.=/ 1990 Oxford University Press
Nucleic Acids Research, Vol. 18, No. 18 5571
A new extraction procedure of autonomous DNA from eucaryotic cells, where DNA could be bound to proteins F.Pognan* and C.Paoletti Unites de Biochimie-Enzymoplogie, U140 INSERM, URA 147 CNRS Institut Gustave-Roussy, Villejuif, France Submitted August 8, 1990
The classical method for extraction of episomes from eucaryotic has been described by B.Hirt (1). It is based on precipitation of proteins associated to cellular DNA by sodium chloride, whereas the episomal DNA remains in the supernatant. However, this method does not allow to purify an eventual fraction of autonomous DNA which would be strongly associated with a protein as it is the case with topoisomerase II in the 'cleavable complex' formation (2). In this reaction, the DNA is cut (apparition of linear DNA in the case of close circular DNA = form HI) by the enzyme which is covalently bound to the DNA and application of the Hirt's procedure in such a case results in losing this DNA associated protein (Fig. la). Here, we describe a new and more rapid extraction method of autonomous DNA from eucaryotic cells, based on the sedimentation of nuclear DNA, which avoids this disadvantage due to the protein association. Fibroblast cell cultures (embryonic rat fibroblasts transformed by Bovine Papilloma Virus type I genome, BPV I) are incubated for one hour at 37°C under CO2 atmosphere with different concentrations of mAMSA (as indicated in figure legend la), a cleavable complex inducing drug (3). Plates (10 cm in diameter, around 6 millions of cells) are washed twice on ice with ice-cold 0.14 m NaCl and immediately lysed with one ml of lysis buffer (50 mM Tris-HCl pH:8; 100 mM Na3-EDTA; 0.5% SDS). The lysate is collected with a tip-cut pipette in order to avoid any breakage of cellular DNA and incubated at 50-55°C at least four hours with 0.5 mg of proteinase K (Boehringer Mannheim), followed by an extraction with chloroform:isoamyl alcohol (24:1). The organic phase is counter-extracted with one ml of lysis buffer. The two aqueous phases are pooled and completed to 14 ml with the lysis buffer and centrifuged one hour at 30,000 rpm in TST 41 Kontron rotor. The cellular DNA sediments as a translucide to almost white and soft pellet. The supernatant is carefully removed and precipitated with ethanol, centrifuged in SW 28 rotor tubes and washed with 70% ethanol. The pellet is suspended in a small volume of TE buffer and loaded on a 0.8% agarose gel. DNA is transferred on a positively charged nylon membrane (Hybond-N+ from Amersham) in order to perform a classical Southern blotting manipulation (4). Figure 1 shows that Hirt's procedure results in losing the cleavable complex form (form HI) unlike the present one. An increase of drug concentration implies an increment of cleavable complex for an identical number of cells. But, a too high amount
cells
*
To whom correspondence should be addressed
of drug, generally induces inhibition of the cleavable complex formation. By scanning the autoradiography on a microdensitometer (Joyce, MK Ill C), the percentage of form HI was measured. A dose dependent curve is obtained, which indicates that this method is quantitative. Identical results are {A}
1 _
Form
2 3 4
5
0
_
--
kb
8 9 10
6 7
WA
23.1
-
9.4 Form ill.--
_ Form
l_
_
_
_
_
_
_
_
_
6.5
_
4.4
__
Flgure la. Each lane corresponds to one plate of cells. Lanes I to 5, cells incubated with several concentrations of mAMSA (respectively: 0; 5; 10; 20 and 50 uM) with BPV I DNA extracted by the Hirt procedure. Lanes 6 to 10, same as 1 to 5 excepted that extraction has been done according to the proposed method.
to
40, -
1 CENTRIFUGATION
30 C-
o LL.
0
20 E~~~~~~~HRT
lO: t v
0
10
20
30
40
50
60
mAMSA concentration (#M) Figure lb. Densitometric scanning quantitation of form III of Fig. la experiment.
5572 Nucleic Acids Research, Vol. 18, No. 18 observed for other drugs such as VP16 (another cleavable complex inducing drug) or other manipulations such as kinetics experiments (data not shown).
ACKNOWLEDGEMENTS We wish to thank Pr. Franqois Cuzin's team for providing the cells, and the support of Institut de Formation Superieure BioM6dicale (IFSBM), Villejuif, France.
REFERENCES 1. 2. 3. 4.
Hirt,B. (1967) J. Mol. Biol. 26, 365-369. Wang,J.C. (1987) Biochem. Biophys. Acta 909, 1-9. Drlica,K. and Franco,R.J. (1988) Biochemistry 27, 2253-2259. Southern,E.M. (1975) J. Mol. Bio. 98, 503-517.
Nucleic Acids Research, Vol. 18, No. 18 5571
A new extraction procedure of autonomous DNA from eucaryotic cells, where DNA could be bound to proteins F.Pognan* and C.Paoletti Unites de Biochimie-Enzymoplogie, U140 INSERM, URA 147 CNRS Institut Gustave-Roussy, Villejuif, France Submitted August 8, 1990
The classical method for extraction of episomes from eucaryotic has been described by B.Hirt (1). It is based on precipitation of proteins associated to cellular DNA by sodium chloride, whereas the episomal DNA remains in the supernatant. However, this method does not allow to purify an eventual fraction of autonomous DNA which would be strongly associated with a protein as it is the case with topoisomerase II in the 'cleavable complex' formation (2). In this reaction, the DNA is cut (apparition of linear DNA in the case of close circular DNA = form HI) by the enzyme which is covalently bound to the DNA and application of the Hirt's procedure in such a case results in losing this DNA associated protein (Fig. la). Here, we describe a new and more rapid extraction method of autonomous DNA from eucaryotic cells, based on the sedimentation of nuclear DNA, which avoids this disadvantage due to the protein association. Fibroblast cell cultures (embryonic rat fibroblasts transformed by Bovine Papilloma Virus type I genome, BPV I) are incubated for one hour at 37°C under CO2 atmosphere with different concentrations of mAMSA (as indicated in figure legend la), a cleavable complex inducing drug (3). Plates (10 cm in diameter, around 6 millions of cells) are washed twice on ice with ice-cold 0.14 m NaCl and immediately lysed with one ml of lysis buffer (50 mM Tris-HCl pH:8; 100 mM Na3-EDTA; 0.5% SDS). The lysate is collected with a tip-cut pipette in order to avoid any breakage of cellular DNA and incubated at 50-55°C at least four hours with 0.5 mg of proteinase K (Boehringer Mannheim), followed by an extraction with chloroform:isoamyl alcohol (24:1). The organic phase is counter-extracted with one ml of lysis buffer. The two aqueous phases are pooled and completed to 14 ml with the lysis buffer and centrifuged one hour at 30,000 rpm in TST 41 Kontron rotor. The cellular DNA sediments as a translucide to almost white and soft pellet. The supernatant is carefully removed and precipitated with ethanol, centrifuged in SW 28 rotor tubes and washed with 70% ethanol. The pellet is suspended in a small volume of TE buffer and loaded on a 0.8% agarose gel. DNA is transferred on a positively charged nylon membrane (Hybond-N+ from Amersham) in order to perform a classical Southern blotting manipulation (4). Figure 1 shows that Hirt's procedure results in losing the cleavable complex form (form HI) unlike the present one. An increase of drug concentration implies an increment of cleavable complex for an identical number of cells. But, a too high amount
cells
*
To whom correspondence should be addressed
of drug, generally induces inhibition of the cleavable complex formation. By scanning the autoradiography on a microdensitometer (Joyce, MK Ill C), the percentage of form HI was measured. A dose dependent curve is obtained, which indicates that this method is quantitative. Identical results are {A}
1 _
Form
2 3 4
5
0
_
--
kb
8 9 10
6 7
WA
23.1
-
9.4 Form ill.--
_ Form
l_
_
_
_
_
_
_
_
_
6.5
_
4.4
__
Flgure la. Each lane corresponds to one plate of cells. Lanes I to 5, cells incubated with several concentrations of mAMSA (respectively: 0; 5; 10; 20 and 50 uM) with BPV I DNA extracted by the Hirt procedure. Lanes 6 to 10, same as 1 to 5 excepted that extraction has been done according to the proposed method.
to
40, -
1 CENTRIFUGATION
30 C-
o LL.
0
20 E~~~~~~~HRT
lO: t v
0
10
20
30
40
50
60
mAMSA concentration (#M) Figure lb. Densitometric scanning quantitation of form III of Fig. la experiment.
5572 Nucleic Acids Research, Vol. 18, No. 18 observed for other drugs such as VP16 (another cleavable complex inducing drug) or other manipulations such as kinetics experiments (data not shown).
ACKNOWLEDGEMENTS We wish to thank Pr. Franqois Cuzin's team for providing the cells, and the support of Institut de Formation Superieure BioM6dicale (IFSBM), Villejuif, France.
REFERENCES 1. 2. 3. 4.
Hirt,B. (1967) J. Mol. Biol. 26, 365-369. Wang,J.C. (1987) Biochem. Biophys. Acta 909, 1-9. Drlica,K. and Franco,R.J. (1988) Biochemistry 27, 2253-2259. Southern,E.M. (1975) J. Mol. Bio. 98, 503-517.
