J. Mol. Biol. (1977) 117, 273-277
LETTERS
TO THE EDITOR
Evidence for Semi-conservative Replication of Mitochondrial DNA from Paramecium aurelia The mode of replication of mitochondrial DNA in Paramecium aurelia w&s Density gradient analysis showed studied using 5-bromouracil incorporation. that 5-BrUra-substituted mitochondrial DNA had a density of 1.702 g/cm3, corresponding to 4% substitution in the duplex. Analysis of monomer length molecules revealed that thest consisted of a mixture of normal density and 5-BrUra-substituted DNA, whereas dimer length molecules consisted of only 5-BrUra-substituted DNA. Analysis of denatured, 5BrUrasubstituted DNA indicated that the 5-BrUra was contained in just one strand, while the other strand had the density expected of normal mitochondrial DNA. It was concluded that the linear molecules from mitochondrial DN.4 of P. art&in replicate via a semi-conservative mode of replication.
work from our laboratory has shown that mitochondrial DNA from Pwaaurelia exists as a linear molecule, 14 pm in length and that replication proceeds via a lariat-molecule inbermediate, terminating in a linear, dimer length molecule (Goddard & Cummings, 1975,1977). The linear dimer molecule consists of two monomer units associated in a head-to-head configuration which allows for rapid snapback renaturation (Goddard & Cummings, 1977). Monomer length molecules also exhibited some snapback renaturation, which indicated that there is a strong linkage at the initiation end of the molecule that is maintained throughout the replication process. Snapback renaturation of monomer molecules but not dimers was prevented by treatment with S, nuclease (Goddard t Cummings, 1977). These results are summarized in the replication scheme depicted in Figure 1. Steps A through D are a direct interpretation of the data; i.e. single-stranded DNA linkage of the initiation end of the linear molecule followed by unidirectional replication resulting in dimer length molecules. It is the processing of dimer molecules into monomer molecules which is at issue. Pathway I would lead to monomers by cleavage at the exact mid-region of the dimer, resulting in two open-ended monomers. Replication would then be started by closure of the initiation end of the molecule. This pathway is supported by our finding that only about 300,b of monomer length molecules were subject to snapback renaturation. However, it is possible that other factors could account for this. Alternatively, processing could occur according to the scheme shown in pathway I1 (steps F and G). Here, unwinding of the dimer molecule followed by snapback renaturation would lead to two closed-end monomer length molecules. While unlikely, unwinding proteips have been characterized, especially in bacteriophage and bacteria (Alberts, 1970; Abdel-Monem et al., I977), so this pathway cannot be ruled out arbitrarily. Both pathways are important for understanding DNA replication of this linear molecule. In pathway I, two enzyme activities would be necessary; a specific endonuclease for cleaving dimers and a ligase for closing the initiation end. Pathway II would require only an unwinding protein. Since dimer molecules accumulate when 18 273 Recent
mecium
271
I).
.I.
('linrhlrNGs
the cells are grown in the presence of chloramphenicol (Goddard & Cummings. 1975). all these possible proteins would presumably be synthesized in the mitochondria. As indicated by the thick and narrow lines in Figure 1. pat’hway I can IJ~ distillreplication(E) guished from pathway I I in that pathway I would IJ~ semi-conservat,ivc whereas pathway T1 would bc conservat,ive (G) (Delbruck & Stent, 1957). To date. DNd from bacteria (Meselson & Stahl, 1958), chloroplasts (Chiang & Sueoka, 1967). mitochondria of Neurospora (Reich & Luck, 1966) and rat liver (Gross & Rabinowitz. 1969), as well as nuclear DNA (Simon. 1961; Gross & Ra,binowit’z. 1969; Luk & Bick. 1977) all undergo semi-conservative replication. Ho~evcr. none ofthrse DN$s possesses t,he unique replication proper&s of mtDNA? from P. nut-din. so it. is of special interest to determine its mode of replication. Because of difficulties encountered in labeling directly mtDNA from Paramecium mutant of Escherichin coli R (Goddard & Cummings. 1975), a thymine-requiring labeled with 5BrUra (Pettijohn & Hanaualt, 1964: Hanawalt, 1967) was used to feed the paramecia. Analysis in the Beckman model E ultracentrifuge indicated that approximately half t’he E. coZi B DNA was of h.vbrid density and half of the fully substituted DNA density. I-‘. aureliu. species 1. stock 513 was grown in scotch grass infusion at 27”C, supplemented with Klebsbellcc aerogenes, to a cell density of about 1000 cells per ml. which corresponds to balanced growth condit’ions (Goddard & Cummings, 1975). The cells wcrt: harvested. concentrated about 50-fold and starved for one hour in unbacterized grass infusion to eliminate the Klrbsiella. These cells were t’hen diluted 50-fold int,o fresh grass infusion containing u.v.-irradiat’ed, 5E. COGB BrUra-labeled E. coli B, plus 100 pg 5-BrUra per ml. The 5BrUra-labeled was u.v.-irradiated to inhibit growth during feeding and, while additional 5-BrUra \vas not completely necessary: control experiments indicated slightly improved labeling in its presence. Growth of the paramecia was continued for 7 to 19 hours. and mitochondria were isolated as previouslv described (Goddard & Cummings. 1975). mt)DNA was purified in CsCl gradiems, and all the DNA contained between t’he Klebsiella and nuclear DNA markers was analyzed by digestion with EcoRI restriction endonuclease to be certain it was of mitochondrial origin (Cummings et al.. 1976). No other DNA was detected in these gradients, nor was mt,DNA of greater density found. The density distribution of the mtDNA was t’hen detormincd in the model E ultracentrifuge (37,020 revs/min at 20°C for 45 h). The first appearance of density labeled mtDNA was noted at 7 hours ; the amount of this DNA increased progressively until about 16 hours, and further growth did not lead to further labeling (Fig. 2). This could be due t’o the growth conditions themselves or it could be a specific property of mtDNA replication. ,4 similar pattern of labeling has been noted for mtDNA from yeast (Leff & Lam, 1976), although increased 5-BrUra labeling occurred after additional growth for 24 hours. Bs can be noted, only two density species of mtDNA were found, rather than the three which would be expected from model TI (Fig. 1). While these results were consistent wit’11 semiconservative replication, it was essential to determine that the 5BrUrasubstituted DNA molecules were hybrids and that hybrid dimer molecules had the same density as monomers. Dimer and monomer mtDNA from 16-hour growth samples were isolated by sucrose gradient cent’rifugation (Goddard & Cummings. 1975) and analyzed. The monomer preparation was a mix of normal and 5-BrUra-substituted DNA and, although just small quantities of dimers were available, only the density of the i Abbreviations
used:
mt,DNA,
mitochondrial
DNA;
5.BrUra,
5.bromouracil.
LETTERS
TO
THE
G
Fro. 1. Schematic diagram of mtDNA processing linear dimer molecules.
replication
275
EDITOR
'
t
with
2 alternate
pathways
(I and
II)
for
FIG. 2. Densitometer tracings of mtDNA CsCl equilibrium gradients after growth using S-BrUralabelptl E. cvli B. Tracings were taken with an Ort,ec scanning densitometer. Normal mtDNA had a density of 1.697 g/cm3 and the 5.BrUra-substituted DNA had a density of 1.702 g/cm3, using macronuclear DSA (1.685 g/cm3) and KZebsieZZa DNA (1.718 g/cmR) a? markers.
5-Br~~ra-subst’it,uted DNA was observed (Fig. 3). Even after 19 hours, some of the mtDNA was unsubstituted, which may indicate some damage to the cells as a result, of harvest’ing, starving and resuspensionz or it may simply be a reflection of some anomaly in 5BrUra substitution, as has been not,ed in bacteria (Hanawalt, 1967). Due to losses in purification, attempts to study denatured DNA from purified monomer and dimer molecules separately were unsuccessful. However, denatured mtDNA from t,he mixed population cont,ained in the 16-hour samples had a bimodal distribu-
276
D. J. (‘UMMIN(:S
16 h monomer
FIG. 3. Densitometer denatured molecules.
tracings
of mtDNA
CsCl equilibrium
gradients
of dimer, monomer
and
tion (Fig. 3). The bulk of the molecules had a density of 1.714 g/cm3, similar to that found for unsubstituted, denatured mtDNA (Goddard, 1976) and the remainder had a density of 1.727 g/cm 3. Based on the equation given by Luk & Bick (1977), the amount of 5BrUra labeling in the denatured DNA was estimated at about lo?;), compared with the 4% substitution estimated in the native DNA. Since approximately two-thirds of the native DNA was 5BrUra-substituted, the relative amounts of the light and heavy denatured DNA, as well as their densities, are consistent with the conclusion that the native 5BrUra-substituted DNA consisted of hybrid molecules. The low percentage of 5BrUra substitution was not surprising, since macronuclear DNA constitutes about 97% of the total cellular DNA (Allen & Gibson, 1972). As expected, macronuclear DNA was substituted to about 200/, 5BrUra (unpublished observations). Based on the general pattern of incorporation of 5-BrUra, the density of dimer and monomer molecules, and the density of 5-BrUra-substituted, denatured DNA, I conclude that mtDNA from P. au&a replicates semi-conservatively. In principle, 19751977) indicating that dimer our earlier conclusion (Goddard & Cummings, molecules are processed directly into two duplex monomer molecules by endonucleolytic cleavage is correct. These results also show that dimer molecules arc intermediates in mtDNA replication, but detailed evidence on the mechanism of replication of this unusual linear DNA must await further experimentabion. I thank It. A. M&i for examining the many DNA fractions wit,11 EcoRI. The work reported hero was supported by grant no. RMS75-11319 from tire National Science Foundation and by grant no. GM21948 from tllc National Institut,c of General
Medical Science. University of Colorado Medical Centw Department of Microbiology and Imm~mology Denver, Col. 80262 U.S.A. Received
19 August
1977
LETTERS
TO
THE
EDITOR
277
REFERENCES Abdel-Monem, J. Mol. Alberts, B. Allen, S. & Chiang, K. Cummings,
M., Lauppe, H., Kartenbeck, J., Diirwald, H. 8: Hoffman-Berling, H. Biol. 110, 667685. M. (1970). Fed. Proc. Fed. Amer. Sot. Exp. Biol. 29, 1154-1163. Gibson, I. (1972). Biochem. Genet. 6, 2933313. S. & Sueoka, N. (1967). Proc. Nat. Acad. Sci., U.S.il. 57, 1506-1521. D. J., Goddard, J. M. & Maki, R. A. (1976). In The Genetic Fulzction of Mitochonckd DNA (Saccone, D. 8: Kroon, A. M., cds), pp. 119.-130, Elsevier/North Holland Biomedical Press, Amsterdam. Delbruck, M. & Stent, G. S. (1957). In The Chemical Basis of Heredity (McElroy, W. D. & Glass, B., eds), pp. 6999736, The Johns Hopkins Press, Baltimore. Goddard, J. M. (1976). Ph.D. thesis, University of Colorado Medical Center. Goddard, J. M. & Cummings, D. J. (1975). J. illoZ. Biol. 97, 593-609. Goddard, J. M. & Cummings, D. J. (1977). J. Mol. BioZ. 109, 327-344. Gross, N. .J. 8c Rahinowitz, M. (1969). J. Biol. Chem. 244, 1563-1566. Hanawalt, 1’. C. (1967). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12X, pp. 702-708, Academic Press, New York. Leff, J. & Lam, K. B. (1976). J. Bacterial. 127, 354-361. Luk, D. C. & Bick, M. D. (1977). Anal. Biochem. 77, 346-349. Meselson, M. & Stahl, F. W. (1958). Proc. Nat. Acad. Xci., U.S.A. 44, 671-682. Pettijoh, D. & Hanawalt, P. C. (1964). J. Mol. BioZ. 9, 395-409. Reich, E. & Luck, D. J. L. (1966). Proc. Nat. Acad. Sci., F.8.A. 55, 1600-1608. Simon, E. H. (1961). J. Mol. BioZ. 3, 101-109.
LETTERS
TO THE EDITOR
Evidence for Semi-conservative Replication of Mitochondrial DNA from Paramecium aurelia The mode of replication of mitochondrial DNA in Paramecium aurelia w&s Density gradient analysis showed studied using 5-bromouracil incorporation. that 5-BrUra-substituted mitochondrial DNA had a density of 1.702 g/cm3, corresponding to 4% substitution in the duplex. Analysis of monomer length molecules revealed that thest consisted of a mixture of normal density and 5-BrUra-substituted DNA, whereas dimer length molecules consisted of only 5-BrUra-substituted DNA. Analysis of denatured, 5BrUrasubstituted DNA indicated that the 5-BrUra was contained in just one strand, while the other strand had the density expected of normal mitochondrial DNA. It was concluded that the linear molecules from mitochondrial DN.4 of P. art&in replicate via a semi-conservative mode of replication.
work from our laboratory has shown that mitochondrial DNA from Pwaaurelia exists as a linear molecule, 14 pm in length and that replication proceeds via a lariat-molecule inbermediate, terminating in a linear, dimer length molecule (Goddard & Cummings, 1975,1977). The linear dimer molecule consists of two monomer units associated in a head-to-head configuration which allows for rapid snapback renaturation (Goddard & Cummings, 1977). Monomer length molecules also exhibited some snapback renaturation, which indicated that there is a strong linkage at the initiation end of the molecule that is maintained throughout the replication process. Snapback renaturation of monomer molecules but not dimers was prevented by treatment with S, nuclease (Goddard t Cummings, 1977). These results are summarized in the replication scheme depicted in Figure 1. Steps A through D are a direct interpretation of the data; i.e. single-stranded DNA linkage of the initiation end of the linear molecule followed by unidirectional replication resulting in dimer length molecules. It is the processing of dimer molecules into monomer molecules which is at issue. Pathway I would lead to monomers by cleavage at the exact mid-region of the dimer, resulting in two open-ended monomers. Replication would then be started by closure of the initiation end of the molecule. This pathway is supported by our finding that only about 300,b of monomer length molecules were subject to snapback renaturation. However, it is possible that other factors could account for this. Alternatively, processing could occur according to the scheme shown in pathway I1 (steps F and G). Here, unwinding of the dimer molecule followed by snapback renaturation would lead to two closed-end monomer length molecules. While unlikely, unwinding proteips have been characterized, especially in bacteriophage and bacteria (Alberts, 1970; Abdel-Monem et al., I977), so this pathway cannot be ruled out arbitrarily. Both pathways are important for understanding DNA replication of this linear molecule. In pathway I, two enzyme activities would be necessary; a specific endonuclease for cleaving dimers and a ligase for closing the initiation end. Pathway II would require only an unwinding protein. Since dimer molecules accumulate when 18 273 Recent
mecium
271
I).
.I.
('linrhlrNGs
the cells are grown in the presence of chloramphenicol (Goddard & Cummings. 1975). all these possible proteins would presumably be synthesized in the mitochondria. As indicated by the thick and narrow lines in Figure 1. pat’hway I can IJ~ distillreplication(E) guished from pathway I I in that pathway I would IJ~ semi-conservat,ivc whereas pathway T1 would bc conservat,ive (G) (Delbruck & Stent, 1957). To date. DNd from bacteria (Meselson & Stahl, 1958), chloroplasts (Chiang & Sueoka, 1967). mitochondria of Neurospora (Reich & Luck, 1966) and rat liver (Gross & Rabinowitz. 1969), as well as nuclear DNA (Simon. 1961; Gross & Ra,binowit’z. 1969; Luk & Bick. 1977) all undergo semi-conservative replication. Ho~evcr. none ofthrse DN$s possesses t,he unique replication proper&s of mtDNA? from P. nut-din. so it. is of special interest to determine its mode of replication. Because of difficulties encountered in labeling directly mtDNA from Paramecium mutant of Escherichin coli R (Goddard & Cummings. 1975), a thymine-requiring labeled with 5BrUra (Pettijohn & Hanaualt, 1964: Hanawalt, 1967) was used to feed the paramecia. Analysis in the Beckman model E ultracentrifuge indicated that approximately half t’he E. coZi B DNA was of h.vbrid density and half of the fully substituted DNA density. I-‘. aureliu. species 1. stock 513 was grown in scotch grass infusion at 27”C, supplemented with Klebsbellcc aerogenes, to a cell density of about 1000 cells per ml. which corresponds to balanced growth condit’ions (Goddard & Cummings, 1975). The cells wcrt: harvested. concentrated about 50-fold and starved for one hour in unbacterized grass infusion to eliminate the Klrbsiella. These cells were t’hen diluted 50-fold int,o fresh grass infusion containing u.v.-irradiat’ed, 5E. COGB BrUra-labeled E. coli B, plus 100 pg 5-BrUra per ml. The 5BrUra-labeled was u.v.-irradiated to inhibit growth during feeding and, while additional 5-BrUra \vas not completely necessary: control experiments indicated slightly improved labeling in its presence. Growth of the paramecia was continued for 7 to 19 hours. and mitochondria were isolated as previouslv described (Goddard & Cummings. 1975). mt)DNA was purified in CsCl gradiems, and all the DNA contained between t’he Klebsiella and nuclear DNA markers was analyzed by digestion with EcoRI restriction endonuclease to be certain it was of mitochondrial origin (Cummings et al.. 1976). No other DNA was detected in these gradients, nor was mt,DNA of greater density found. The density distribution of the mtDNA was t’hen detormincd in the model E ultracentrifuge (37,020 revs/min at 20°C for 45 h). The first appearance of density labeled mtDNA was noted at 7 hours ; the amount of this DNA increased progressively until about 16 hours, and further growth did not lead to further labeling (Fig. 2). This could be due t’o the growth conditions themselves or it could be a specific property of mtDNA replication. ,4 similar pattern of labeling has been noted for mtDNA from yeast (Leff & Lam, 1976), although increased 5-BrUra labeling occurred after additional growth for 24 hours. Bs can be noted, only two density species of mtDNA were found, rather than the three which would be expected from model TI (Fig. 1). While these results were consistent wit’11 semiconservative replication, it was essential to determine that the 5BrUrasubstituted DNA molecules were hybrids and that hybrid dimer molecules had the same density as monomers. Dimer and monomer mtDNA from 16-hour growth samples were isolated by sucrose gradient cent’rifugation (Goddard & Cummings. 1975) and analyzed. The monomer preparation was a mix of normal and 5-BrUra-substituted DNA and, although just small quantities of dimers were available, only the density of the i Abbreviations
used:
mt,DNA,
mitochondrial
DNA;
5.BrUra,
5.bromouracil.
LETTERS
TO
THE
G
Fro. 1. Schematic diagram of mtDNA processing linear dimer molecules.
replication
275
EDITOR
'
t
with
2 alternate
pathways
(I and
II)
for
FIG. 2. Densitometer tracings of mtDNA CsCl equilibrium gradients after growth using S-BrUralabelptl E. cvli B. Tracings were taken with an Ort,ec scanning densitometer. Normal mtDNA had a density of 1.697 g/cm3 and the 5.BrUra-substituted DNA had a density of 1.702 g/cm3, using macronuclear DSA (1.685 g/cm3) and KZebsieZZa DNA (1.718 g/cmR) a? markers.
5-Br~~ra-subst’it,uted DNA was observed (Fig. 3). Even after 19 hours, some of the mtDNA was unsubstituted, which may indicate some damage to the cells as a result, of harvest’ing, starving and resuspensionz or it may simply be a reflection of some anomaly in 5BrUra substitution, as has been not,ed in bacteria (Hanawalt, 1967). Due to losses in purification, attempts to study denatured DNA from purified monomer and dimer molecules separately were unsuccessful. However, denatured mtDNA from t,he mixed population cont,ained in the 16-hour samples had a bimodal distribu-
276
D. J. (‘UMMIN(:S
16 h monomer
FIG. 3. Densitometer denatured molecules.
tracings
of mtDNA
CsCl equilibrium
gradients
of dimer, monomer
and
tion (Fig. 3). The bulk of the molecules had a density of 1.714 g/cm3, similar to that found for unsubstituted, denatured mtDNA (Goddard, 1976) and the remainder had a density of 1.727 g/cm 3. Based on the equation given by Luk & Bick (1977), the amount of 5BrUra labeling in the denatured DNA was estimated at about lo?;), compared with the 4% substitution estimated in the native DNA. Since approximately two-thirds of the native DNA was 5BrUra-substituted, the relative amounts of the light and heavy denatured DNA, as well as their densities, are consistent with the conclusion that the native 5BrUra-substituted DNA consisted of hybrid molecules. The low percentage of 5BrUra substitution was not surprising, since macronuclear DNA constitutes about 97% of the total cellular DNA (Allen & Gibson, 1972). As expected, macronuclear DNA was substituted to about 200/, 5BrUra (unpublished observations). Based on the general pattern of incorporation of 5-BrUra, the density of dimer and monomer molecules, and the density of 5-BrUra-substituted, denatured DNA, I conclude that mtDNA from P. au&a replicates semi-conservatively. In principle, 19751977) indicating that dimer our earlier conclusion (Goddard & Cummings, molecules are processed directly into two duplex monomer molecules by endonucleolytic cleavage is correct. These results also show that dimer molecules arc intermediates in mtDNA replication, but detailed evidence on the mechanism of replication of this unusual linear DNA must await further experimentabion. I thank It. A. M&i for examining the many DNA fractions wit,11 EcoRI. The work reported hero was supported by grant no. RMS75-11319 from tire National Science Foundation and by grant no. GM21948 from tllc National Institut,c of General
Medical Science. University of Colorado Medical Centw Department of Microbiology and Imm~mology Denver, Col. 80262 U.S.A. Received
19 August
1977
LETTERS
TO
THE
EDITOR
277
REFERENCES Abdel-Monem, J. Mol. Alberts, B. Allen, S. & Chiang, K. Cummings,
M., Lauppe, H., Kartenbeck, J., Diirwald, H. 8: Hoffman-Berling, H. Biol. 110, 667685. M. (1970). Fed. Proc. Fed. Amer. Sot. Exp. Biol. 29, 1154-1163. Gibson, I. (1972). Biochem. Genet. 6, 2933313. S. & Sueoka, N. (1967). Proc. Nat. Acad. Sci., U.S.il. 57, 1506-1521. D. J., Goddard, J. M. & Maki, R. A. (1976). In The Genetic Fulzction of Mitochonckd DNA (Saccone, D. 8: Kroon, A. M., cds), pp. 119.-130, Elsevier/North Holland Biomedical Press, Amsterdam. Delbruck, M. & Stent, G. S. (1957). In The Chemical Basis of Heredity (McElroy, W. D. & Glass, B., eds), pp. 6999736, The Johns Hopkins Press, Baltimore. Goddard, J. M. (1976). Ph.D. thesis, University of Colorado Medical Center. Goddard, J. M. & Cummings, D. J. (1975). J. illoZ. Biol. 97, 593-609. Goddard, J. M. & Cummings, D. J. (1977). J. Mol. BioZ. 109, 327-344. Gross, N. .J. 8c Rahinowitz, M. (1969). J. Biol. Chem. 244, 1563-1566. Hanawalt, 1’. C. (1967). In Methods in Enzymology (Grossman, L. & Moldave, K., eds), vol. 12X, pp. 702-708, Academic Press, New York. Leff, J. & Lam, K. B. (1976). J. Bacterial. 127, 354-361. Luk, D. C. & Bick, M. D. (1977). Anal. Biochem. 77, 346-349. Meselson, M. & Stahl, F. W. (1958). Proc. Nat. Acad. Xci., U.S.A. 44, 671-682. Pettijoh, D. & Hanawalt, P. C. (1964). J. Mol. BioZ. 9, 395-409. Reich, E. & Luck, D. J. L. (1966). Proc. Nat. Acad. Sci., F.8.A. 55, 1600-1608. Simon, E. H. (1961). J. Mol. BioZ. 3, 101-109.
