Volume 6 Number 51979
Nucleic Acids Research
Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture
Jean Benard*, Guy Riou* and Jean-Marie Saucier**
*Laboratoire de Pharmacologie Moleculaire and **Groupe de Recherches sur la Pharmacologie des Medicaments Anticancereux (INSERM U 140), Institut Gustave Roussy, 94800 Villejuif, France Received 22 February 1979 ABSTRACT The kinetoplast DNA rietworks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.
INTRODUCTION The kinetoplast is the specialized portion of the mitochondrial apparatus which contains in the culture form of Trypanosoma cruzi about 25 % of the
total DNA (1) organized in a complex structure (2). The biological role of the kinetoplast DNA (kDNA) has not yet been elucidated. In the electron microscope the kDNA appears as a network composed of about 25 000 double stranded nminicircular molecules (1440 base pairs) topologically interlocked or covalently bound, and of long linear molecules in urndertermined amount (2). Two different forms of kDNA networks are fractionated from T. cruzi (2), form I networks containing supercoiled minicircular molecules and form II networks in which the minicircles have single strand interruption(s). We have measured by analytical ultracentrifugation the sedimentation coefficient of the two native forms of T. cruzi kDNA, of form I relaxed by pancreatic DNase and closed in vitro with DNA ligase. Form II networks which have a higher molecular weight than form I networks are not observed in the stationary phase. These observations suggest that form II is a replicative
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1941
Nucleic Acids Research intermediate. The superhelix density of the supercoiled minicircles composing the form I kDNA networks has been determined by sedimentation velocity ethidium bromide (EthBr) titration. Very low values of the superhelix density,varying with the physiological state of the trypanosomes, were observed. In addition, when the trypanosomes were treated with the trypanocidal drug EthBr, a significant increase of the number of negative superhelical turns of the
supercoiled minicircles was detected.
MATERIALS AND METHODS Trypanosomes culture. Culture form of T. cruzi, originated from Tehuantepec strain of Institut Pasteur, was grown in a diphasic medium in conditions previously described (3). In a typical culture inoculated with 5xlO6
cells/ml, the number of trypanosomes determined using a Coultronic Counter, was at 3 days, 3.4x107; at 4 days, 6.4x10 7; at 5 days, 8.2x10 ; at 7 days, l.0xlO ; at 9 days, 9.0x10 per ml of medium. kDNA purification. Trypanosomes were harvested and immediately lysed by sarkosyl (final concentration 1 %) in 0.15 M NaCl-0.015 M sodium citrate, pH 7.2 (SSC) for 15 min. at 370C. The lysate was incubated with pronase (1 rrg/ml) for 2 h at 370C. The kDNA networks were purified by several slow speed centrifugations (9000 g for 30 min. at 40C in SSC). The two forms were separated in a CsCl gradient containing 500 1ug/ml propidium (3). The bands were collected and propidium extracted with isopropanol (4). Conversion of kDNA form I into the relaxed form and resealing with DNA ligase. kDNA form I was nicked by pancreatic DNase in the presence of an excess of EthBr to limit the endonucleolytic reaction to one single strand break per DNA minicircle (5). The incubation mixture contained 0.1 M NaCl, 2 rrM MgCl2, 1 mM Na3 EDTA, 10 mM Tris-HCl pH 8.0, 0.1 mg/ml of bovine plasma albumin, 40,pg/ml kDNA form I and 60)pg/ml EthBr. After addition of 10 g of pancreatic DNase (Worthington) per ml of solution, the reaction was monitor-ed at room temperature using a Zeiss PMv2 III spectrophotometer equipped with its fluorescence attachment. Excitation was set at 545 nm and emission at 600 nm. When more than 99 % of the minicircles were nicked, the reaction was stopped by phenol extraction and the phenol removed by dialysis. The kDNA relaxed form was closed in vitro using T4 DNA ligase (Bethesda Research Laboratories, Inc). The reaction mixture contained 4 mfM MgCl2, 1.2 rrM Na3-EDTA, 30 rrM Tris-HCl pH 8.0, 50 1g/ml of bovine plasma albumin, 0.2 mM ATP, 10 mrM dithiotreitol and 17 units of enzyme per ml. The incubation 1942
Nucleic Acids Research was carried out for 1 hr at 300C. When the in vitro closure of kDNA was performed in the presence of EthBr, the time of incubation was 2 hrs. The con-
centration of free EthBr in the incubation mixture was evaluated by fluorescence measurement of the supernatant obtained by slow speed centrifugation after completion of the reaction. The closed kDNA was phenol extracted and the phenol removed by dialysis. Analytical ultracentrifugation. A Spinco model E analytical uJltracentrifuge was used. Sedimentation coefficients were usually measured with an ANlJ rotor at 4000 rpm by band sedimentation analysis at 200C using 3 M CsCl0.01 M Na3-EDTA (pH 8.0) as bulk solvent. Each cell was filled with 15 i4 of the DNA solution previously dialysed against 2 M NaCl-0.01 M Na3-EDTA. The corrected values of sedimentation coefficients were calculated according to Bruner and Vinngrad (6). The critical EthBr concentration needed to remove all the superhelical turns of the DNA closed circles was determined by band sedimentation velocity EthBr titration. The number of bound ethidium per DNA nucleotide in these conditions ( j) c ) was calculated according to Wang (7). Isopycnic ultracentrifugation in CsCl gradient containing EthBr was carried out as described by Wang (8). The solution had an initial density of 1.56 g/cm3 and an initial EthBr concentration of 1001pg/ml. Centrifugation was performed at 40 Krpm at 200C for 48 hours. Scans were obtained at 345 nm using an ultracentrifuge equipped with a reflective mirror assembly and the Beckman photoelectric scanning system.
RESULTS
Analysis of kDNA networks by equilibrium density centrifugation in CsCl-EthBr gradient. The total kDNA purified from trypanosomes grown for 4, 7 and 9 days have been analysed by equilibrium density centrifugation in CsCl gradient containing EthBr. As shown in figure 1, the kDNA networks exist under two forms which are well resolved in these conditions. We also observed small amounts of networks of intermediate density. The two bands corresponding to form I and II of kDNA are sharp as a consequence of the high molecular weight of the networks. We have evaluated the relative amount of DNA present in each band as explained in the legend of figure 1, without taking into account the networks of intermediate density: 34 % of the 4 days kDNA and 25
% of the 7 days
are
under form II. There is no detectable form II
after 9 days (figure 1 c). The separation between the closed form of PM2 DNA and the kDNA form II (0.360 cm, figure 1 d) is nearly equal to the sepa1943
Nucleic Acids Research 11
k DNA 4 days
a 1
k DNA 7 days
b k DNA 9 days
k DNA 7days
PM2
d 1t
PM22
e 6.40
6.60
6.80
7.00
Distance to rotor axis (cm)
Figure 1. Equilibrium density centrifugation of kDNA networks in CsCl-EthBr gradients. Total kDNAs purified from trypanosomes harvested after 4 days (a), 7 days (b) and 9 days of culture (c) ; kDNA form II and covalently closed Fv2 DNA (d) and a mixture of the two forms of PM2 DNA (e). The pen excursion of the photoelectric scanner is proportional to the concentration of EthBr bound to DNA. Using the measurements of Bauer and Vinograd (9), we estimated from the distance between the two bands that A\ = 0.064 (where ; expresses the bound dye/nucleotide molar ratio). Since for an EthBr concentration of 100 g/ml, j) f= 0.168 according to Bauer and Vinograd (10), therefore
=I 0.104. form of kDNA
These values were used to estimate the relative amount of each network in the mixtures.
ration between the nicked and the closed forms of PM2 DNA (0.346 cm, figure 1 e). As PM2 DNA and kDNA differ in their base composition by only 2 % (11), this result indicates that the kDNA form II network contains essentially open circles. We have measured the separation between the two forms of kDNA 1944
Nucleic Acids Research networks in different kDNA preparations and obtained inconstant results. the
same
sample, the separation decreases
as a
This is presumably due to the nicking of the
rage.
form I networks. For the sanples analysed
sible to derive
)
as
For
function of the time of stoclosed circles in the
shown in figure 1, it is
values using the calibration
pos-
of Wang (12) after
curve
correction for the slight difference of rotor argular velocity. values obtained by this procedure
are
The -c systematically higher than the values
obtained by sedimentation velocity analysis (see below) by
a
factor of
up
to 3.
Sedimentation velocity analysis of form II networks. When analysed by band sedimentation in the analytical ultracentrifuge, the two forms of kDNA
networks exhibit
an
unimodal distribution. Linear log
obtained in spite of of the
run
a
r vs
time plots
progressive spreading of the bands during the
presumably due to
a
are
course
size heterogeneity of the networks. The sedi-
mentation coefficient of high molecular weight DNA usually exhibits a strong dependence on centrifuge speed (13). This effect is not observed with kDNA
networks: the sedimentation coefficient of a 7 days kDNA form II was measured in the absence of EthBr at 3.0, 3.4, 4.0, 4.4 Krpm. The same result = 7430 350. The sedimentation within experimental error was obtained, S 20,w
coefficient of kDNA form II was measured in the presence of various EthBr concentrations. The results plotted i n figure 3 show that the sedimentation coefficient of kDNA under this form first decreases as a function of the free EthBr concentration, then levels off at about 25 % below its initial value free EthBr concentration higher than about
1M.
for
a
was
Sedimentation velocity analysis of relaxed kDNA networks. kDNA form I relaxed by the action of pancreatic DNase and the sedimentation coeffi-
cient of the rEelaxed networks
was
measured
as a
10
function of free EthBr con-
centration (figure 3). The sedimentation coefficient first decreases of about 12 % below its initial value ,then remains constant for EthBr concentration higher than about 10 VM. The
curve is
qualitatively similar to those
obtained with kDNA form II. Sedimentation velocity analysis of kDNA networks closed in vitro with DNA
ligase.
kDNA
was
closed in vitro with DNA ligase as described in Mate-
rials and Methods and its degree of superhelicity was determined by sedimentation velocity EthBr titration. The results are plotted in figure 2 and the
; values presented in table 1
were calculated from the critical free
c
EthBr concentrations at the minimum of the curves (7). One kDNA sample was
closed in the presence of 2.5x10
mole EthBr added per mole of DNA nuclec-
1945
Nucleic Acids Research
C',
0 c
0 4-
c
0 0
E
C',D1
2
4
6
8
10
15
Free ethidium (xlO M)
Figure 2. Sedimentation coefficient (uncorrected value) as a function of free EthBr concentration of kDNA closed in vitro with DNA ligase, in the absence (-0-O-) or in the presence of 1.7x10-2 mole of EthBr bound per mole of DNA nucleotide (-0--).
tide, the amount of EthBr bound in the closure medium was found equal to 1.7xlO mole/mole of DNA nucleotide. We observe a difference of 1.8xlO between the 9 c values obtained for this sample and the kDNA closed in the absence of EthBr. This result indicates that the sedimentation velocity EthBr titration is a suitable method to measure the superhelix density of kDNA form I minicircles. Sedimentation velocity analysis of form I networks. In the absence of forrp I EthBr, the sedimentation coefficient of kDNA extracted after 7 days of cul= ture is equal to S 20,w 5460 - 150. The sedimentation coefficient of form I networks extracted after 4 days and 7 days of culture was measured as a function of free EthBr concentration (figure 3) and the Q values calculated from these data are presented in table 1. The results show that the ;) measurements performed in duplicate are reproducible within 10 %. To rule out the possibility of superhelix density changes during the extraction procedure, we have purified a kDNA sample in the presence of 250 rmtM EDTA 1946
Nucleic Acids Research
C.,
0 x c
.2_ 0
u c
0
E c D
cn
20 0
2
4
Free ethidium
6
8
10
(xl06M )
Figure 3. Sedimentation coefficient (uncorrected value) of kDNA networks as a function of free EthBr concentration. Form I (-@v@-)and form II (--0C-) were isolated from kDNA samples extracted after 4 days and 7 days of culture. kDNA form I extracted after 7 days of culture and converted to relaxed form by pancreatic DNase treatment (-A-ti-).
Table 1 kDNA
saffples
closed in vitro at 300C closed in vitro at 300C in the presence of EthBr? ( =T1.7xl0-2) 3 days kDNA form I 3 days kDNA form I extracted in presence of EDTA 4 days kDNA form I 7 days kDNA form I 7 days kDNA form I from EthBr treated trypanosomes
Free EthBr 6 (x 10 M)
)
(x
2 10)
3.3
1.9
7.2
3.7
4.1
2.3
4.3
2.4
4.3 4.0 2.2 2.3
2.2 1.3 1.3
5.7
3.0
Values of 9c of kDNA samples determined by sedimentation EthBr titration.
2.4
velocity 1947
Nucleic Acids Research (14) to inhibit hypothetical nicking-closing activities. As shown in table 1 the
same
result within experimental
fied in the
presence
error
is obtained for 3 days kDNA puri-
and in the absence of EDTA. For kDNA samples extracted
at different stages of culture,
we
observe that the superhelix density is
significantly larger during the exponential phase than during the stationary phase.
Superhelix density of form I networks from EthBr treated trypanosomes. Riou has observed that shortly after addition of EthBr to
a
culture of try-
the drug binds selectively to the kinetoplast (15) and causes subsequently the appearance of abnormal forms of kinetoplast (16), then the progressive loss of kDNA by inhibition of its replication (17). Since EthBr
panosomes,
is
a
DNA intercalating dye, it presumably induces conformational changes of
kDNA form I networks when added to
an
in vitro culture of trypanosomes. As
shown in figure 4 and table 1, the superhelix density of kDNA extracted from
trypanosomes grown
in the
presence
higher than that of trypanosomes
T
grown
of
1
,Pg/ml
of
EthBr
is
significantly
for 7 days in the absence of EthBr.
40
0
3.5
0
0
E
Free ethidium (xlO M)
Figure 4. Superhelix density measurerrent of form I networks from EthBr treated trypanosomes. 1 jig per ml of EthBr was added to a culture of trypanosomes at the stationary phase. The trypanosomes were harvested 24 hours later and after purification of kDNA form I its sedimentation coefficient was measured as a function of the free EthBr concentration. 1948
Nucleic Acids Research DISCUSSION
The buoyant density at equilibrium of kDNA networks in
a
CsCl gradient
containing EthBr is determined by the amount of dye intercalated which itself varies as a function of the superhelix free energy of the closed circular molecules (10) associated in the networks. According to Wang (12),
the separation between monomeric closed DNA circles of superhelix density
9c
= 0.02 and nicked circles is 0.505 cm. We attribute corresponding to the smaller separations actually observed between form I and II networks to the presence of linear or nicked circular DNA species in the form I networks. The nicked circular molecules might not exist in vivo, and appear during the purification and the storage of the networks, causing a gradual shift of the band of form I networks to the regions of lower density in the CsCl/EthBr gradient. Kleisen et al. (18) have observed that the separation between the two forms of kDNA networks of Crithidia luciliae is larger than the separation between nicked and closed monomeric minicircles and suggest that topological constraint between interlocked minicircles further limits the dye binding to the networks, in addition to the restriction due to the closed circular structure of the molecules. For these reasons, it seems inappropriate to use the buoyant density method of Gray et al. (19) to determine c
the superhelix density of the closed minicircles in the kDNA networks. However our measurement of the separation between form I and form II networks shows that form II networks of T. cruzi bind the same amount of ethidium as FM2 DNA form II and consequently do not contain closed circles. Another observation provided by centrifugation in a CsCl/EthBr gradient is that networks sediment as sharp bands: this indicates a great homogeneity in the relative amount of closed circles in these networks as well as in the average superhelix density of the closed minicircles in the form I networks. The two forms of kDNA networks exhibit very high sedimentation coefficients as measured by analytical ultracentrifugation. A better accuracy
is obtained with this method than by zone centrifugation in sucrose gradient (20). These high sedimentation coefficients, of the same order of magnitude as those observed previously for the folded chrmnosome of Drosophila melanogaster (21) are due to the high molecular weight of the networks and to their
comrpact structure. The higher sedimentation coefficient measured was obtained with kDNA form II in the absence of EthBr in the sedimentation medium. kDNA form I networks, after relaxation with pancreatic DNase, have a sedicoefficient of about 70 % that of form II networks. This obser-
mentation
vation indicates that form II networks have a higher molecular weight than
1949
Nucleic Acids Research form I networks, in agreement with the observations of Englund (22,23) proposes that kDNA form II is
a
replicative intermediate. In the
who
presence
increasing concentrations of EthBr, the sedimentation coefficient relaxed networks decreases as it has been previously reported for
of
of kDNA free
nicked circles (24). A much steeper decrease of the sedimentation coefficient of kDNA form II was observed, and we have no reasonable explanation for this behavior. As shown in figure 3, native kDNA form I and kDNA networks relaxed by DNase have the
same
sedimentation coefficient for
a
critical EthBr
concen-
tration. This indicates that at this point, all the superhelical turns of
removed, and justify the use of the sedimentation velocity EthBr titration in the case of kDNA networks. Using this method, we = 1.3xlO ) have found that the superhelix density of the 7 days kDNA ( is slightly higher than that of the free minicircles liberated from the the minicircles
are
c
Crithidia luciliae networks at the stationary phase (18). It is interesting
to note that this value is
very
low compared to SV40 DNA (
(19) and mitochondrial DNA from HeLa cells
(
=
9,~ =
5.8xlO 2)
4.2xlO) (25). However,
c
Rubenstein et al. (26) have reported the existence in Drosophila melanogaster of covalently closed mitochondrial DNA of superhelix density practically equal to zero. When a covalently closed circular molecule is transfered from medium containing 0.13 M NaCl to 3 M CsCl, its superhelix density changes a value corresponding to 9c = 9xlO 3 as measured by Saucier and Wang (27). Most of the superhelix density of the 7 days kDNA measured in 3 M CsCl
a
by
c
is therefore introduced by the solvent used, suggesting the absence of interaction with binding proteins able to modify the DNA structure inside the
kinetoplast. This observation could explain the extreme accessibility of kDNA to various DNA binding drugs endowed with trypanocidal activity (reviewed by Simpson, 28). The superhelix density of the networks is higher during the exponential phase of growth. This presumably reflects the interactions with the proteins involved in kDNA replication, possibly DNA gyrase (29), and in the kDNA transcription and recombination if they occur. We have not detected kDNA form II in trypanosomes in stationary phase, and this ob-
servation agrees with the results of Englund (23) showing that kDNA form II is
a
mass
replicative intermediate. Furthermore
we have found at most 35 % in
of kDNA form II during the exponential phase. From this result, we es-
timate that about 21 % of the cells at mid-exponential phase have their kDNA as
form II networks, assuming that the DNA content of form II network is
twice the DNA content of form I network.
1950
Nucleic Acids Research For trypanosomes
grown
in the
presence
of EthBr,
we
found
a
signifi-
cantly higher superhelix density of the kDNA closed circles than for trypanosomes
grown
Smith et al. (25)
without EthBr. A comparable effect has with the mitochondrial DNA of human and
the
been observed by mouse
cells in
cul-
ture, but with a much higher shift of Qc in the presence of 1 ,p/ml of EthBr. Our result supports the hypothesis that kDNA is a target for trypanocidal drugs. The determination of the superhelix density of kDNA form I by sedimentation analysis in the presence of EthBr provides a valuable method to study quantitatively the interaction in vivo of intercalating drugs with this target.
ACKNOWLEDGEMENTS We thank J. Inacio and M. Re for their skilful technical assistance.
We
are
also indebted to Dr. F. Schaeffer for his help in using the photo-
electric
scanner.
REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25. 26.
Riou, G. and Paoletti, C. (1967) J. Mol. Biol. 28, 377-382. Riou, G. and Delain, E. (1969) Proc. Natl. Acad. Sci. USA 62, 210-217. Riou, G. and Yot, P. (1977) Biochemistry 16, 2390-2396. Ostrander, D.A. and Gray, H.B. (1973) Biopolymers 12, 1387-1419. Barzilai, R. (1973) J. Mol. Biol. 74, 739-742. Bruner, R. and Vinograd, J. (1965) Biochim. Biophys. Acta 108, 18-29. Wang, J.C. (1969) J. Mol. Biol. 43, 263-272. Wang, J.C. (1974) J. Mol. Biol. 87, 797-816. Bauer, W. and Vinograd, J. (1968) J. Mol. Biol. 33, 141-171. Bauer, W. and Vinograd, J. (1970) J. Mol. Biol. 47, 419-435. Riou, G. and Pautrizel, R. (1969) J. Protozool. 16, 509-513. Wang, J.C. (1974) J. Mol. Biol. 89, 783-801. Kavenoff, R.J. (1972) J. Mol. Biol. 72, 801-806. Fairlamb, A.H., Weislogel, P.O., Hoeijmakers, J.H.J. and Borst, P. (1978) J. Cell Biol. 76, 293-309. Riou, G. (1967) C. R. Acad. Sci. Paris 265, 2004-2007. Delain, E. and Riou, G. (1969) C. R. Acad. Sci. Paris 268, 1327-1330. Riou, G. (1970) Biochemical Pharmacol. 19, 1524-1526. Kleisen, C.M., Borst, P. and Weijers, P.J. (1975) Biochim. Biophys. Acta 390, 155-167. Gray, H.B., Upholt, W.B. and Vinograd, J. (1971) J. Mol. Biol. 62, 1-19. Simpson, L. and Berliner, J. (1974) J. Protozool. 21, 382-393. Benyajati, C. and Worcel, A. (1976) Cell 9, 393-407. Englund, P.T., Di-fNlaio, D.C. and Price, S.S. (1977) J. Biol. Chem. 252, 6208-6216. Englund, P.T. (1978) Cell 14, 157-168. Crawford, L.V. and Waring, M.J. (1967) J. Mol. Biol. 25, 23-30. Smith, C.A., Jordan, J.M. and Vinograd, J. (1971) J. Mol. Biol. 59, 255-272. Rubenstein, J.L.R., Brutlag, D. and Clayton, D.A. (1977) Cell 12, 471-482.
1951
Nucleic Acids Research 27. 28.
29.
1952
Saucier, J.M. and Warg, J.C. (1973) Biochemistry 12, 2755-2758. Siffpson, L. in International Review of Cytology. Academic Press, Inc. New York and London (1972) pp. 139-207. Gellert, M., Ntizuuchi, K., O'Dea, M.H. and Nash, H.A. (1976) Proc. Natl. Acad. Sci. USA 73, 3872-3876.
Nucleic Acids Research
Characterization by sedimentation analysis of kinetoplast DNA from Trypanosoma cruzi at different stages of culture
Jean Benard*, Guy Riou* and Jean-Marie Saucier**
*Laboratoire de Pharmacologie Moleculaire and **Groupe de Recherches sur la Pharmacologie des Medicaments Anticancereux (INSERM U 140), Institut Gustave Roussy, 94800 Villejuif, France Received 22 February 1979 ABSTRACT The kinetoplast DNA rietworks of Trypanosoma cruzi exist under two forms which have been studied by equilibrium density centrifugation in CsCl gradients containing ethidium bromide and by band sedimentation analysis. The relative proportion of the two forms has been measured and varies significantly between the exponential and stationary phase of growth, suggesting that one of these forms is a replicative intermediate. Both forms exhibit very high sedimentation coefficients. The sedimentation velocity ethidium titration was used to measure the superhelix density of the kinetoplast DNA after having established the validity of the method with in vitro closed DNA networks. The superhelix density of the native form of the kinetoplast DNA minicircles is very low and varies according to the physiological state of the trypanosomes. Furthermore, we observed a significant increase of the superhelix density of the kinetoplast DNA of trypanosomes grown in the presence of ethidium.
INTRODUCTION The kinetoplast is the specialized portion of the mitochondrial apparatus which contains in the culture form of Trypanosoma cruzi about 25 % of the
total DNA (1) organized in a complex structure (2). The biological role of the kinetoplast DNA (kDNA) has not yet been elucidated. In the electron microscope the kDNA appears as a network composed of about 25 000 double stranded nminicircular molecules (1440 base pairs) topologically interlocked or covalently bound, and of long linear molecules in urndertermined amount (2). Two different forms of kDNA networks are fractionated from T. cruzi (2), form I networks containing supercoiled minicircular molecules and form II networks in which the minicircles have single strand interruption(s). We have measured by analytical ultracentrifugation the sedimentation coefficient of the two native forms of T. cruzi kDNA, of form I relaxed by pancreatic DNase and closed in vitro with DNA ligase. Form II networks which have a higher molecular weight than form I networks are not observed in the stationary phase. These observations suggest that form II is a replicative
C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1941
Nucleic Acids Research intermediate. The superhelix density of the supercoiled minicircles composing the form I kDNA networks has been determined by sedimentation velocity ethidium bromide (EthBr) titration. Very low values of the superhelix density,varying with the physiological state of the trypanosomes, were observed. In addition, when the trypanosomes were treated with the trypanocidal drug EthBr, a significant increase of the number of negative superhelical turns of the
supercoiled minicircles was detected.
MATERIALS AND METHODS Trypanosomes culture. Culture form of T. cruzi, originated from Tehuantepec strain of Institut Pasteur, was grown in a diphasic medium in conditions previously described (3). In a typical culture inoculated with 5xlO6
cells/ml, the number of trypanosomes determined using a Coultronic Counter, was at 3 days, 3.4x107; at 4 days, 6.4x10 7; at 5 days, 8.2x10 ; at 7 days, l.0xlO ; at 9 days, 9.0x10 per ml of medium. kDNA purification. Trypanosomes were harvested and immediately lysed by sarkosyl (final concentration 1 %) in 0.15 M NaCl-0.015 M sodium citrate, pH 7.2 (SSC) for 15 min. at 370C. The lysate was incubated with pronase (1 rrg/ml) for 2 h at 370C. The kDNA networks were purified by several slow speed centrifugations (9000 g for 30 min. at 40C in SSC). The two forms were separated in a CsCl gradient containing 500 1ug/ml propidium (3). The bands were collected and propidium extracted with isopropanol (4). Conversion of kDNA form I into the relaxed form and resealing with DNA ligase. kDNA form I was nicked by pancreatic DNase in the presence of an excess of EthBr to limit the endonucleolytic reaction to one single strand break per DNA minicircle (5). The incubation mixture contained 0.1 M NaCl, 2 rrM MgCl2, 1 mM Na3 EDTA, 10 mM Tris-HCl pH 8.0, 0.1 mg/ml of bovine plasma albumin, 40,pg/ml kDNA form I and 60)pg/ml EthBr. After addition of 10 g of pancreatic DNase (Worthington) per ml of solution, the reaction was monitor-ed at room temperature using a Zeiss PMv2 III spectrophotometer equipped with its fluorescence attachment. Excitation was set at 545 nm and emission at 600 nm. When more than 99 % of the minicircles were nicked, the reaction was stopped by phenol extraction and the phenol removed by dialysis. The kDNA relaxed form was closed in vitro using T4 DNA ligase (Bethesda Research Laboratories, Inc). The reaction mixture contained 4 mfM MgCl2, 1.2 rrM Na3-EDTA, 30 rrM Tris-HCl pH 8.0, 50 1g/ml of bovine plasma albumin, 0.2 mM ATP, 10 mrM dithiotreitol and 17 units of enzyme per ml. The incubation 1942
Nucleic Acids Research was carried out for 1 hr at 300C. When the in vitro closure of kDNA was performed in the presence of EthBr, the time of incubation was 2 hrs. The con-
centration of free EthBr in the incubation mixture was evaluated by fluorescence measurement of the supernatant obtained by slow speed centrifugation after completion of the reaction. The closed kDNA was phenol extracted and the phenol removed by dialysis. Analytical ultracentrifugation. A Spinco model E analytical uJltracentrifuge was used. Sedimentation coefficients were usually measured with an ANlJ rotor at 4000 rpm by band sedimentation analysis at 200C using 3 M CsCl0.01 M Na3-EDTA (pH 8.0) as bulk solvent. Each cell was filled with 15 i4 of the DNA solution previously dialysed against 2 M NaCl-0.01 M Na3-EDTA. The corrected values of sedimentation coefficients were calculated according to Bruner and Vinngrad (6). The critical EthBr concentration needed to remove all the superhelical turns of the DNA closed circles was determined by band sedimentation velocity EthBr titration. The number of bound ethidium per DNA nucleotide in these conditions ( j) c ) was calculated according to Wang (7). Isopycnic ultracentrifugation in CsCl gradient containing EthBr was carried out as described by Wang (8). The solution had an initial density of 1.56 g/cm3 and an initial EthBr concentration of 1001pg/ml. Centrifugation was performed at 40 Krpm at 200C for 48 hours. Scans were obtained at 345 nm using an ultracentrifuge equipped with a reflective mirror assembly and the Beckman photoelectric scanning system.
RESULTS
Analysis of kDNA networks by equilibrium density centrifugation in CsCl-EthBr gradient. The total kDNA purified from trypanosomes grown for 4, 7 and 9 days have been analysed by equilibrium density centrifugation in CsCl gradient containing EthBr. As shown in figure 1, the kDNA networks exist under two forms which are well resolved in these conditions. We also observed small amounts of networks of intermediate density. The two bands corresponding to form I and II of kDNA are sharp as a consequence of the high molecular weight of the networks. We have evaluated the relative amount of DNA present in each band as explained in the legend of figure 1, without taking into account the networks of intermediate density: 34 % of the 4 days kDNA and 25
% of the 7 days
are
under form II. There is no detectable form II
after 9 days (figure 1 c). The separation between the closed form of PM2 DNA and the kDNA form II (0.360 cm, figure 1 d) is nearly equal to the sepa1943
Nucleic Acids Research 11
k DNA 4 days
a 1
k DNA 7 days
b k DNA 9 days
k DNA 7days
PM2
d 1t
PM22
e 6.40
6.60
6.80
7.00
Distance to rotor axis (cm)
Figure 1. Equilibrium density centrifugation of kDNA networks in CsCl-EthBr gradients. Total kDNAs purified from trypanosomes harvested after 4 days (a), 7 days (b) and 9 days of culture (c) ; kDNA form II and covalently closed Fv2 DNA (d) and a mixture of the two forms of PM2 DNA (e). The pen excursion of the photoelectric scanner is proportional to the concentration of EthBr bound to DNA. Using the measurements of Bauer and Vinograd (9), we estimated from the distance between the two bands that A\ = 0.064 (where ; expresses the bound dye/nucleotide molar ratio). Since for an EthBr concentration of 100 g/ml, j) f= 0.168 according to Bauer and Vinograd (10), therefore
=I 0.104. form of kDNA
These values were used to estimate the relative amount of each network in the mixtures.
ration between the nicked and the closed forms of PM2 DNA (0.346 cm, figure 1 e). As PM2 DNA and kDNA differ in their base composition by only 2 % (11), this result indicates that the kDNA form II network contains essentially open circles. We have measured the separation between the two forms of kDNA 1944
Nucleic Acids Research networks in different kDNA preparations and obtained inconstant results. the
same
sample, the separation decreases
as a
This is presumably due to the nicking of the
rage.
form I networks. For the sanples analysed
sible to derive
)
as
For
function of the time of stoclosed circles in the
shown in figure 1, it is
values using the calibration
pos-
of Wang (12) after
curve
correction for the slight difference of rotor argular velocity. values obtained by this procedure
are
The -c systematically higher than the values
obtained by sedimentation velocity analysis (see below) by
a
factor of
up
to 3.
Sedimentation velocity analysis of form II networks. When analysed by band sedimentation in the analytical ultracentrifuge, the two forms of kDNA
networks exhibit
an
unimodal distribution. Linear log
obtained in spite of of the
run
a
r vs
time plots
progressive spreading of the bands during the
presumably due to
a
are
course
size heterogeneity of the networks. The sedi-
mentation coefficient of high molecular weight DNA usually exhibits a strong dependence on centrifuge speed (13). This effect is not observed with kDNA
networks: the sedimentation coefficient of a 7 days kDNA form II was measured in the absence of EthBr at 3.0, 3.4, 4.0, 4.4 Krpm. The same result = 7430 350. The sedimentation within experimental error was obtained, S 20,w
coefficient of kDNA form II was measured in the presence of various EthBr concentrations. The results plotted i n figure 3 show that the sedimentation coefficient of kDNA under this form first decreases as a function of the free EthBr concentration, then levels off at about 25 % below its initial value free EthBr concentration higher than about
1M.
for
a
was
Sedimentation velocity analysis of relaxed kDNA networks. kDNA form I relaxed by the action of pancreatic DNase and the sedimentation coeffi-
cient of the rEelaxed networks
was
measured
as a
10
function of free EthBr con-
centration (figure 3). The sedimentation coefficient first decreases of about 12 % below its initial value ,then remains constant for EthBr concentration higher than about 10 VM. The
curve is
qualitatively similar to those
obtained with kDNA form II. Sedimentation velocity analysis of kDNA networks closed in vitro with DNA
ligase.
kDNA
was
closed in vitro with DNA ligase as described in Mate-
rials and Methods and its degree of superhelicity was determined by sedimentation velocity EthBr titration. The results are plotted in figure 2 and the
; values presented in table 1
were calculated from the critical free
c
EthBr concentrations at the minimum of the curves (7). One kDNA sample was
closed in the presence of 2.5x10
mole EthBr added per mole of DNA nuclec-
1945
Nucleic Acids Research
C',
0 c
0 4-
c
0 0
E
C',D1
2
4
6
8
10
15
Free ethidium (xlO M)
Figure 2. Sedimentation coefficient (uncorrected value) as a function of free EthBr concentration of kDNA closed in vitro with DNA ligase, in the absence (-0-O-) or in the presence of 1.7x10-2 mole of EthBr bound per mole of DNA nucleotide (-0--).
tide, the amount of EthBr bound in the closure medium was found equal to 1.7xlO mole/mole of DNA nucleotide. We observe a difference of 1.8xlO between the 9 c values obtained for this sample and the kDNA closed in the absence of EthBr. This result indicates that the sedimentation velocity EthBr titration is a suitable method to measure the superhelix density of kDNA form I minicircles. Sedimentation velocity analysis of form I networks. In the absence of forrp I EthBr, the sedimentation coefficient of kDNA extracted after 7 days of cul= ture is equal to S 20,w 5460 - 150. The sedimentation coefficient of form I networks extracted after 4 days and 7 days of culture was measured as a function of free EthBr concentration (figure 3) and the Q values calculated from these data are presented in table 1. The results show that the ;) measurements performed in duplicate are reproducible within 10 %. To rule out the possibility of superhelix density changes during the extraction procedure, we have purified a kDNA sample in the presence of 250 rmtM EDTA 1946
Nucleic Acids Research
C.,
0 x c
.2_ 0
u c
0
E c D
cn
20 0
2
4
Free ethidium
6
8
10
(xl06M )
Figure 3. Sedimentation coefficient (uncorrected value) of kDNA networks as a function of free EthBr concentration. Form I (-@v@-)and form II (--0C-) were isolated from kDNA samples extracted after 4 days and 7 days of culture. kDNA form I extracted after 7 days of culture and converted to relaxed form by pancreatic DNase treatment (-A-ti-).
Table 1 kDNA
saffples
closed in vitro at 300C closed in vitro at 300C in the presence of EthBr? ( =T1.7xl0-2) 3 days kDNA form I 3 days kDNA form I extracted in presence of EDTA 4 days kDNA form I 7 days kDNA form I 7 days kDNA form I from EthBr treated trypanosomes
Free EthBr 6 (x 10 M)
)
(x
2 10)
3.3
1.9
7.2
3.7
4.1
2.3
4.3
2.4
4.3 4.0 2.2 2.3
2.2 1.3 1.3
5.7
3.0
Values of 9c of kDNA samples determined by sedimentation EthBr titration.
2.4
velocity 1947
Nucleic Acids Research (14) to inhibit hypothetical nicking-closing activities. As shown in table 1 the
same
result within experimental
fied in the
presence
error
is obtained for 3 days kDNA puri-
and in the absence of EDTA. For kDNA samples extracted
at different stages of culture,
we
observe that the superhelix density is
significantly larger during the exponential phase than during the stationary phase.
Superhelix density of form I networks from EthBr treated trypanosomes. Riou has observed that shortly after addition of EthBr to
a
culture of try-
the drug binds selectively to the kinetoplast (15) and causes subsequently the appearance of abnormal forms of kinetoplast (16), then the progressive loss of kDNA by inhibition of its replication (17). Since EthBr
panosomes,
is
a
DNA intercalating dye, it presumably induces conformational changes of
kDNA form I networks when added to
an
in vitro culture of trypanosomes. As
shown in figure 4 and table 1, the superhelix density of kDNA extracted from
trypanosomes grown
in the
presence
higher than that of trypanosomes
T
grown
of
1
,Pg/ml
of
EthBr
is
significantly
for 7 days in the absence of EthBr.
40
0
3.5
0
0
E
Free ethidium (xlO M)
Figure 4. Superhelix density measurerrent of form I networks from EthBr treated trypanosomes. 1 jig per ml of EthBr was added to a culture of trypanosomes at the stationary phase. The trypanosomes were harvested 24 hours later and after purification of kDNA form I its sedimentation coefficient was measured as a function of the free EthBr concentration. 1948
Nucleic Acids Research DISCUSSION
The buoyant density at equilibrium of kDNA networks in
a
CsCl gradient
containing EthBr is determined by the amount of dye intercalated which itself varies as a function of the superhelix free energy of the closed circular molecules (10) associated in the networks. According to Wang (12),
the separation between monomeric closed DNA circles of superhelix density
9c
= 0.02 and nicked circles is 0.505 cm. We attribute corresponding to the smaller separations actually observed between form I and II networks to the presence of linear or nicked circular DNA species in the form I networks. The nicked circular molecules might not exist in vivo, and appear during the purification and the storage of the networks, causing a gradual shift of the band of form I networks to the regions of lower density in the CsCl/EthBr gradient. Kleisen et al. (18) have observed that the separation between the two forms of kDNA networks of Crithidia luciliae is larger than the separation between nicked and closed monomeric minicircles and suggest that topological constraint between interlocked minicircles further limits the dye binding to the networks, in addition to the restriction due to the closed circular structure of the molecules. For these reasons, it seems inappropriate to use the buoyant density method of Gray et al. (19) to determine c
the superhelix density of the closed minicircles in the kDNA networks. However our measurement of the separation between form I and form II networks shows that form II networks of T. cruzi bind the same amount of ethidium as FM2 DNA form II and consequently do not contain closed circles. Another observation provided by centrifugation in a CsCl/EthBr gradient is that networks sediment as sharp bands: this indicates a great homogeneity in the relative amount of closed circles in these networks as well as in the average superhelix density of the closed minicircles in the form I networks. The two forms of kDNA networks exhibit very high sedimentation coefficients as measured by analytical ultracentrifugation. A better accuracy
is obtained with this method than by zone centrifugation in sucrose gradient (20). These high sedimentation coefficients, of the same order of magnitude as those observed previously for the folded chrmnosome of Drosophila melanogaster (21) are due to the high molecular weight of the networks and to their
comrpact structure. The higher sedimentation coefficient measured was obtained with kDNA form II in the absence of EthBr in the sedimentation medium. kDNA form I networks, after relaxation with pancreatic DNase, have a sedicoefficient of about 70 % that of form II networks. This obser-
mentation
vation indicates that form II networks have a higher molecular weight than
1949
Nucleic Acids Research form I networks, in agreement with the observations of Englund (22,23) proposes that kDNA form II is
a
replicative intermediate. In the
who
presence
increasing concentrations of EthBr, the sedimentation coefficient relaxed networks decreases as it has been previously reported for
of
of kDNA free
nicked circles (24). A much steeper decrease of the sedimentation coefficient of kDNA form II was observed, and we have no reasonable explanation for this behavior. As shown in figure 3, native kDNA form I and kDNA networks relaxed by DNase have the
same
sedimentation coefficient for
a
critical EthBr
concen-
tration. This indicates that at this point, all the superhelical turns of
removed, and justify the use of the sedimentation velocity EthBr titration in the case of kDNA networks. Using this method, we = 1.3xlO ) have found that the superhelix density of the 7 days kDNA ( is slightly higher than that of the free minicircles liberated from the the minicircles
are
c
Crithidia luciliae networks at the stationary phase (18). It is interesting
to note that this value is
very
low compared to SV40 DNA (
(19) and mitochondrial DNA from HeLa cells
(
=
9,~ =
5.8xlO 2)
4.2xlO) (25). However,
c
Rubenstein et al. (26) have reported the existence in Drosophila melanogaster of covalently closed mitochondrial DNA of superhelix density practically equal to zero. When a covalently closed circular molecule is transfered from medium containing 0.13 M NaCl to 3 M CsCl, its superhelix density changes a value corresponding to 9c = 9xlO 3 as measured by Saucier and Wang (27). Most of the superhelix density of the 7 days kDNA measured in 3 M CsCl
a
by
c
is therefore introduced by the solvent used, suggesting the absence of interaction with binding proteins able to modify the DNA structure inside the
kinetoplast. This observation could explain the extreme accessibility of kDNA to various DNA binding drugs endowed with trypanocidal activity (reviewed by Simpson, 28). The superhelix density of the networks is higher during the exponential phase of growth. This presumably reflects the interactions with the proteins involved in kDNA replication, possibly DNA gyrase (29), and in the kDNA transcription and recombination if they occur. We have not detected kDNA form II in trypanosomes in stationary phase, and this ob-
servation agrees with the results of Englund (23) showing that kDNA form II is
a
mass
replicative intermediate. Furthermore
we have found at most 35 % in
of kDNA form II during the exponential phase. From this result, we es-
timate that about 21 % of the cells at mid-exponential phase have their kDNA as
form II networks, assuming that the DNA content of form II network is
twice the DNA content of form I network.
1950
Nucleic Acids Research For trypanosomes
grown
in the
presence
of EthBr,
we
found
a
signifi-
cantly higher superhelix density of the kDNA closed circles than for trypanosomes
grown
Smith et al. (25)
without EthBr. A comparable effect has with the mitochondrial DNA of human and
the
been observed by mouse
cells in
cul-
ture, but with a much higher shift of Qc in the presence of 1 ,p/ml of EthBr. Our result supports the hypothesis that kDNA is a target for trypanocidal drugs. The determination of the superhelix density of kDNA form I by sedimentation analysis in the presence of EthBr provides a valuable method to study quantitatively the interaction in vivo of intercalating drugs with this target.
ACKNOWLEDGEMENTS We thank J. Inacio and M. Re for their skilful technical assistance.
We
are
also indebted to Dr. F. Schaeffer for his help in using the photo-
electric
scanner.
REFERENCES 1. 2. 3. 4. 5.
6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
23. 24. 25. 26.
Riou, G. and Paoletti, C. (1967) J. Mol. Biol. 28, 377-382. Riou, G. and Delain, E. (1969) Proc. Natl. Acad. Sci. USA 62, 210-217. Riou, G. and Yot, P. (1977) Biochemistry 16, 2390-2396. Ostrander, D.A. and Gray, H.B. (1973) Biopolymers 12, 1387-1419. Barzilai, R. (1973) J. Mol. Biol. 74, 739-742. Bruner, R. and Vinograd, J. (1965) Biochim. Biophys. Acta 108, 18-29. Wang, J.C. (1969) J. Mol. Biol. 43, 263-272. Wang, J.C. (1974) J. Mol. Biol. 87, 797-816. Bauer, W. and Vinograd, J. (1968) J. Mol. Biol. 33, 141-171. Bauer, W. and Vinograd, J. (1970) J. Mol. Biol. 47, 419-435. Riou, G. and Pautrizel, R. (1969) J. Protozool. 16, 509-513. Wang, J.C. (1974) J. Mol. Biol. 89, 783-801. Kavenoff, R.J. (1972) J. Mol. Biol. 72, 801-806. Fairlamb, A.H., Weislogel, P.O., Hoeijmakers, J.H.J. and Borst, P. (1978) J. Cell Biol. 76, 293-309. Riou, G. (1967) C. R. Acad. Sci. Paris 265, 2004-2007. Delain, E. and Riou, G. (1969) C. R. Acad. Sci. Paris 268, 1327-1330. Riou, G. (1970) Biochemical Pharmacol. 19, 1524-1526. Kleisen, C.M., Borst, P. and Weijers, P.J. (1975) Biochim. Biophys. Acta 390, 155-167. Gray, H.B., Upholt, W.B. and Vinograd, J. (1971) J. Mol. Biol. 62, 1-19. Simpson, L. and Berliner, J. (1974) J. Protozool. 21, 382-393. Benyajati, C. and Worcel, A. (1976) Cell 9, 393-407. Englund, P.T., Di-fNlaio, D.C. and Price, S.S. (1977) J. Biol. Chem. 252, 6208-6216. Englund, P.T. (1978) Cell 14, 157-168. Crawford, L.V. and Waring, M.J. (1967) J. Mol. Biol. 25, 23-30. Smith, C.A., Jordan, J.M. and Vinograd, J. (1971) J. Mol. Biol. 59, 255-272. Rubenstein, J.L.R., Brutlag, D. and Clayton, D.A. (1977) Cell 12, 471-482.
1951
Nucleic Acids Research 27. 28.
29.
1952
Saucier, J.M. and Warg, J.C. (1973) Biochemistry 12, 2755-2758. Siffpson, L. in International Review of Cytology. Academic Press, Inc. New York and London (1972) pp. 139-207. Gellert, M., Ntizuuchi, K., O'Dea, M.H. and Nash, H.A. (1976) Proc. Natl. Acad. Sci. USA 73, 3872-3876.
