y r i b o n u c l e a s e activity.
S.M. Jenns and J.A. Bryant: Deoxyribonuclease Activity in Germinating Peas
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