Physicochemical studies on the minicircular DNA in Escherichia coli 15

285

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96456

PHYSICOCHEMICAL STUDIES ON T H E MINICIRCULAR DNA IN E S C H E R I C H I A COLI 15 C H O N G S U N G L E E * AND N O R M A N DAVIDSON

Gates and Grellin Laboratories o/ Chemistry, and Church Laboratory o] Chemical Biology", Cali/ornia Institute o/ Technology, Pasadena, Call/. (U.S.A.) (Received N o v e m b e r 3rd, 1969)

SUMMARY

The properties of the minute circular plasmid DNA that occurs in Escherichia coli 15 have been studied. Its weight average length is 0.670/z, corresponding to a molecular weight of 1.47. lO 6. Dimers and higher integral multimeric forms are found in plasmid DNA preparations from Escherichia coli 15 wild type. These species amount to about 5 ~/o b y number of the total plasmid molecules. Renaturation kinetics measurements show that all of the approx. 15 plasmids in a bacterium contain the same base sequences. Two presumed replicating species, the a form and the 0 form, of the minicircular DNA are observed in electron micrographs of cell lysates.

INTRODUCTION

There have been several recent discoveries of covalently closed minute circular DNA species, of unknown biological function, as cytoplasmic elements in microorganisms (refs. 1-4; and H. S. JANSZ, personal communication). Micrococcus lysodeikticus contains about I plasmid per bacterium ~. Escherichia coli 15 contains about 15 plasmid molecules, all with the same molecular length, per chromosome 1. Shigella dysenteriae also harbors multiple copies of a plasmid (ref. 4 and H. S. JANsz, personal communication).Thus, the latter plasmids differ from the well-known cytoplasmic elements, such as sex factors, colicinogenic factors, and drug resistance factors, for each of which there is usually one copy per bacterial chromosome. The question of greatest interest is the function ot these minute plasmids. Because of their small size, they can code for only a few proteins. In this connection, for those cases where m a n y plasmid molecules occur per bacterial chromosome, it is conceivable that the several plasmids are not identical, but contain different base sequences, and code for different proteins. In the present communication, we have studied the question of whether all the minicircles in E. coli 15 contain the same base sequence by the method of ienaturation kinetics. We have also looked for clues as to factors which affect the replication and morphology of the E. coli 15 minicircles b y careful observation of electron micro* P r e s e n t address: D e p a r t m e n t of Biological C h e m i s t r y , H a r v a r d Medical School, B o s t o n , Mass. o2115, U.S.A. *~ C o n t r i b u t i o n No. 3968.

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scope grids of lysates of E. coll. We observe multimeric forms of the minicircles, amounting to about 5 ~o b y number of the total plasmid molecules. We also find two structures which we presume to be replicating circular forms: one with two fork points and no free end (0 form) and one with a protruding linear DNA branch (a form).

MATERIALS AND METHODS

Bacterial strains E. coli 15 wild type was obtained from Professor S. S. Cohen. E. coli 15 TAU and TAU-bar were gifts from Dr. F. Funk and Professor P. C. Hanawalt. Tile auxotroph E. coli 15 TAU requires thymine, uracil, arginine, methionine and tryptophan; TAU-bar requires all these and proline. The geneology of these strains is described by LEE et al. 5. Growth media E. coli I5 wild type was grown in M9 buffer supplemented with o. 4 °/o glucose, I mM MgS04, and o.I mM CaCla. M 9 buffer consists of 7 g of Na2HPO 4, 3 g of KH2P04, I g of NH4CI , and 0.5 g of NaC1 in I 1 of water. E. coli 15 TAU and TAU-bar were grown in the above medium supplemented with IO #g/ml of thymine, 30 #g/ml of uracil and of cytosine, and 1/2o vol. of an amino acid solution which contains 7.6 mg/ml of arginine, 6 mg/ml of methionine, 2.5 mg/ml of tryptophan, and 7.5 mg/ml of proline. Preparation o/minicircles on a large scale E. coli 15 TAU-bar was grown in 5 1 of the medium described above at 37 ° with vigorous aeration. When Ae00 m~ m 2, cells were pelleted b y centrifuging in a Sorvall centrifuge at 8000 rev./min for IO rain. The pelleted cells were resuspended in 15o ml of IO mM Tris-OH, IO mM E D T A (disodium salt) (pH 7) (Tris-EDTA buffer) ; 3o mg of lysozyme (Worthington) was added to the suspension followed by an incubation at 37 ° for 15 min. To the resulting spheroplast suspension, 1. 5 ml of pronase stock solution (IO mg/ml, preincubated at 37 ° for I h) was added. After another 5-rain incubation, 15 ml of 5 % sodium dodecyl sulfate was added and the lysate incubated at 37 ° for I h with occasional gentle stirring. Solid CsC1 was added to this viscous cell lysate to give a concentration of I M. After standing in the cold for 2 h or longer, the lysate was centrifuged in a Spinco model L2-5o centrifuge with an SW-25.2 rotor at 22 ooo rev./min for 2 h. This procedure removes the sodium dodecyl sulfate precipitate, the bacterial debris and most of the large pieces of host DNA. The supernatant was carefully withdrawn and deproteinized b y chloroform-isoamyl alcohol extraction s. These solvents were removed by ether extraction; the remaining ether was evaporated b y bubbling N 2 gas through the solution. In order to reduce the volume, the DNA was pelleted by centrifuging at 45 ooo rev./min for 24 h with a Ti-5o rotor. The DNA pellet was resuspended in 20-30 ml of T r i s - E D T A buffer. This DNA solution was treated with ribonuclease and extracted with freshly distilled phenol. The DNA was then banded in a CsCl-ethidium bromide medium by the method of RAI)LOFF et al. 7. The concentration of ethidium bromide was 0.2 mg/ml and the l~iochim. Biophys. Acta, 204 (197o) 285-295

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density of the medium was around 1.55 g/ml. The centrifugation was done with an SW-4I-Ti rotor at 31 ooo rev./min or an SW-5o.I rotor at 40 ooo rev./min for 48 h. The lower band containing the closed circular DNA was collected and subjected to a second banding for further purification. For optical studies such as renaturation kinetics, a third banding was necessary to obtain an optically clean preparation. After the second banding, the contamination of host (linear) DNA was less than 5% b y weight, as judged b y electron microscope observation. A typical second banding profile is shown in Fig. I. After removal of ethidium bromide, either b y isoamyl alcohol extraction or b y Dowex-5o column chromatography 7, the DNA was dialyzed vs. a desired buffer and stored at --20 °.

Sonication o] DNA Ar gas was bubbled through 1-2 ml of DNA solution for 15 min before sonication. The small sonicator tip was cleaned b y sonicating redistilled water and then the same buffer as the DNA solution until no more ultraviolet-absorbing material was released. A Bronson sonicator was used at a power level setting of 2. DNA samples were sonicated for 3 rain at o ° under an Ar atmosphere (repeats of 15 sec sonication and 15 sec rest). The sonicated DNA samples were characterized b y electron microscopy and analytical band sedimentation in alkali. Electron microscopy The moditied basic protein film technique was used 5,s-1°. Analytical ultracentri/ugation Band velocity centrifugation was done essentially as previously described Ix except that a photoelectric scanner was used. Measurements o/renaturation rates The method employed for renaturation rate measurements of sonicated, denatured DNA is in principle the same as previously described TM. The purpose of the experiment is to compare the renaturation rate of the minicircular DNA to that of a standard DNA, preferably of similar base composition and similar complexity (genome size). For the standard DNA, we used # X I 7 4 replicative form. Both DNA's were sheared, b y sonication, to pieces of similar length. In order to slow down the rates to a range that is conveniently measured, the ionic strength of the renaturation medium was rather low. In order to achieve optimal accuracy in the ratio of renaturation rates for the minicircular DNA and the # X I 7 4 replicative form, the two samples were dialyzed against the same buffer and the renaturation rates were measured simultaneously in a thermostatted cell compartment. A copper cell-holder for four cells, with its temperature controlled b y circulating water, and with good thermal contact to the quartz cells, was constructed. Stoppered quartz microcells, 3 m m wide, with a I o - m m light p a t h were used. A small thermistor with a response time of less than IO sec was immersed in water in one of the cells. B y means of two three-way teflon stopcocks, t a p water or the fluid from either one of two water baths could be rapidly circulated through the holder, o.5-ml Biochim. Biophys. Acta, 204 (197o) 285-295

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s a m p l e s in t h e q u a r t z microcells were degassed b y suction for IO min a n d inserted in the cell-holder. One b a t h was m a i n t a i n e d at a b o u t 95 ° which is ( T m + I 5 ° ) , a n d one at 55 ° which is (Tm--25°). A f t e r IO min a t 95 °, b y a p p r o p r i a t e switching of t h e flow from t h e first b a t h to t h e t a p w a t e r to t h e second b a t h , t h e t e m p e r a t u r e in a cell was r e d u c e d from 95 to 55 ° w i t h i n 5 min. This t i m e is short c o m p a r e d to t h e half r e n a t u r a t i o n times used of a b o u t 2 h. The decrease in A~60 ms as a function of t i m e was t h e n m e a s u r e d . I t should be recalled t h a t t h e r e is a b r o a d m a x i m u m in the r e n a t u r a t i o n r a t e a t Tin--25 ° so t h a t precise control of the t e m p e r a t u r e is u n n e c e s s a r y ~. A t t h e end of the reaction, the D N A was again d e n a t u r e d a n d its s p e c t r u m was t a k e n to verify t h a t t h e h y p e r c h r o m i c i t y was solely due to d e n a t u r a t i o n . The r a t e m e a s u r e m e n t s were t h e n r e p e a t e d w i t h t h e same s a m p l e s a n d gave i d e n t i c a l results.

D N A - R N A hybridization D N A - R N A h y b r i d i z a t i o n s were p e r f o r m e d b y Mr. Don R o b b e r s o n b y the s t a n d a r d m e m b r a n e filter procedure la. A s a m p l e of 14C-labeled E. coli K I 2 23-S + I6-S r i b o s o m a l R N A p r e p a r e d b y h i m was used.

RESULTS AND DISCUSSION

General properties o/the minicircles and other circular DNA species in E. coli I5 The minicircles isolated from t h e lower b a n d in a C s C l - e t h i d i u m b r o m i d e cent r i f u g a t i o n (Fig. I) show b o t h t w i s t e d a n d open circular s t r u c t u r e s in t h e electron microscope. T h e r a t i o of m o l e c u l a r lengths of ~bXI74 r e p l i c a t i v e form D N A a n d the minicircles was m e a s u r e d from a n u m b e r of molecules m o u n t e d on t h e s a m e grid to be 2.32 ( ± o . I o ) .

(b)

rninicircle

1 hosiDNA *--P

~---~3.3T~ ~

Fig. i. Banding profile of minicircles in a CsCl-ethidium bromide medium, a. Photographs of fluorescent bands were taken with a Polaroid camera using a Type 46-L film. The position of the minicircle band is indicated by an arrow, b. The photograph was traced with a microdensitometer (magnification, × io). The separation of the two bands is 3-37 mm in a i/e inch × 2 inch centrifuge tube. This s t a n d a r d d e v i a t i o n is a b o u t as e x p e c t e d for homogeneous D N A molecules of t h e size of t h e m i n i c i r c l e sS, 10 . If we t a k e 3.4" IO e for t h e m o l e c u l a r weight of g}XI74 r e p l i c a t i v e form D N A 14, the m o l e c u l a r weight of t h e m i n i c i r c u l a r D N A is 1.47" lO 6,

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in good agreement with the value previously reportedl. The weight average contour length of the minicircles was measured as 0.670 (fo.030) ,u. However, the contour length of a given DNA depends somewhat on the conditions for preparing the electron microscope grid; the length is less significant than the ratio of lengths with a standard DNAlO.

Fig. 2. Minicircles and higher multimeric forms in E. coli 15 wild type. The basic-protein film technique was used. Electron microscope grids were rotary-shadowed with Pt/Pd after uranyl staining. A, supercoiled and open minicircles; B, dimer; C, trimer; D, tetramer; E, pentamer. Biochim.

Biophys.

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(‘970)

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The sedimentation coefficient, S°2o,~, of the minicircles at pH 7.4 has been measured as 17.2 S. The ratio of uncorrected S values at pH 13 and at pH 7.4 in 3 M CsC1 is 2.56 which agrees very well with the ratio obtained for polyoma DNA 15. The uncorrected S value of 27.2 at pH 13 is as expected for this size closed circular DNA 16. The snperhelical density of a closed circular DNA, as defined by BAUER AND VINOGRAD 17, is the ratio of the number of superhelical turns to the number of ordinary Watson-Crick helical turns. This can be measured for the nfinicircular DNA from the separation of the two bands shown in Fig. Ib, by comparison with the corresponding separation for a standard DNA. Such a comparison was made in collaboration with Dr. H. B. Gray, using his data for SV 40 DNA with a superhelical density of --0.030. The calculated superhelical density for the minicircular DNA is --o.o38. This value is in good agreement with the value of --0.039 previously reported by WANGis from sedimentation velocity measurements. In addition to the minicircle size species, there occur circular molecules with contour lengths that are integral multiples of the minicircle size up to the pentamer in E. coli 15 wild type. Electron micrographs and length histograms are shown in Figs. 2 and 3. The amount of the multimers is about 5 % of total circular DNA species by number. As seen in Fig. 3, the frequency of the occurrence of different multimeric forms decreases as the order of the multimer increases. Preparations from E. coli 15 TAU and TAU-bar show, however, much less frequent multimeric forms of the order of O.l-O.5 % of the total circular DNA species. Furthermore, some molecules 1. 5 times the size of a monomer were observed in the TAU preparation and some with half the size of a monomer in the TAU-bar preparation. I

I

i

7-

f

~x~

4C

, i tt x RF 0.129(+0.050)/~

I

,

minicircle

0 123(i.0,050) F

o

o. . . . . . . '.

. 2.0 .

.

.

!5

O~ 40

L i / ' ( L > me

415

50

o 2 0 4.0 6 0

,oo

n 20

4.0 6.0

t~ , 100

length

Fig. 3. L e n g t h h i s t o g r a m s of the minicircle and its multimeric forms. The n u m b e r of molecules are plotted vs. the ratio of length, Lt, to the weight average length of the minicircle sample ((g)wme). The n u m b e r s of molecules in the m o n o m e r b a n d have been reduced by a factor of 3 ° for convenience in plotting. Fig. 4. L e n g t h h i s t o g r a m s of sonicated D N A samples. The lengths s h o w n are in cm on tracing paper. The lengths are converted to/~ and the weight-average molecular weights calculated using ~ X I 7 4 replicative form as a standard. The calculated molecular weights are: o.281, lO 6 for sonicated # X I 7 4 replicative form and 0.269- IOe for sonicated minicircle.

Molecular weights oI sonicated D N A The rate of renaturation of a DNA depends on its molecular weight 12. Therefore the molecular weights of the sonicated minicircles and ~bXI74 replicative form prepared as described in MATERIALSAND METHODS were measured by electron microscopy and by band velocity sedimentation. Biochim. Biophys. Acta, 2o 4 (i97 o) 285-295

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The distributions of contour lengths of the sonicated DNA's are shown in Fig. 4. The distributions are rather heterogeneous with standard deviations of 40 %. The expected standard deviation of the length distribution b y electron microscopy for a population of homogeneous double-stranded DNA molecules of this size is IO % (refs. 5, IO); thus, there is a true length heterogeneity in each sonicated sample. However, the important point for our purpose is that the shapes of the distributions are the same for the two sonicated DNA samples. The molecular weights given in Table I for single-stranded DNA's are half the values for the double-stranded DNA fragments, evaluated from the contour lengths in Fig. 4. TABLE

I

MOLECULAR

WEIGHTS

OF SONICATED

DNA

#X replicative form Minicircle

DENATURED

DNA's

Electron microscope

Sedimentation coefficients

(L}w* (I~ )

Mol. wt. of single-stranded D N A × 20 -6 **

S°~o,w (pH I3)

Mol. wt. × zo -e***

o.129 o.123

o.14o o.135

5.39 5.26

o.II o.io

* Weight-average molecular length. ** Molecular weights of double-stranded DNA's based on (L)w were divided by two. *** Based on the relation 1~, s%0,w (pH 13) = 0.0528 M°'4° Sedimentation coefficients of sonicated DNA's in the single strand form were determined by alkaline band velocity centrifugations. The sedimentation coefficients (s°~0,,, (pH 13)) and the molecular weights evaluated by the relation of STUDIER19 are summarized in Table I. The agreement betweeen the weight-average molecular weights determined from contour lengths ((L}w) of double-stranded DNA and irom the sedimentation coefficients is satisfactory. The small difference observed could easily be due to the inappropriateness of the relation of STUDIERTM in this range of molecular weight. Another possibility is that there are a small number of single-strand breaks on the double-stranded DNA fragments. These would not be revealed in the electron microscope. If the latter possibility is assumed correct, the number of breaks per single-stranded DNA can be calculated by the following relation ~0, Mw

2(e-*+ s--I)

Mw0

s2

where Mw0 is the weight-average molecular weight of original polymer, Mw the weightaverage molecular weight of degraded polymer, and s the average number of breaks in the original polymer. In our case, Mwo = 0.14"106 from the contour length measurements and Mw = 0.i0.106 from alkaline sedimentation coefficients. The above relation yields I . i single-strand breaks per strand. Complexity

o~ m i n i c i r c l e

There appear to be about 12-15 covalently closed minicircles per chromosome in E . coli 15 (ref. I). All of these molecules have the same molecular length. Normally Biochim. Biophys. Acta, 2o4 (197o) 285-295

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one copy of a plasmid or an episome exists per host chromosome, the number being somehow regulated b y the host 21. The question is whether all of the minieircles in a bacterium are the same, or whether there are several different kinds. This question can be answered b y measuring the renaturation rate. The complexity of a DNA m a y be defined as the molecular weight in daltons of the total number of base pairs required to include all of the different base sequences that occur in this particular class of DNA molecules 12. The second-order renaturation rate constant is inversely proportional to the complexity of the DNA 12,22 since the concentration of a given base sequence in the population is inversely proportional to the complexity. Thus, for instance, if there were 15 different kinds of minicircles in a bacterium, the rate of renaturation would be I5-fold slower than if there were only one. Renaturation of sonicated denatured minicircle and ~ X I 7 4 replicative form DNA has been performed as described in MATERIALS AND METHODS. The following equation was used to determine the second-order rate constants ~2. Ao--Aeo A --Aoo

7.35" lO-5 Aookzt+ i

where A o is the absorbance at 260 m/~ of fully denatured DNA, Aoo the absorbance of native DNA, A the absorbance during renaturation, k 2 the second-order rate constant (1.mole-l.sec-1), and t the time (see). The rate constant, k 2, was determined from the slope of the change in absorbance v s . time plot. Such plots are shown in Fig. 5- The observed rate constants are 3.23 and 1.55 1.mole-l.sec -1, respectively,

j

• • ; ¢xRF O; rninici~cle

~,~.~t ~

~I"o7 .o/

,o

io

,6o

,50

Time (rain)

Fig. 5. Second-order rate plots for r e n a t u r a t i o n runs. The of experimental points. Minicircle a n d ~ X I 7 4 replicative IO mM Na~HPO a (pH 7.0). Minicircle: Aoo = o.41o, slope ~ X I 7 4 replicative form DNA; Aoo ~ 0.578, slope = 3.94"

s t r a i g h t ]ines are least-squares plots form D N A are in io mM NaH~PO 4= 5.9O.lO -3 min-l~ intercept = 1.o 5. IO-3 rain-l, intercept -- 1.14.

for minicircle and ~bXI74 replicative form DNA in a salt medium with a cation concentration of 0.03 M Na +. The ratio of rate constants is 2.09. A slight correction should be made for the fact that the single-strand molecular weight of the sonicated ~ X I 7 4 replicative form is 5-1o °/o greater than that of the minicircular DNA (Table I). Since the second-order renaturation rate constant ot a sheared sample of a DNA oi given complexity is inversely proportional to the square root of the single-strand molecular weight 12, the corrected ratio of renaturation rates of the minicircular DNA and # X I 7 4 replicative form for sheared samples of the same molecular weight is 2.17 (4-0.05). If the complexity of # X I 7 4 replicative form is taken as 3.4" IO6, that of minicircles is calculated as 1.56" lO 6. This is in agreement within experimental Biochim. Biophys. Acta, 204 (197 o) 285-295

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error with the measured molecular weight of 1.47. IOe. Thus, we conclude t h a t all the minicircles contain the same base sequences; the molecules are presumably all identical.

DNA-ribosomal R N A hybridization It might be speculated that the presence of multiple copies in a plasmid of a unique base sequence might be due to some sort of gene amplification, for instance, as observed for ribosomal RNA genes in oocytes 2a. Direct D N A - R N A hybridization with 2 # g of minicircular DNA in the membrane filter showed that the DNA is not significantly ( < o.2 °/o) complementary to E. coli ribosomal RNA. Comparable experiments with 20/~g of whole E. coli DNA give the expected hybridization level of 0.3 %. Replicating/orms o/minicircles Replicating forms of DNA's from several microorganisms have been observed directly in the electron microscope ~-29. The following three species of replicating DNA have been visualized: a long concatenate, a circular DNA molecule with a long protruding linear double-stranded DNA (a form) zS, and a circular molecule with two fork points, one presumably corresponding to the origin of replication and the other corresponding to the replicating point (0 form) 24. When the main DNA band containing host linear DNA, nicked minicircles, and presumably replicating forms of the minicircles, was carefully examined by electron microscopy, it was observed that there occur two kinds (a form and 0 form) that we tentatively identify as of replicating minicircles. Of course, long concatenates, if any, could not be distinguished from host DNA fragments in this examination. Some examples of these two species are shown in Fig. 6. The average length of the circular portions of the replicating forms agrees with the average minicircle length previously measured. The standard deviation was 0.04/~ comparable to 0.03 # given already. Out of 28 molecules examined, 4 molecules were the 0 form and 23 molecules were the a form. The lengths of branches in the a form varied from o.I to IO#. In fact, 7 out of 23 a forms had branch lengths longer than the size of the minicircle. Replicating forms in the main DNA band represent about 5 % of the nicked minicircles. Since the amount of nicked minicircles was less than IO % of the total minicircles, the replicating forms amount to 0.5 % or less of the total. This amount is about as expected if the rate of replication is the same in both minicircle and host, and if each minicircle replicates once on the average per one round ot host replication. It might be argued that the a forms could be accidentally formed from linear molecules. Therefore, E. coli S/6, which does not carry the minicircle, was treated and examined under identical conditions. No such species in which the length of the circular part is the size of the minicircle was detectable. However, the possibility still remains that a forms would be formed by an accidental overlap of a minicircle and a linear molecule. We regard this possibility as unlikely, but it has not been rigorously excluded. The a form, as a replicating form, is expected for the "rolling circle" model of replication a°. It has been observed in replicating ~t DNA 25. The 0 form is conBiochim. Biophys. Acta, 204 (197o) 285-295

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Fig. 6. Replicating forms of minicircles. After b a n d i n g in a CsCl-ethidium bromide medium, the u p p e r b a n d was collected and the CsC1 and ethidium bromide r e m o v e d as described in MATERIALS AND METHODS. This p r e p a r a t i o n was carefully and laboriously examined u n d e r an electron microscope. F o u r a forms and two 0 forms are s h o w n here.

s i s t e n t w i t h t h e CAIRNS 31 m o d e l , a n d c h o n d r i a l D N A 24,27.

Biochim. Biophys. Acta, 204 (197 o) 285 295

has been observed

i n ~t D N A

a n d in m i t o -

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DNA

IN

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295

ACKNOWLEDGMENTS

We are indebted

to Professor

S. S. Cohen, Professor

P. C. Hanawalt

and Dr.

F. Funk for girts of E. coli strains, and to Mr. J. Sedat for a gift of @x174 replicative form DNA. We thank Mr. R. D. Bowman for help in the sedimentation velocity experiments,

Mr. D. Robberson

for performing

the RNA-DNA

hybridization

test,

and Dr. H. B. Gray for the calculation of superhelical density. This research was supported by a grant GM roggr from the U.S. Public Health Service.

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