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Cyclization of eucaryotic deoxyribonucleic acid fragments
J. Mol. Bid. (1970) 51, 621-632
Cyclization of Eucaryotic Deoxyribonucleic C. A. THOMAS JR., B. A. liLumuot,D.N.MIs~a$
Acid Fragments AND C.S. LEE
Department of Biological Chmistry Harvard Medical S&o01 Boston, Ma.w. 02115, U.S.A. (Received 10 March 1970, and in revised form 24 April 1970) Highly purified, shear-broken fragments of DNA extracted from certain euoaryotes (salmon, trout, Necturus, calf thymus and others) can form circles and circular structures by either “folding” or “slipping”. Procaryotic DNA fragments (from T7 bacteriophage, Escherid& coli and Bacillus aubtdi.~) do not form circles or circular structures by this treatment. “Folding” involves partial degradation with exonucleases (exonuclease III or r\ exonuclease) and annealing. Up to 35% of all Necturw, DNA fragments seen in the electron microscope are folded into circular structures. The same treatment applied to trout and salmon sperm DNA fragments results in about 20% circular structures. “Slipping” involves chain separation and subsequent annealing. In this case circular struotures are easy to find, but their frequency cannot be estimated because much of the DNA remains denatured and invisible. We conolude that a large fraction of the eucaryotic genomes that we have studied is composed of regions containing tandemly-repeating sequences. At this time it is not known whether or not these tandemly-repeating regions function in the genetic sense (to specify amino acid sequences). Either way this question is resolved, the finding that such a large fraction of the eucaryotic chromosome is constructed in this manner raises some paradoxical questions.
1. Introduction In order to interpret his cytological studies of lampbrush chromosomes, Callan suggested that the euoaryotic chromatid contains a single DNA double helix, along which each gene is represented by a specific number, perhaps hundreds or even thousands, of identical, functional copies arranged in tandem (Callan & Lloyd, 1960; Callan, 1967). Within the framework of this theory, it is possible to account for many unexplained features of eucaryotic chromosomes (Thomas, 1970). This theory can be rejected or contirmed by the answers supplied to the following questions : (1) Does a large fraction of a eucaryotic DNA consist of regions of tandemlyrepeating sequences ? (2) Is each of the tandem copies genetically functional ? (3) If the answers to the foregoing questions prove to be affirmative, then one must ask: What mechanism allows the synthesis of protein molecules of apparently-identical amino acid sequence from many copies of a gene, if each copy is free to mutate and evolve independently! The experiments reported here were devised to answer the 6rst of these questions. t Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Term., U.S.A. $ Present address: Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Cal-37, Calcutta, India.
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If a large fraction of the eucaryotic DNA were composed of tandemly-repeating sequences, then it should be possible to form rings and circular structures by a process In accord with this expectation, eucaryotic DNA called “folding” or “slipping”. fragments are readily cyclized by these treatments, while procaryotic DNA fragments are not. These experiments are described below.
2. The Method of Folded Circle@ Eucaryotic DNA’s were purified and fragmented by shear into segments ranging from 1 to 10 TVin length. These duplex fragments were then degraded to various extents (5 to 25%) with either exonuclease III (Richardson, Inman & Kornberg, 1964) or h exonuclease (Little, 1967 ; Radding, 1965). The action of these nucleases exposes either 5’-ended (exo III) or 3’-ended (h exonuclease) single polynucleotide chains at the terminals of the fragment. If these chains are sufficiently complementary in
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Exon$ease
Chain diagram
Chain diagram 12
First folding
- 1 node, 1 edge
Strand diagram Second folding - 2 nodes, 3 edges
Strand diagram Third folding - 2 nodes,
4 edges
FIG. 1. Formation of “folded” circular structures from tandemly-repeating sequences. The diagrems illustrate how randomly-broken, tandemly-repetitious DNA fragments would be expected to form circular structures if their terminals had been partly degraded by exonuclet~~~~ III. The double helix is depicted by two parallel lines, and repetitious sequences by the broad arrows -b. The numbers - 1, 2, 3 - represent the nucleotide sequence, and the primed numbers - l’, 2’, 3’ - the complementary sequence. The numbers written between the chains )ix)‘) - represent the different copies of the identical sequences. The arrow represents a 5’ end and the bar ---I a 3’ end of a polynuoleotide chain. The strand diagrams show the double helix as a single line. The contour length of each closed loop shown should be a simple multiple, N, of a common value, 8, the length of the identical copy (see caption to Fig. 4). It is these figures, and their degraded products, that are to be expected in the electron microscope (see Plate II). For clarity, the digestion and folding have been presented in rr stepwise manner, however, the same structures could be produced in a single cycle of digestion and annealing. t “Folding” is a term first used by Hershey, Burgi & Ingraham (1963) to describe the analogous process of cyclization displayed by h bacteriophage DNA molecules which possess natural, mutually-complementary single chain terminals.
$ J $
A fraction corresponding to the center of the band I’ortions were removed at various times, mixed to were prepared and phot,ographed. (a) Sample removed Table 1, Figs 2 and 3 are all derived from a single
(4
(‘4
I. Nectwms 1)NA fragments. from a companion tube to that shown in Fig. 3 was dialyzed and treat4 with X exonucleasr. 2 times standard saline citrate and incubated for 2 hr at 60°C. Electron microscope specimens Thsso pirtjnres, the dat,a shown in at, t,ime zero, (b) sample removed a,ftPr 10~~ degmdation. experiment. PLATE
Example
Strand
diagram
0 Ring
Chain diagram
a
Lariat
8 Double
ring
I’I.A~~ 11. Examples of foltlvtl circular structures and tht,ir possiblr~ mtwpwtat ions 111tomb of tantlcmly-repeating sequences. The most. abundant struct,ures are “rings” which make up &bout are easy to find, particu~arlp with ~sonuclease IT1 half of all circular structures swn. “Lariats” degraded fragments. Double rings are infrequent, but not surprising. Polyrings are raw and often difficult to identify with confidence. The oxamplr sho\vn is the clearest, but \ve prrsumc it to t)(, broken once. The drawings follow the same rules as drswibcd brlow Fig. I Thr length of th(a scale bar in the micrographs is 0.3 p.
PLATE III. Some examples of “slipped” circular structures. Salmon or trout sperm DNA was denatured by heat or alkali, then annealed for 12 hr or more at 60°C. Alternatively, samples were annealed for several days at room temperature in standard saline citrate containing 50”/” formamide. Many of the circular structures shown here came from the thermal chromatography experiment depicted in Fig. 5.
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TAEZLE~ Frequency
of
C~TCU~T
DNA structures seen in electron microscope
Source of DNA fragments length 1 to 10~
Not digested, annealed
Procaryotic DNA (T7 Phage)
= <0*2% > 600
Eucaryotic DNA (Nffiturw)
= <0*3% > 300
Partly exomlcleasedigested, annealed
0
1
(5)
64 -= -4000
N l*aO/o (4) (6)
This Table shows that a significant fraction of eucaryotic DNA fragments will anneal to form circular structures if they are iirst partly degraded by exonucleases that are known to expose single ohain terminals. T7 and bacterial DNA fragments will form very few circular struotures bv this treatment. Neither DNA will form any_ significant number of circular structures if not degraded or annealed. The DNA’s shown were isolated and purified as described earlier. The Necturuu DNA fragments were further fractionated bv sucrose aadient centrifugation and then by CsCl banding. Each time the modal fractions we& aelected, dialysed and partly degraded (lo%)-by X exonuole&e. The frequency of circular structures was determined by counting the molecules having closed loops. About one-half to three-quarters of all circular structures appear to be unblemished circular duplex molecules. A number of other experiments have been performed that indicate the generality of these findings. These are listed below, keyed by number to the Table above. (1) Necturua DNA fragments, 13% degraded by exonuclease III give 23% ciroular structures, after 18 hr annealing at 60°C. Undigested oontrols suffering the same annealing give 6% circular structures. Extended amealing treatment increases the frequency of circles in undegraded controls, perhaps by exposing te rminal single chains. Briefer (36 hr) exposure to 60°C results in fewer circles in controls (0.8%), yet produoes 20% (14/71) circular struotures if 6 to 8% degraded by exonuclease III. Long (> 10~) Nectum-s DNA fragments produced the lowest observed frequency (N 6%) of circular structures. (2) Salmon sperm DNA fragments 1 to 10 p long produce 17% (110/837) oimular struotures if 10% degraded by exonuclease III and annealed for 18 hr. Storing the degraded samples for 2 days in the cold yields as many circular structures, 20% (61/311). Undegraded, muumealed oontrols yielded 2.3% circular structures. Nearly identical results were obtained with rainbow trout sperm DNA fragments. We suspect that additional purifioation (sedimentation and CsCl banding), that was applied to the Necturus DNA fragments shown in the Table, may result in removal of some substance that blocks the action of the exonuclease. (3) Calf-thymus DNA fragments, partly-degraded by exonuclease III will produce circular moleoules that are easy to find in the electron microscope. No systematic inventories have yet been made. (4) The &o&r molecules found with T7 DNA fragments are unexpeoted, rare, yet born&de and unblemished in appearance. More frequent than circles are “T-joints” that we suspeot to be or “gap” opened by the known the union of a single chain terminal with an interior “window” a&ion of exonuclease III. (6) Fragments of J. 01% DNA that were 10% degraded by h exonuclease gave 2 to 7/386 ciroles. (This notation meana 2 bon&de ciroles plus 6 additional doubtful circles were found among 386 fragments total.) B. sub& fragments, 16% and 20% degraded, gave 4 to 61709 and 3 to 61668 aimlee. Undegraded oontrols showed no (O/422) cimular structures. Taken together, this cyclization frequency, 0.6 to 1% is somewhat lower than seen with T7 DNA fragments.
mquenoe, an annealing treatment will unite them to form a duplex, and in the process form a circular molecule. When the fragments of eucaryotic DNA’s were treated in this manner and examined by electron microscopy, a large fraction (up to 36% by number) of the fragments were found in circular form (Plate I). These results are summarized in Table 1, together with the procaryotic DNA controls which show a very low frequency (O45o/o)of circular structures.
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From these experiments we conclude that a large fraction (significantly more than 35%, depending upon the unknown efficiency of the cyclization process) of the duplex DNA fragments are ternainally-repetitious. That is, the nucleotide sequence found at one end of a given fragment is repeated at or near the other end. Unless this were the case, the exonuclease treatment would not expose single-chain terminals of mutually-complementary sequence and circles would not be formed by annealing. In general, procaryotic DNA fragments are not terminally-repetitious. On the other hand, experiments of this kind have led to the conclusion that intact, linear DNA molecules from bacteriophage are terminally-repetitious (see Thomas, 1967; Thomas, Kelly C%Rhoades, 1968). If the shear-breakages generally occur at random with respect to nucleotide sequence, the most plausible interpretation of these experiments is that the fragments are tundemly-repetitious. This would assure that most fragments will be terminally-
(a)
20~1
12.4~
i-l
-
Linear fragments
-
Circular fragments 1 exe 10% degraded
-
Linear
--
Circular fragments, alkali denatured, annealed
fragment
Length $1
FIQ. 2. Length distributions of circular fragments. (a) Folded circular structures. The lengths of the linear fragments of Necturus DNA were measured from photographs such as that shown in Plate I(a). The contour lengths of the circular fragments were measured around the closed portion of the figures found in photographs equivalent to Plate I(b). The mean length of the linear fragments is 2.38 p (corresponding to 6.25 million with this spreading technique). A 10% degradation should produce circles of 2.1 p whereas the mean value observed is 2.0 p. In histograms not shown, all the Nectuncs DNA circles can be seen to range from 0.2 p to 15 p with the greatest density occurring between 0.2 and 1 p. Essentially the same distributions are produced by salmon and trout sperm DNA fragments, but the upper limit is about 5 ~1.Almost all of the rare circles produced by T7 and baoterial DNA are less than 1 p around. The mean lengths of fragments are indicated by arrows. (b) Slipped circular structures. These were measured from Plates such as those shown in Plate III. Salmon sperm DNA was denatured with 0.20 M-NaOH, reneutralized and annealed at room temperature for several days in standard saline citrate containing 60% (v/v) formamide. It can be seen that the contour lengths of the slipped circles are generally shorter than those of the folded circles. However, this could be due to the fact that the parent linear fragments were shorter.
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repetitious even though the shear-breakages occurred at random. This interpretation is diagrammed in Figure 1. The contour lengths of the closed loops of the circular structures are shown by histograms in Figure 2. Most rings are less than 2 TVaround. However, many have contour lengths in excess of 6 p, and some exceed 20 p. About half of the circular structures seen are not rings but “lariats”, “double rings” and “polyrings”. As shown in Plate II and diagrammed in Figure 1, these structures can easily be interpreted on the basis of tandemly-repeating sequences and are diflicult to interpret under other models. However, the most definitive structures, double-rings and polyrings, are more rare. In view of the existence of “satellite” and “ribosomal” DNA that are thought to consist of multiple tandem copies (Flamm, Walker & McCallum, 1969o,b; Brown & Weber, 196&b) we desired to learn whether fragments of different buoyant density in CsCl were equally susoeptible to cyclization after exonuclease treatment, even though trout and Necturus DNA display no visible “satellites” in sedimentation equilibrium experiments in the analytical ultracentrifuge. Necturw DNA fragments (band sedimentation S,,*w = 20 8) were crudely fractionated by sedimentation through sucrose. The peak tubes were pooled and centrifuged to equilibrium in CsCl. Small fractions were collected, dialyzed and degraded with h exonuclease to various
-II*O R T
I.5
Fraction number
FIU. 3. Frequency of oyclization of fragments of different density. by zone sedimentation, then banded Ne&urw DNA fragments (6& = 20 8) were fraotionated in C&l for 48 hr at 35,000 rev./min. Fraotions (10 drops) were collected in the usual manner, their absorbance measured, then dialyzed against 0*067 M-Tris, pH 9.6. Selected fraotiona were degraded with A exonuclease in the speotrophotometer and portiona mixed to 2 times standard ealine citrate and annealed for 2 hr at 6O’C. The number of linear and circular molecules was counted on the fluorescent soreen as the gride were searched in a systematic fashion. The shaded freotions were examined. The numbers near the data points refer to the extent of X exonucleaae degradation. on this band sign&ant, because the aompanion We do not oonsider the apparent “shoulders” tube and the analytical U.V. photographs displayed none. Density decreases to the left.
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extents in spectrophotometer cuvettes. After a two-hour annealing, the samples were observed in the electron microscope and the number of linear and circular molecules counted on the fluorescent screen. The results are shown in Figure 3. Here one sees about 20% circular structures which are found in all regions of the band, provided the extent of exonuclease degradation is greater than 5%. From this it would appear that the ability to cyclize is not restricted to a special density (or compositional) species of fragments.
3. The Method of Slipped Circles Another kind of experiment can be used to produce circular structures from DNA’s containing tandemly-repetitious regions. It differs from the first approach in the way single-chained ends are “exposed” for annealing. In this second approach, the sheared DNA fragments, of same size as used in the nuclease experiments, are totally denatured by treatment with heat or alkali, then annealed. If complementary chains are tandemly-repeating, they are likely to unite in a complementary manner, yet out of register (“slipped”) by one or more repeating units. Such molecules have terminal single chains that can proceed to form circular structures as shown in Figure 4. Some representative circular structures are shown in the electron micrographs reproduced in Plate III. When these experiments are repeated with E. coli DNA or T7 phage DNA fragments of equivalent length, electron micrographs reveal linear segments of renatured DNA that are strikingly free of circular structures or other entanglements. They provide a sharp contrast to renatured eucaryotic DNA where circular structures and more complicated entanglements (called “mops”) are the rule. As diagrammed in Figure A, this is in accord with expectaton for segments of DNA consisting of tandemly-repeating sequences. This experimental strategy has previously been applied to intact phage DNA molecules, and has led to the conclusion that certain species of phage contain DNA molecules that are circular permutations of each other (Thomas t MacHattie, 1964; Rhoades, MacHattie & Thomas, 1968). The contour lengths of the slipped circles range from 0.20 to 2.0 p as shown by the dotted histogram in Figure 2(b). The slipped circles are smaller than the folded circles, the lengths of which can be compared in the same Figure. While “slipped” circles are easy to find, it is nearly impossible to measure their frequency, because an uncertain fraction of DNA is denatured, invisible, or present in “mops”. Again, we were concerned whether this ability to form slipped circular structures was a general property of DNA fragments of all compositions, or whether it was restricted to some special component, analogous to the “satellite” DNA mentioned above. To answer this question, salmon sperm DNA fragments were loaded on a thermostatically controlled hydroxyapatite column which was then washed with a phosphate buffer that would elute single chains but not duplex segments. As the temperature is raised, the washing continues. As shown previously (Miyazawa & Thomas, 1965), this process, called “thermal chromatography”, fractionates DNA segments with respect to melting temperature and mole fraction G + C. Fractions collected at various temperatures are then annealed and examined in the electron microscope for the presence of circular structures. The results shown in Figure 5 indicate that all thermal fractions contain circular structures. Thus, the conclusions are the same as that made from the experiment described in Figure 3: the ability to oyclize is not restricted to a special compositional species of DNA segments.
CYCLIZATION
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FRAGMENTS
Shear >
627
Shear
1.123.123.123~S-‘l23.723.12/. I, > > ,
>
>
>
I>
,
+ Denature,
separate
chains, anneal
4 Ma 8’8 g,,-
7;
, 8A NGJ
El
Chain diagrams
Strand
. 2 Nodes, 3 Edges E,+E2=NS E2 + E3 = NS E,+E,#NS
diagrams
FICA 4. Formation of “slipped” circular structures from tandemly-repeating sequences. When a double helix consisting of repeating sequences is denatured and the component single chains are separated, then allowed to anneal, it is probable that they will reunite out of register or “slipped” by one or more unit lengths. The resulting duplex molecule has complementary terminals that may anneal repeatedly to form rings, double rings and polyringe, that are nearly equivalent to those depicted in Fig. 1 even though they were formed by a different route. The segments of length, El, Ea, etc. that form closed loops consisting of an integral number, N, of identical copies of length 8, are indicated by the equations shown near the related diagram.
4. Some Experimental Details (a) DNA
sources and purification
Salmon sperm DNA from Sigma Biochemical Co. was dissolved with stirring overnight in 0.006 M-Tris buffer, pH 7.5, to a flnal concentration of 1 mg/ml. About 6 O.D. units of DNA were purified by being loaded on a hydroxyapatite (Tiselius, Hjerten & Levin, 1966; Miyazawa BE Thomas, 1965) column (2 to 3 ml. packed volume) and then wsshed with 2.0 M-NaCl, 0.02 m-phosphate buffer (the phosphate buffer consisted of equal moles of NasHPOd and NaHzP04, pH 6-S). After washing the NaCl out with additional 0.20 M-phosphate buffer, the DNA was eluted with 0.30 M-phosphate buffer, and dialyzed against 0.007 Irr-Tris buffer, pH 7.5 (for experiments with exonuclease III) or pH 9.6 (for h exonuclease experiments). Trout sperm DNA was prepared as follows : freshly shed rainbow trout milt was filtered with dry through a nylon cloth, mixed with glycerol to 15% ( v / v ) and frozen promptly
028
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97 % recovered
f
Are circular structures
Temperature
found?
(“Cl
Fro. 6. Presence of “slipped” circular structures in the thermal-chromatographic fractions of salmon sperm DNA fragments. Salmon sperm DNA fragments (z = 1.5 p, see Fig. 2) were loaded onto a thermally-jacketed chromatographic column and washed with 0.10 M-phosphate buffer. As the temperature was increased stepwise, more and more DNA was eluted in a denatured condition. The amount recovered in this manner is plotted as a cumulative distribution. Each thermal fraction was annealed and seerahed for circular structures. Circular structures could be readily found in all thermal fractions.
ice. The milt contained 7.6 x log cells/ml. and Burton diphenylamine assays indicated 4.0 pg of DNA per cell, which is somewhat more than the 2.45 pg reported earlier by Vendrely (1958). The glycerolated milt was thawed, a 100 pl. portion diluted to 10 ml. in standard saline citrate (0.15 M-N&I + 0.015 M-sodium citrate), and sodium lauryl sulfate was added to 1%. Solid pronase (Calbiochem Grade B) was added to 1 mg/ml., and the mixture was incubated at 37’C overnight. After phenol extraction and dialysis (Thomas, Berns & Kelly, 1966), the DNA was purified by high salt hydroxyapatite chromatography as described above. As an alternative preparation scheme, the glycerolated milt was diluted loo-fold in 8.0 ~-urea, 1-O i+d-NaC1G4, 0.24 M-phosphate buffer (Britten, Pavich & Smith, 1970, personal communication). This caused prompt lysis of the cells and rendered the solution very viscous. This solution was then stirred in a Virtis homogenizer at a speed that would just begin to break intact T7 DNA molecules. At this point about 4 ml. packed volume of hydroxyapatite (previously washed with the urea solution) was added and the mixture, and washed with 10 to 20 slurried about for 10 to 15 mm, then poured into a column column volumes of the urea solution, After the column was washed with an equal volume of 0.014 &f-phosphate buffer, then 0.2 M-buffer containing 2.0 ~-N&cl, then 0.20 M-buffer alone, the DNA was eluted with 0.40 M-phosphate buffer and dialyzed against Tris buffer as described. DNA could be isolated from Nectumce liver by the urea method. T7 phage and DNA was prepared as described previously (Thomas & Abelson, 1966), but it too was purified by high salt hydroxyapatite chromatography. (b) Analytical
information
RNA contents were estimated by the fraction perchloric acid soluble by alkali treatment. All RNA. Although no protein determinations were matography yielded preparations with Aaso/Aaso
of the absorbance at 260 mn rendered preparations contained less than 1% made, high salt-hydroxyapatite chroratios often exceeding 2.5, a fact that
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indicates the absence of protein and other substances with high end-absorption. Comparative sedimentation velocity experiments were made in sucrose gradients (containing 1.0 or 0.10 M-N&~, 0.01 aa-Tris, 0.001 M-EDTA) with labeled T7 DNA as a marker. Alternatively, the method of band sedimentation (Vinograd, Bruner, Kent & Weigle, 1963) was employed using 30 mm cells in the analytical ultracentrifuge. CsCl equilibrium centrifugation was conducted either in the analytical or preparative ultraoentrifuge in solutions containing 0.01 na-Tris (pH 7), 0.001 M-EDTA. In the latter case, 0.30 ml. fractions were assayed by Ass,., in miniature IO-mm cuvettes. (c) Sheuti~ Shearing ww performed crudely by pressing the DNA twice through a no. 27 hypodermic needle.
solutions
(10 to 20 &ml.)
Exonuclease digestions were followed in either of two ways: (1) A trace of 3aP-labeled T7 phago DNA was added to the unlabeled eucaryotic DNA before shearing. If it is assumed that both species of DNA are attacked by the exonuclease at equal rates, the extent of digestion can be estimated by the fraction of label rendered trichloroacetic acid soluble as previously described (Richardson et al., 1964). Other experiments measuring perchloric acid soluble optical density and radioactivity showed this assumption to be acceptable. (2) The reaction was conducted in a miniature, 10 mm, spectrophotometer ouvette containing 300 ~1. The increase in absorbance, A -A,,, associated with digestion can be measured and the fraction digested,f, is calculated from the equationf = 0877 (A-A,)/ A,. This equation assumes that nucleotides and single oh&ins are 1.82 and 1.32 times more absorptive than duplex DNA. It is possible to use DNA solutions with es little as 6 pg/ml. (A, = 0.10) and still enjoy approximately 10% precision in the determination off. The reaction mixtures for exonuclease III contained 0.067 M-Tris buffer (pH 76), O*OOl M-MgC12, 0.02 M-mercaptoethtLno1 and 30 to 160 mqoles DNA/ml. Exonuclease HI (20 to 50 units/ml.) was added and the solution stirred with a glass fiber. The reaction mixtures for h exonuclease contained 0.067 M--Tris buffer (pH 96), 0.002 rd-MgC12, 30 to 150 mpmoles DNA/ml. and O-1 to 1.0 unit h exonuclease/ml. (Radding, 1966). Small samples (20 ~1.) were removed, added to 2 $. 20 times standard saline citrate and incubated at 60°C for 1 to 3 hr. In some cases annealing wee continued overnight, however, such prolonged annealing proved unnecessary. The samples could be stored for many days at 4°C during which time many electron microscope grids could be prepared. (f) Electrm ??&3'08CO$ly Speoimens were prepared by several variations of the Kleinschmidt procedure (Ritchie, Thomas, MaeHattie & Wensink, 1967; Davis, Simon & Davidson, 1970). Molecules were generally photographed at a magni&&ion of 6000 to 9000 and projected and traced at a total magni6cation of 100,000.
5. Discussion In this account, we have presented evidence that a large fraction of the shearbroken fragments of eucaryotic DNA’s will form circles and circular structures when partly degraded by exonuclease and annealed. Since bacterial and bacteriophage
DNA fragments will produce very few circular structures by this treatment, we conclude that the ability to cyclize is a special property of eucaryotic DNA fragments. We assume that the exposed terminal polynucleotide chains of eucaryotic DNA
fragments unite because they are complementary, just as the exposed terminals of intact phage DNA molecules unite. However, it might be conjectured that the
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terminals of eucaryotic DNA fragments have some residual protein(s) or other substance(s) that interact to form circles. To maintain this conjecture, one must explain why the “substance” becomes sticky only after treatment with either exonuclease. This objection might survive, if one imagines that the substance is only bound to single chains. But if it does not associate with duplex fragments, it should have been separated from the DNA during the extensive purification, which in some cases included all of the following: sodium lauryl sulphate treatment, pronase digestion, phenol extraction, hydroxyapatite chromatography, sedimentation through sucrose, banding in CsCl. The conjecture of non-DNA, or non-specific sticky substances might still be maintained by further special assumptions. However, these would not explain why only circles and not end-to-end enchainments of fragments are produced. In sum, the notion of non-specific joining of molecular ends must be rejected. If we agree that the circles are formed because the exposed polynucleotides are sufficiently complementary in sequence, then we conclude that the shear-broken fragments are terminally-repetitious. If the shear-breaks occurred at or in the neighborhood of special sequences, then our experiments reveal something unexpected about the shear fracture of eucaryotic DNA. However, such need not be the case, and is not the case with T7 DNA. Shear breakages are more probable near the middle of DNA molecules (Burgi & Hershey, 1961), and single chain discontinuities, “nicks”, may be slightly more sensitive to shear (Burgi, Hershey & Ingraham, 1966 ; Abelson & Thomas, 1966). If eucaryotic DNA could be shown to have specifically-located nicks, this argument may have some relevance. However, at this point, there is no evidence that such nicks exist, or if they did, that most shear-breakages would occur at them. If the shear-breaks occur randomly, the question arises : “How can random breaks accidentally form fragments whose terminals are repetitious ?” The most plausible interpretation is that the eucaryotic DNA’s studied consist of regions of tandemlyrepeating sequences in accord with Callan’s suggestion. However, other models for the organization of sequences might be considered. It might be supposed that a fraction of the eucaryotic genome is composed of (‘spacer” DNA. Within a region extending over 10 to 20 II, the spacer sequences must be considered nearly-identical. The relevant calculations have not yet been performed, but certain limiting cases can be recognized. If the number of nucleotides removed by exonuclease is somewhat smaller than the length of the individual “spacer” segment, then the chance that both ends of a given fragment fall in a spacer region is P2 if P is the fraction of the DNA composed of “spacer”. In order to account for the frequency of circles presented in Table 1, namely 35%, we must suppose that 59% of the DNA is of this character. If we assume that the efficiency of circle formation is a generous 50o/ot, then 84% of the genome must be of this character. Even this becomes an underestimate, unless the “spacer” itself is considered to be internally highly repetitious so that simultaneous exposure of any part of the spacer region will form a cyclizable fragment. The large amount of this “spacer” DNA, together with the requirements as to its internal structure means that this alternative model must resemble the original model. Apart t Phagge DNA molecules, all of which are thought to be terminally-repetitious often display considerably less than 60% circles after these treatments (see Thomas et al., 1968). This is likely to result from inhibition of the exonuclease degradation. From what is known of the specificity of h exonuclease, molecular ends bearing Y-OH, or having overlapping 5’-ended single chains are refractory. Likewise, ends having overlapping 3’-ended single chains are resistant to exonuoleaae III.
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FRAUMENTS
631
from these calculations, models of this kind cannot easily account for the polyrings (Plate II) or the slipped circular structures (Plate III). If the “spacer” is distributed into short lengths that are longer than 100 nucleotides, yet smaller than the length exposed by the action of exonuclease, circular structures could conceivably be formed, particularly if the density of these hypothetical regions was high enough. However, this model demands intermittent regions of non-complementarity that could be detected in the electron microscope at intervals around the rings. In contrast, most circular structures appear unblemished, except for occasional single chain appendages. In partial denaturation studies, we find that it is possible to detect regions of single chains that are 0.02 p long, corresponding to 59 nucleotides. Therefore, if mismatched regions of less than this length were distributed over long (1 to 2 t.~)regions, they would probably escape detection. To reject this unlikely model, the thermal stability of the rings must be determined, and the relative contour lengths of polyring structures measured. Thus, it may be possible to devise models that cannot be rejected by the evidence available to date. However, the original model of regional, tandem repetition suggested by Callan proves to be in exact accord with these results. What fraction of Necturus and fish sperm DNA is tandemly repetitious ? At this writing all that is known is that 20 to 35% of the fragments form circular structures. If we assume (as before) a generous 50% over-all efficiency of cyclization, then 40 to 70% (by number) or 36 to 56% (by weight) of the fragments are tandemly-repetitious. Clearly if the exposed ends of a fragment terminate outside a region of tandem repetition, or in regions containing tandem repetitions of a substantially different sequence, then cyclization would be impossible. If the tandemly-repetitious copies of one type or family (Thomas, 1970) span a length P, then the fraction of segments touching this region that are capable of cyclizing will be (F- L)/P when L is the length of the shear broken fragment. This primitive idea suggests that some gene families? are at least 4 to 5 p long or more in trout DNA and 8 to 10 p or more in Necturu.s DNA. However, the histograms shown in Figure 2 are in no sense indicative of the length distribution of tandemly-repeating sections because the distribution of linear fragments resulting from shear is not uniform. Moreover, longer fragments should cyclize more slowly for kinetic reasons (Wang & Davidson, 1966) and therefore be under-represented. What is needed is a systematic study of the frequency of circular structures as a function of fragment length and extent of exonuclease action. It is to be expected that the results would depend upon the species from which the DNA was extracted. We are pleased to thank a series of skillful research assistants. M. Lucinde Kelly obtained the first photographs (Plate III) that encouraged us. Barbara V. Otto wa8 instrumental in the tist folded-circle experiments and finally Carolyn Compton supplied much of the effort and pragmatic intelligence that brought the work to this stage. We have benefited from many conversations with Dr Oscar L. Miller Jr. We are grateful to Dr Charles Radding for hi8 excellent A exonuclease. We benefited from many‘discu88ion8 with Dr L. M. Okun. Mr Reed Pyeritz has performed the cyclization experiments with calf thymus DNA. We thank R. A. Schlegel for advising us on controlled shearing of DNA solutions. We thank our benefactors, the National Science Foundation (GB-6082) and the National Institutes of Health (Gm-08186) without whose support this work could not have been done. One of us (B. A. H.) was the recipient of a National Institutes of Health Postt It must be remembered, been shown to be functions1
that the tsndemly-repetitious in the genetic seme.
DNA
identified
by this work
has not
632
C. A. THOMAS
JR. ET AL.
doctoral Fellowship no. 5-F02-CA41226-02 from the National Cancer Institute. and another (C. S. L.) is a Fellow of the Jane Coffi Childs Memorial Fund for Medical Research. Finally, we express our appreciation to the Harvard Medical School where this work was done. REFERENCES Abelson, J. A. & Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Brown, D. D. & Weber, C. S. (1968a). J. Mol. Biol. 34, 661. Brown, D. D. & Weber, C. S. (19688). J. Mol. BioZ. 34, 681. Burgi, E. & Hershey, A. D. (1961). J. Mol. BioZ. 3, 458. Burgi, E., Hershey, A. D. & Ingraham, L. (1966). Virology, 28, 11. Callan, H. G. (1967). J. Cell. Sci. 2, 1. Callan, H. G. & Lloyd, L. (1960). Phil. Trans. Roy. Sot. B, 243, 135. Davis, R. W., Simon, M. & Davidson, N. (1970). In Methods in Enzymology, ed. by L. Grossman & K. Moldave, vol. 12, part C. New York: Academic Press, in the press. Flamm, W. G., Walker, P. M. B. & McCallum, M. (1969a). J. Mol. BioZ. 40, 423. Flamm, W. G., Walker, P. M. B. & McCallum, M. (19693). J. Mol. BioZ. 42, 441. Hershey, A. D., Burgi, E. & Ingraham, L. (1963). Proc. Nat. Acad. Sk., Wmh. 49, 748. Little, J. W. (1967). J. BioZ. Chem. 242, 679. Miyazawa, Y. & Thomas, C. A., Jr. (1965). J. MOE. BioZ. 11, 223. Radding, C. M. (1965). J. Mol. BioZ. 18, 235. Rhoades, M., MacHattie, L. A. & Thomas, C. A. (1968). J. Mol. BioZ. 37, 21. Richardson, C. C., Inman, R. B. & Kornberg, A. (1964). J. Mol. BioZ. 9, 46. Ritchie, D. A., Thomas, C. A., Jr., MacHattie, L. A. & Wensink, P. C. (1967). J. Mol. Biol. 23, 365. Thomas, C. A., Jr. (1967). J. Cell Physiol. 70, (Suppl. 1) 13. Thomas, C. A., Jr. (1970). In The Neurosciences: Second Study Program, ed. by F. 0. Schmitt. New York: Rockefeller University Press, in the press. Thomas, C. A., Jr. & Abelson, J. A. (1966). In Procedures in Nucleic Acid Research, ed. by G. L. Cantoni & D. R. Davies, p. 553. New York: Harper & Row. Thomas, C. A., Jr., Berns, K. I. & Kelly, T. J., Jr. (1966). In Procedures in Nucleic Acid Reaeurch, ed by G. L. Cantoni & D. R. Davies, p. 535. New York: Harper & Row. Thomas, C. A., Jr., Kelly, T. J., Jr. & Rhoades, M. (1968). Cold Spr. Hark Syrnp. @amt. Biol.
33, 417.
Thomas, C. A., Jr. & MacHattie, L. A. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1297. Tiselius, A., Hjerten, S. & Levin, 0. (1966). Arch. Biochem. Biophys. 65, 132. Vendrely, R. (1958). Ann. Inst. Pmteur, 94, 142. Vinograd, J., Bnmer, R., Kent, R. & Weigle, J. (1963). Proc. ANat. Acad. Sk., Wash. 49, 902. Wang, J. C. BEDavidson, N. (1966). J. Mol. BioZ. 19, 469.
Cyclization of Eucaryotic Deoxyribonucleic C. A. THOMAS JR., B. A. liLumuot,D.N.MIs~a$
Acid Fragments AND C.S. LEE
Department of Biological Chmistry Harvard Medical S&o01 Boston, Ma.w. 02115, U.S.A. (Received 10 March 1970, and in revised form 24 April 1970) Highly purified, shear-broken fragments of DNA extracted from certain euoaryotes (salmon, trout, Necturus, calf thymus and others) can form circles and circular structures by either “folding” or “slipping”. Procaryotic DNA fragments (from T7 bacteriophage, Escherid& coli and Bacillus aubtdi.~) do not form circles or circular structures by this treatment. “Folding” involves partial degradation with exonucleases (exonuclease III or r\ exonuclease) and annealing. Up to 35% of all Necturw, DNA fragments seen in the electron microscope are folded into circular structures. The same treatment applied to trout and salmon sperm DNA fragments results in about 20% circular structures. “Slipping” involves chain separation and subsequent annealing. In this case circular struotures are easy to find, but their frequency cannot be estimated because much of the DNA remains denatured and invisible. We conolude that a large fraction of the eucaryotic genomes that we have studied is composed of regions containing tandemly-repeating sequences. At this time it is not known whether or not these tandemly-repeating regions function in the genetic sense (to specify amino acid sequences). Either way this question is resolved, the finding that such a large fraction of the eucaryotic chromosome is constructed in this manner raises some paradoxical questions.
1. Introduction In order to interpret his cytological studies of lampbrush chromosomes, Callan suggested that the euoaryotic chromatid contains a single DNA double helix, along which each gene is represented by a specific number, perhaps hundreds or even thousands, of identical, functional copies arranged in tandem (Callan & Lloyd, 1960; Callan, 1967). Within the framework of this theory, it is possible to account for many unexplained features of eucaryotic chromosomes (Thomas, 1970). This theory can be rejected or contirmed by the answers supplied to the following questions : (1) Does a large fraction of a eucaryotic DNA consist of regions of tandemlyrepeating sequences ? (2) Is each of the tandem copies genetically functional ? (3) If the answers to the foregoing questions prove to be affirmative, then one must ask: What mechanism allows the synthesis of protein molecules of apparently-identical amino acid sequence from many copies of a gene, if each copy is free to mutate and evolve independently! The experiments reported here were devised to answer the 6rst of these questions. t Present address: Biology Division, Oak Ridge National Laboratory, Oak Ridge, Term., U.S.A. $ Present address: Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Cal-37, Calcutta, India.
621
C. A.
622
THOMAS
JR.
ET
AL.
If a large fraction of the eucaryotic DNA were composed of tandemly-repeating sequences, then it should be possible to form rings and circular structures by a process In accord with this expectation, eucaryotic DNA called “folding” or “slipping”. fragments are readily cyclized by these treatments, while procaryotic DNA fragments are not. These experiments are described below.
2. The Method of Folded Circle@ Eucaryotic DNA’s were purified and fragmented by shear into segments ranging from 1 to 10 TVin length. These duplex fragments were then degraded to various extents (5 to 25%) with either exonuclease III (Richardson, Inman & Kornberg, 1964) or h exonuclease (Little, 1967 ; Radding, 1965). The action of these nucleases exposes either 5’-ended (exo III) or 3’-ended (h exonuclease) single polynucleotide chains at the terminals of the fragment. If these chains are sufficiently complementary in
31231
Exon$ease
Chain diagram
Chain diagram 12
First folding
- 1 node, 1 edge
Strand diagram Second folding - 2 nodes, 3 edges
Strand diagram Third folding - 2 nodes,
4 edges
FIG. 1. Formation of “folded” circular structures from tandemly-repeating sequences. The diagrems illustrate how randomly-broken, tandemly-repetitious DNA fragments would be expected to form circular structures if their terminals had been partly degraded by exonuclet~~~~ III. The double helix is depicted by two parallel lines, and repetitious sequences by the broad arrows -b. The numbers - 1, 2, 3 - represent the nucleotide sequence, and the primed numbers - l’, 2’, 3’ - the complementary sequence. The numbers written between the chains )ix)‘) - represent the different copies of the identical sequences. The arrow represents a 5’ end and the bar ---I a 3’ end of a polynuoleotide chain. The strand diagrams show the double helix as a single line. The contour length of each closed loop shown should be a simple multiple, N, of a common value, 8, the length of the identical copy (see caption to Fig. 4). It is these figures, and their degraded products, that are to be expected in the electron microscope (see Plate II). For clarity, the digestion and folding have been presented in rr stepwise manner, however, the same structures could be produced in a single cycle of digestion and annealing. t “Folding” is a term first used by Hershey, Burgi & Ingraham (1963) to describe the analogous process of cyclization displayed by h bacteriophage DNA molecules which possess natural, mutually-complementary single chain terminals.
$ J $
A fraction corresponding to the center of the band I’ortions were removed at various times, mixed to were prepared and phot,ographed. (a) Sample removed Table 1, Figs 2 and 3 are all derived from a single
(4
(‘4
I. Nectwms 1)NA fragments. from a companion tube to that shown in Fig. 3 was dialyzed and treat4 with X exonucleasr. 2 times standard saline citrate and incubated for 2 hr at 60°C. Electron microscope specimens Thsso pirtjnres, the dat,a shown in at, t,ime zero, (b) sample removed a,ftPr 10~~ degmdation. experiment. PLATE
Example
Strand
diagram
0 Ring
Chain diagram
a
Lariat
8 Double
ring
I’I.A~~ 11. Examples of foltlvtl circular structures and tht,ir possiblr~ mtwpwtat ions 111tomb of tantlcmly-repeating sequences. The most. abundant struct,ures are “rings” which make up &bout are easy to find, particu~arlp with ~sonuclease IT1 half of all circular structures swn. “Lariats” degraded fragments. Double rings are infrequent, but not surprising. Polyrings are raw and often difficult to identify with confidence. The oxamplr sho\vn is the clearest, but \ve prrsumc it to t)(, broken once. The drawings follow the same rules as drswibcd brlow Fig. I Thr length of th(a scale bar in the micrographs is 0.3 p.
PLATE III. Some examples of “slipped” circular structures. Salmon or trout sperm DNA was denatured by heat or alkali, then annealed for 12 hr or more at 60°C. Alternatively, samples were annealed for several days at room temperature in standard saline citrate containing 50”/” formamide. Many of the circular structures shown here came from the thermal chromatography experiment depicted in Fig. 5.
CYCLIZATION
OF
EUCARYOTIC
DNA
FRAGMENTS
623
TAEZLE~ Frequency
of
C~TCU~T
DNA structures seen in electron microscope
Source of DNA fragments length 1 to 10~
Not digested, annealed
Procaryotic DNA (T7 Phage)
= <0*2% > 600
Eucaryotic DNA (Nffiturw)
= <0*3% > 300
Partly exomlcleasedigested, annealed
0
1
(5)
64 -= -4000
N l*aO/o (4) (6)
This Table shows that a significant fraction of eucaryotic DNA fragments will anneal to form circular structures if they are iirst partly degraded by exonucleases that are known to expose single ohain terminals. T7 and bacterial DNA fragments will form very few circular struotures bv this treatment. Neither DNA will form any_ significant number of circular structures if not degraded or annealed. The DNA’s shown were isolated and purified as described earlier. The Necturuu DNA fragments were further fractionated bv sucrose aadient centrifugation and then by CsCl banding. Each time the modal fractions we& aelected, dialysed and partly degraded (lo%)-by X exonuole&e. The frequency of circular structures was determined by counting the molecules having closed loops. About one-half to three-quarters of all circular structures appear to be unblemished circular duplex molecules. A number of other experiments have been performed that indicate the generality of these findings. These are listed below, keyed by number to the Table above. (1) Necturua DNA fragments, 13% degraded by exonuclease III give 23% ciroular structures, after 18 hr annealing at 60°C. Undigested oontrols suffering the same annealing give 6% circular structures. Extended amealing treatment increases the frequency of circles in undegraded controls, perhaps by exposing te rminal single chains. Briefer (36 hr) exposure to 60°C results in fewer circles in controls (0.8%), yet produoes 20% (14/71) circular struotures if 6 to 8% degraded by exonuclease III. Long (> 10~) Nectum-s DNA fragments produced the lowest observed frequency (N 6%) of circular structures. (2) Salmon sperm DNA fragments 1 to 10 p long produce 17% (110/837) oimular struotures if 10% degraded by exonuclease III and annealed for 18 hr. Storing the degraded samples for 2 days in the cold yields as many circular structures, 20% (61/311). Undegraded, muumealed oontrols yielded 2.3% circular structures. Nearly identical results were obtained with rainbow trout sperm DNA fragments. We suspect that additional purifioation (sedimentation and CsCl banding), that was applied to the Necturus DNA fragments shown in the Table, may result in removal of some substance that blocks the action of the exonuclease. (3) Calf-thymus DNA fragments, partly-degraded by exonuclease III will produce circular moleoules that are easy to find in the electron microscope. No systematic inventories have yet been made. (4) The &o&r molecules found with T7 DNA fragments are unexpeoted, rare, yet born&de and unblemished in appearance. More frequent than circles are “T-joints” that we suspeot to be or “gap” opened by the known the union of a single chain terminal with an interior “window” a&ion of exonuclease III. (6) Fragments of J. 01% DNA that were 10% degraded by h exonuclease gave 2 to 7/386 ciroles. (This notation meana 2 bon&de ciroles plus 6 additional doubtful circles were found among 386 fragments total.) B. sub& fragments, 16% and 20% degraded, gave 4 to 61709 and 3 to 61668 aimlee. Undegraded oontrols showed no (O/422) cimular structures. Taken together, this cyclization frequency, 0.6 to 1% is somewhat lower than seen with T7 DNA fragments.
mquenoe, an annealing treatment will unite them to form a duplex, and in the process form a circular molecule. When the fragments of eucaryotic DNA’s were treated in this manner and examined by electron microscopy, a large fraction (up to 36% by number) of the fragments were found in circular form (Plate I). These results are summarized in Table 1, together with the procaryotic DNA controls which show a very low frequency (O45o/o)of circular structures.
C. A.
624
THOMAS
JR.
ET
dL
From these experiments we conclude that a large fraction (significantly more than 35%, depending upon the unknown efficiency of the cyclization process) of the duplex DNA fragments are ternainally-repetitious. That is, the nucleotide sequence found at one end of a given fragment is repeated at or near the other end. Unless this were the case, the exonuclease treatment would not expose single-chain terminals of mutually-complementary sequence and circles would not be formed by annealing. In general, procaryotic DNA fragments are not terminally-repetitious. On the other hand, experiments of this kind have led to the conclusion that intact, linear DNA molecules from bacteriophage are terminally-repetitious (see Thomas, 1967; Thomas, Kelly C%Rhoades, 1968). If the shear-breakages generally occur at random with respect to nucleotide sequence, the most plausible interpretation of these experiments is that the fragments are tundemly-repetitious. This would assure that most fragments will be terminally-
(a)
20~1
12.4~
i-l
-
Linear fragments
-
Circular fragments 1 exe 10% degraded
-
Linear
--
Circular fragments, alkali denatured, annealed
fragment
Length $1
FIQ. 2. Length distributions of circular fragments. (a) Folded circular structures. The lengths of the linear fragments of Necturus DNA were measured from photographs such as that shown in Plate I(a). The contour lengths of the circular fragments were measured around the closed portion of the figures found in photographs equivalent to Plate I(b). The mean length of the linear fragments is 2.38 p (corresponding to 6.25 million with this spreading technique). A 10% degradation should produce circles of 2.1 p whereas the mean value observed is 2.0 p. In histograms not shown, all the Nectuncs DNA circles can be seen to range from 0.2 p to 15 p with the greatest density occurring between 0.2 and 1 p. Essentially the same distributions are produced by salmon and trout sperm DNA fragments, but the upper limit is about 5 ~1.Almost all of the rare circles produced by T7 and baoterial DNA are less than 1 p around. The mean lengths of fragments are indicated by arrows. (b) Slipped circular structures. These were measured from Plates such as those shown in Plate III. Salmon sperm DNA was denatured with 0.20 M-NaOH, reneutralized and annealed at room temperature for several days in standard saline citrate containing 60% (v/v) formamide. It can be seen that the contour lengths of the slipped circles are generally shorter than those of the folded circles. However, this could be due to the fact that the parent linear fragments were shorter.
CYCLIZATION
OF
EUCARYOTIC
DNA
FRAGMENTS
625
repetitious even though the shear-breakages occurred at random. This interpretation is diagrammed in Figure 1. The contour lengths of the closed loops of the circular structures are shown by histograms in Figure 2. Most rings are less than 2 TVaround. However, many have contour lengths in excess of 6 p, and some exceed 20 p. About half of the circular structures seen are not rings but “lariats”, “double rings” and “polyrings”. As shown in Plate II and diagrammed in Figure 1, these structures can easily be interpreted on the basis of tandemly-repeating sequences and are diflicult to interpret under other models. However, the most definitive structures, double-rings and polyrings, are more rare. In view of the existence of “satellite” and “ribosomal” DNA that are thought to consist of multiple tandem copies (Flamm, Walker & McCallum, 1969o,b; Brown & Weber, 196&b) we desired to learn whether fragments of different buoyant density in CsCl were equally susoeptible to cyclization after exonuclease treatment, even though trout and Necturus DNA display no visible “satellites” in sedimentation equilibrium experiments in the analytical ultracentrifuge. Necturw DNA fragments (band sedimentation S,,*w = 20 8) were crudely fractionated by sedimentation through sucrose. The peak tubes were pooled and centrifuged to equilibrium in CsCl. Small fractions were collected, dialyzed and degraded with h exonuclease to various
-II*O R T
I.5
Fraction number
FIU. 3. Frequency of oyclization of fragments of different density. by zone sedimentation, then banded Ne&urw DNA fragments (6& = 20 8) were fraotionated in C&l for 48 hr at 35,000 rev./min. Fraotions (10 drops) were collected in the usual manner, their absorbance measured, then dialyzed against 0*067 M-Tris, pH 9.6. Selected fraotiona were degraded with A exonuclease in the speotrophotometer and portiona mixed to 2 times standard ealine citrate and annealed for 2 hr at 6O’C. The number of linear and circular molecules was counted on the fluorescent soreen as the gride were searched in a systematic fashion. The shaded freotions were examined. The numbers near the data points refer to the extent of X exonucleaae degradation. on this band sign&ant, because the aompanion We do not oonsider the apparent “shoulders” tube and the analytical U.V. photographs displayed none. Density decreases to the left.
626
C. A.
THOMAS
JR.
ET
AL.
extents in spectrophotometer cuvettes. After a two-hour annealing, the samples were observed in the electron microscope and the number of linear and circular molecules counted on the fluorescent screen. The results are shown in Figure 3. Here one sees about 20% circular structures which are found in all regions of the band, provided the extent of exonuclease degradation is greater than 5%. From this it would appear that the ability to cyclize is not restricted to a special density (or compositional) species of fragments.
3. The Method of Slipped Circles Another kind of experiment can be used to produce circular structures from DNA’s containing tandemly-repetitious regions. It differs from the first approach in the way single-chained ends are “exposed” for annealing. In this second approach, the sheared DNA fragments, of same size as used in the nuclease experiments, are totally denatured by treatment with heat or alkali, then annealed. If complementary chains are tandemly-repeating, they are likely to unite in a complementary manner, yet out of register (“slipped”) by one or more repeating units. Such molecules have terminal single chains that can proceed to form circular structures as shown in Figure 4. Some representative circular structures are shown in the electron micrographs reproduced in Plate III. When these experiments are repeated with E. coli DNA or T7 phage DNA fragments of equivalent length, electron micrographs reveal linear segments of renatured DNA that are strikingly free of circular structures or other entanglements. They provide a sharp contrast to renatured eucaryotic DNA where circular structures and more complicated entanglements (called “mops”) are the rule. As diagrammed in Figure A, this is in accord with expectaton for segments of DNA consisting of tandemly-repeating sequences. This experimental strategy has previously been applied to intact phage DNA molecules, and has led to the conclusion that certain species of phage contain DNA molecules that are circular permutations of each other (Thomas t MacHattie, 1964; Rhoades, MacHattie & Thomas, 1968). The contour lengths of the slipped circles range from 0.20 to 2.0 p as shown by the dotted histogram in Figure 2(b). The slipped circles are smaller than the folded circles, the lengths of which can be compared in the same Figure. While “slipped” circles are easy to find, it is nearly impossible to measure their frequency, because an uncertain fraction of DNA is denatured, invisible, or present in “mops”. Again, we were concerned whether this ability to form slipped circular structures was a general property of DNA fragments of all compositions, or whether it was restricted to some special component, analogous to the “satellite” DNA mentioned above. To answer this question, salmon sperm DNA fragments were loaded on a thermostatically controlled hydroxyapatite column which was then washed with a phosphate buffer that would elute single chains but not duplex segments. As the temperature is raised, the washing continues. As shown previously (Miyazawa & Thomas, 1965), this process, called “thermal chromatography”, fractionates DNA segments with respect to melting temperature and mole fraction G + C. Fractions collected at various temperatures are then annealed and examined in the electron microscope for the presence of circular structures. The results shown in Figure 5 indicate that all thermal fractions contain circular structures. Thus, the conclusions are the same as that made from the experiment described in Figure 3: the ability to oyclize is not restricted to a special compositional species of DNA segments.
CYCLIZATION
OF
EUCARYOTIC
DNA
FRAGMENTS
Shear >
627
Shear
1.123.123.123~S-‘l23.723.12/. I, > > ,
>
>
>
I>
,
+ Denature,
separate
chains, anneal
4 Ma 8’8 g,,-
7;
, 8A NGJ
El
Chain diagrams
Strand
. 2 Nodes, 3 Edges E,+E2=NS E2 + E3 = NS E,+E,#NS
diagrams
FICA 4. Formation of “slipped” circular structures from tandemly-repeating sequences. When a double helix consisting of repeating sequences is denatured and the component single chains are separated, then allowed to anneal, it is probable that they will reunite out of register or “slipped” by one or more unit lengths. The resulting duplex molecule has complementary terminals that may anneal repeatedly to form rings, double rings and polyringe, that are nearly equivalent to those depicted in Fig. 1 even though they were formed by a different route. The segments of length, El, Ea, etc. that form closed loops consisting of an integral number, N, of identical copies of length 8, are indicated by the equations shown near the related diagram.
4. Some Experimental Details (a) DNA
sources and purification
Salmon sperm DNA from Sigma Biochemical Co. was dissolved with stirring overnight in 0.006 M-Tris buffer, pH 7.5, to a flnal concentration of 1 mg/ml. About 6 O.D. units of DNA were purified by being loaded on a hydroxyapatite (Tiselius, Hjerten & Levin, 1966; Miyazawa BE Thomas, 1965) column (2 to 3 ml. packed volume) and then wsshed with 2.0 M-NaCl, 0.02 m-phosphate buffer (the phosphate buffer consisted of equal moles of NasHPOd and NaHzP04, pH 6-S). After washing the NaCl out with additional 0.20 M-phosphate buffer, the DNA was eluted with 0.30 M-phosphate buffer, and dialyzed against 0.007 Irr-Tris buffer, pH 7.5 (for experiments with exonuclease III) or pH 9.6 (for h exonuclease experiments). Trout sperm DNA was prepared as follows : freshly shed rainbow trout milt was filtered with dry through a nylon cloth, mixed with glycerol to 15% ( v / v ) and frozen promptly
028
C. A.
THOMAS
JR.
ET AL.
97 % recovered
f
Are circular structures
Temperature
found?
(“Cl
Fro. 6. Presence of “slipped” circular structures in the thermal-chromatographic fractions of salmon sperm DNA fragments. Salmon sperm DNA fragments (z = 1.5 p, see Fig. 2) were loaded onto a thermally-jacketed chromatographic column and washed with 0.10 M-phosphate buffer. As the temperature was increased stepwise, more and more DNA was eluted in a denatured condition. The amount recovered in this manner is plotted as a cumulative distribution. Each thermal fraction was annealed and seerahed for circular structures. Circular structures could be readily found in all thermal fractions.
ice. The milt contained 7.6 x log cells/ml. and Burton diphenylamine assays indicated 4.0 pg of DNA per cell, which is somewhat more than the 2.45 pg reported earlier by Vendrely (1958). The glycerolated milt was thawed, a 100 pl. portion diluted to 10 ml. in standard saline citrate (0.15 M-N&I + 0.015 M-sodium citrate), and sodium lauryl sulfate was added to 1%. Solid pronase (Calbiochem Grade B) was added to 1 mg/ml., and the mixture was incubated at 37’C overnight. After phenol extraction and dialysis (Thomas, Berns & Kelly, 1966), the DNA was purified by high salt hydroxyapatite chromatography as described above. As an alternative preparation scheme, the glycerolated milt was diluted loo-fold in 8.0 ~-urea, 1-O i+d-NaC1G4, 0.24 M-phosphate buffer (Britten, Pavich & Smith, 1970, personal communication). This caused prompt lysis of the cells and rendered the solution very viscous. This solution was then stirred in a Virtis homogenizer at a speed that would just begin to break intact T7 DNA molecules. At this point about 4 ml. packed volume of hydroxyapatite (previously washed with the urea solution) was added and the mixture, and washed with 10 to 20 slurried about for 10 to 15 mm, then poured into a column column volumes of the urea solution, After the column was washed with an equal volume of 0.014 &f-phosphate buffer, then 0.2 M-buffer containing 2.0 ~-N&cl, then 0.20 M-buffer alone, the DNA was eluted with 0.40 M-phosphate buffer and dialyzed against Tris buffer as described. DNA could be isolated from Nectumce liver by the urea method. T7 phage and DNA was prepared as described previously (Thomas & Abelson, 1966), but it too was purified by high salt hydroxyapatite chromatography. (b) Analytical
information
RNA contents were estimated by the fraction perchloric acid soluble by alkali treatment. All RNA. Although no protein determinations were matography yielded preparations with Aaso/Aaso
of the absorbance at 260 mn rendered preparations contained less than 1% made, high salt-hydroxyapatite chroratios often exceeding 2.5, a fact that
CYCLIZATION
OF
EUCARYOTIC
DNA
FRAGMENTS
629
indicates the absence of protein and other substances with high end-absorption. Comparative sedimentation velocity experiments were made in sucrose gradients (containing 1.0 or 0.10 M-N&~, 0.01 aa-Tris, 0.001 M-EDTA) with labeled T7 DNA as a marker. Alternatively, the method of band sedimentation (Vinograd, Bruner, Kent & Weigle, 1963) was employed using 30 mm cells in the analytical ultracentrifuge. CsCl equilibrium centrifugation was conducted either in the analytical or preparative ultraoentrifuge in solutions containing 0.01 na-Tris (pH 7), 0.001 M-EDTA. In the latter case, 0.30 ml. fractions were assayed by Ass,., in miniature IO-mm cuvettes. (c) Sheuti~ Shearing ww performed crudely by pressing the DNA twice through a no. 27 hypodermic needle.
solutions
(10 to 20 &ml.)
Exonuclease digestions were followed in either of two ways: (1) A trace of 3aP-labeled T7 phago DNA was added to the unlabeled eucaryotic DNA before shearing. If it is assumed that both species of DNA are attacked by the exonuclease at equal rates, the extent of digestion can be estimated by the fraction of label rendered trichloroacetic acid soluble as previously described (Richardson et al., 1964). Other experiments measuring perchloric acid soluble optical density and radioactivity showed this assumption to be acceptable. (2) The reaction was conducted in a miniature, 10 mm, spectrophotometer ouvette containing 300 ~1. The increase in absorbance, A -A,,, associated with digestion can be measured and the fraction digested,f, is calculated from the equationf = 0877 (A-A,)/ A,. This equation assumes that nucleotides and single oh&ins are 1.82 and 1.32 times more absorptive than duplex DNA. It is possible to use DNA solutions with es little as 6 pg/ml. (A, = 0.10) and still enjoy approximately 10% precision in the determination off. The reaction mixtures for exonuclease III contained 0.067 M-Tris buffer (pH 76), O*OOl M-MgC12, 0.02 M-mercaptoethtLno1 and 30 to 160 mqoles DNA/ml. Exonuclease HI (20 to 50 units/ml.) was added and the solution stirred with a glass fiber. The reaction mixtures for h exonuclease contained 0.067 M--Tris buffer (pH 96), 0.002 rd-MgC12, 30 to 150 mpmoles DNA/ml. and O-1 to 1.0 unit h exonuclease/ml. (Radding, 1966). Small samples (20 ~1.) were removed, added to 2 $. 20 times standard saline citrate and incubated at 60°C for 1 to 3 hr. In some cases annealing wee continued overnight, however, such prolonged annealing proved unnecessary. The samples could be stored for many days at 4°C during which time many electron microscope grids could be prepared. (f) Electrm ??&3'08CO$ly Speoimens were prepared by several variations of the Kleinschmidt procedure (Ritchie, Thomas, MaeHattie & Wensink, 1967; Davis, Simon & Davidson, 1970). Molecules were generally photographed at a magni&&ion of 6000 to 9000 and projected and traced at a total magni6cation of 100,000.
5. Discussion In this account, we have presented evidence that a large fraction of the shearbroken fragments of eucaryotic DNA’s will form circles and circular structures when partly degraded by exonuclease and annealed. Since bacterial and bacteriophage
DNA fragments will produce very few circular structures by this treatment, we conclude that the ability to cyclize is a special property of eucaryotic DNA fragments. We assume that the exposed terminal polynucleotide chains of eucaryotic DNA
fragments unite because they are complementary, just as the exposed terminals of intact phage DNA molecules unite. However, it might be conjectured that the
630
C. A. THOMAS
JR.
ET
AL.
terminals of eucaryotic DNA fragments have some residual protein(s) or other substance(s) that interact to form circles. To maintain this conjecture, one must explain why the “substance” becomes sticky only after treatment with either exonuclease. This objection might survive, if one imagines that the substance is only bound to single chains. But if it does not associate with duplex fragments, it should have been separated from the DNA during the extensive purification, which in some cases included all of the following: sodium lauryl sulphate treatment, pronase digestion, phenol extraction, hydroxyapatite chromatography, sedimentation through sucrose, banding in CsCl. The conjecture of non-DNA, or non-specific sticky substances might still be maintained by further special assumptions. However, these would not explain why only circles and not end-to-end enchainments of fragments are produced. In sum, the notion of non-specific joining of molecular ends must be rejected. If we agree that the circles are formed because the exposed polynucleotides are sufficiently complementary in sequence, then we conclude that the shear-broken fragments are terminally-repetitious. If the shear-breaks occurred at or in the neighborhood of special sequences, then our experiments reveal something unexpected about the shear fracture of eucaryotic DNA. However, such need not be the case, and is not the case with T7 DNA. Shear breakages are more probable near the middle of DNA molecules (Burgi & Hershey, 1961), and single chain discontinuities, “nicks”, may be slightly more sensitive to shear (Burgi, Hershey & Ingraham, 1966 ; Abelson & Thomas, 1966). If eucaryotic DNA could be shown to have specifically-located nicks, this argument may have some relevance. However, at this point, there is no evidence that such nicks exist, or if they did, that most shear-breakages would occur at them. If the shear-breaks occur randomly, the question arises : “How can random breaks accidentally form fragments whose terminals are repetitious ?” The most plausible interpretation is that the eucaryotic DNA’s studied consist of regions of tandemlyrepeating sequences in accord with Callan’s suggestion. However, other models for the organization of sequences might be considered. It might be supposed that a fraction of the eucaryotic genome is composed of (‘spacer” DNA. Within a region extending over 10 to 20 II, the spacer sequences must be considered nearly-identical. The relevant calculations have not yet been performed, but certain limiting cases can be recognized. If the number of nucleotides removed by exonuclease is somewhat smaller than the length of the individual “spacer” segment, then the chance that both ends of a given fragment fall in a spacer region is P2 if P is the fraction of the DNA composed of “spacer”. In order to account for the frequency of circles presented in Table 1, namely 35%, we must suppose that 59% of the DNA is of this character. If we assume that the efficiency of circle formation is a generous 50o/ot, then 84% of the genome must be of this character. Even this becomes an underestimate, unless the “spacer” itself is considered to be internally highly repetitious so that simultaneous exposure of any part of the spacer region will form a cyclizable fragment. The large amount of this “spacer” DNA, together with the requirements as to its internal structure means that this alternative model must resemble the original model. Apart t Phagge DNA molecules, all of which are thought to be terminally-repetitious often display considerably less than 60% circles after these treatments (see Thomas et al., 1968). This is likely to result from inhibition of the exonuclease degradation. From what is known of the specificity of h exonuclease, molecular ends bearing Y-OH, or having overlapping 5’-ended single chains are refractory. Likewise, ends having overlapping 3’-ended single chains are resistant to exonuoleaae III.
CYCLIZATION
OF
EUCARYOTIC
DNA
FRAUMENTS
631
from these calculations, models of this kind cannot easily account for the polyrings (Plate II) or the slipped circular structures (Plate III). If the “spacer” is distributed into short lengths that are longer than 100 nucleotides, yet smaller than the length exposed by the action of exonuclease, circular structures could conceivably be formed, particularly if the density of these hypothetical regions was high enough. However, this model demands intermittent regions of non-complementarity that could be detected in the electron microscope at intervals around the rings. In contrast, most circular structures appear unblemished, except for occasional single chain appendages. In partial denaturation studies, we find that it is possible to detect regions of single chains that are 0.02 p long, corresponding to 59 nucleotides. Therefore, if mismatched regions of less than this length were distributed over long (1 to 2 t.~)regions, they would probably escape detection. To reject this unlikely model, the thermal stability of the rings must be determined, and the relative contour lengths of polyring structures measured. Thus, it may be possible to devise models that cannot be rejected by the evidence available to date. However, the original model of regional, tandem repetition suggested by Callan proves to be in exact accord with these results. What fraction of Necturus and fish sperm DNA is tandemly repetitious ? At this writing all that is known is that 20 to 35% of the fragments form circular structures. If we assume (as before) a generous 50% over-all efficiency of cyclization, then 40 to 70% (by number) or 36 to 56% (by weight) of the fragments are tandemly-repetitious. Clearly if the exposed ends of a fragment terminate outside a region of tandem repetition, or in regions containing tandem repetitions of a substantially different sequence, then cyclization would be impossible. If the tandemly-repetitious copies of one type or family (Thomas, 1970) span a length P, then the fraction of segments touching this region that are capable of cyclizing will be (F- L)/P when L is the length of the shear broken fragment. This primitive idea suggests that some gene families? are at least 4 to 5 p long or more in trout DNA and 8 to 10 p or more in Necturu.s DNA. However, the histograms shown in Figure 2 are in no sense indicative of the length distribution of tandemly-repeating sections because the distribution of linear fragments resulting from shear is not uniform. Moreover, longer fragments should cyclize more slowly for kinetic reasons (Wang & Davidson, 1966) and therefore be under-represented. What is needed is a systematic study of the frequency of circular structures as a function of fragment length and extent of exonuclease action. It is to be expected that the results would depend upon the species from which the DNA was extracted. We are pleased to thank a series of skillful research assistants. M. Lucinde Kelly obtained the first photographs (Plate III) that encouraged us. Barbara V. Otto wa8 instrumental in the tist folded-circle experiments and finally Carolyn Compton supplied much of the effort and pragmatic intelligence that brought the work to this stage. We have benefited from many conversations with Dr Oscar L. Miller Jr. We are grateful to Dr Charles Radding for hi8 excellent A exonuclease. We benefited from many‘discu88ion8 with Dr L. M. Okun. Mr Reed Pyeritz has performed the cyclization experiments with calf thymus DNA. We thank R. A. Schlegel for advising us on controlled shearing of DNA solutions. We thank our benefactors, the National Science Foundation (GB-6082) and the National Institutes of Health (Gm-08186) without whose support this work could not have been done. One of us (B. A. H.) was the recipient of a National Institutes of Health Postt It must be remembered, been shown to be functions1
that the tsndemly-repetitious in the genetic seme.
DNA
identified
by this work
has not
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JR. ET AL.
doctoral Fellowship no. 5-F02-CA41226-02 from the National Cancer Institute. and another (C. S. L.) is a Fellow of the Jane Coffi Childs Memorial Fund for Medical Research. Finally, we express our appreciation to the Harvard Medical School where this work was done. REFERENCES Abelson, J. A. & Thomas, C. A., Jr. (1966). J. Mol. Biol. 18, 262. Brown, D. D. & Weber, C. S. (1968a). J. Mol. Biol. 34, 661. Brown, D. D. & Weber, C. S. (19688). J. Mol. BioZ. 34, 681. Burgi, E. & Hershey, A. D. (1961). J. Mol. BioZ. 3, 458. Burgi, E., Hershey, A. D. & Ingraham, L. (1966). Virology, 28, 11. Callan, H. G. (1967). J. Cell. Sci. 2, 1. Callan, H. G. & Lloyd, L. (1960). Phil. Trans. Roy. Sot. B, 243, 135. Davis, R. W., Simon, M. & Davidson, N. (1970). In Methods in Enzymology, ed. by L. Grossman & K. Moldave, vol. 12, part C. New York: Academic Press, in the press. Flamm, W. G., Walker, P. M. B. & McCallum, M. (1969a). J. Mol. BioZ. 40, 423. Flamm, W. G., Walker, P. M. B. & McCallum, M. (19693). J. Mol. BioZ. 42, 441. Hershey, A. D., Burgi, E. & Ingraham, L. (1963). Proc. Nat. Acad. Sk., Wmh. 49, 748. Little, J. W. (1967). J. BioZ. Chem. 242, 679. Miyazawa, Y. & Thomas, C. A., Jr. (1965). J. MOE. BioZ. 11, 223. Radding, C. M. (1965). J. Mol. BioZ. 18, 235. Rhoades, M., MacHattie, L. A. & Thomas, C. A. (1968). J. Mol. BioZ. 37, 21. Richardson, C. C., Inman, R. B. & Kornberg, A. (1964). J. Mol. BioZ. 9, 46. Ritchie, D. A., Thomas, C. A., Jr., MacHattie, L. A. & Wensink, P. C. (1967). J. Mol. Biol. 23, 365. Thomas, C. A., Jr. (1967). J. Cell Physiol. 70, (Suppl. 1) 13. Thomas, C. A., Jr. (1970). In The Neurosciences: Second Study Program, ed. by F. 0. Schmitt. New York: Rockefeller University Press, in the press. Thomas, C. A., Jr. & Abelson, J. A. (1966). In Procedures in Nucleic Acid Research, ed. by G. L. Cantoni & D. R. Davies, p. 553. New York: Harper & Row. Thomas, C. A., Jr., Berns, K. I. & Kelly, T. J., Jr. (1966). In Procedures in Nucleic Acid Reaeurch, ed by G. L. Cantoni & D. R. Davies, p. 535. New York: Harper & Row. Thomas, C. A., Jr., Kelly, T. J., Jr. & Rhoades, M. (1968). Cold Spr. Hark Syrnp. @amt. Biol.
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Thomas, C. A., Jr. & MacHattie, L. A. (1964). Proc. Nat. Acad. Sci., Wash. 52, 1297. Tiselius, A., Hjerten, S. & Levin, 0. (1966). Arch. Biochem. Biophys. 65, 132. Vendrely, R. (1958). Ann. Inst. Pmteur, 94, 142. Vinograd, J., Bnmer, R., Kent, R. & Weigle, J. (1963). Proc. ANat. Acad. Sk., Wash. 49, 902. Wang, J. C. BEDavidson, N. (1966). J. Mol. BioZ. 19, 469.