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Stability and fine structure of eukaryotic DNA rings in formamide
J. Mol. Ba’ol. (1973) 77, 75-84
tability and Fine Structure of Eukaryotic DNA Rings in Formamide M. D. BICK, H. L. HUANCJ AND C. A. THOMAS JE Harvard Medical School Depadment of Biological Chemistry Boston, Mass. 02115, U.X.A.
(Received 18 September 29’iZ) Folded rings formed from Drosophila and Necturus DNA fragments
were examined by electron microscopy in increasing concentrations of formamide, in an effort t,o identify regions of non-homology within the closure region. Unusual closure regions of this type were not found, in spite of an extensive search. If such regions exist, they must be too short to be detectable (<50 nucleotides), or longer than 1000 nucleotides. In this latter case, they could no% be contained within the overlap region of the ring. A study of the thermal (formamide) stability of these rings in relation to the observed closure lengths suggests that extensive (>2 to 3%) mismatching is not possible. At higher formamide conoentrations, some rings will partly denature, yet remain circular because the closure region remains intact.
1. Introduction IEvidence presented in the two preceding papers (Lee 8~ Thomas, 1973; Pyeritz 8r. Thomas, 1973) indicates that the ability to form rings seems to be a general feature of eukaryotic, but not of prokaryotic, DNA fragments. A study of the frequency of folded rings as a function of fragment length and degree of resection has led to the conclusions that repetitious sequences of a given type must be densely clustered into relatively short regions, called “g-regions”, that are about 5 to 10 pm (15,000 to 30,000 nucleotide pairs) in length. Paradoxically, fragments shorter than 1 pm cyclize less efficiently, suggesting that the repeating sequences are spaced at intervals of 600 to 6000 nucleotides, yet the absolute frequency of ring formation, together with the observed thermal stability of the rings, demands at least 50% of the g-region be represented in repeating blocks that are more than 200 units long. These observations can be explained by supposing that repetitious sequences are regularly, or nearly regularly, spaced at intervals ranging from 600 to 6000 nucleotide pairs. In other words, it, is suggested that the g-regions may be composed of approximately regularly spaced, intermittently repetitious sequences or tandemly-repeating sequences in which some portion, less than half, is non-repetitious. At this point, it appears that debate can profitably be focused on the following question: do non-repetitious blocks exist 76
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between the repetitious sequences within g-regions, or are g-regions composed of purely tandemly-repetitious sequences? If non-repetitious sequences were located between the repetitious blocks, the closure region of folded fragment rings might be expected to contain regions of double helix interrupted by regions of unassociated single polynucleotide chains. On the other hand, if the g-regions contained only repetitious sequences, the closure regions should not contain mismatched single-chain regions. The competing models and consequences are shown in Figure 1. In the experiments recounted here, we have examined folded rings (and some slipped rings) that have been mounted for electron microscopy in various concentrations of formamide. Under these conditions, the non-complementary single chains should be visible. Nothing we have found can be taken as evidence to support the view that g-regions contain non-repetitious sequences. It might plausibly be argued that the repetitious blocks, or the hypothetical nonrepetitious blocks, within the g-regions contain only somewhat different sequences. In this way, the closure region might contain very short mismatched chains that could be below the ability of formamide electron microscopy to detect. This limit is about 50 nucleotides. To approach this problem, we have attempted to measure the closure length in the electron microscope. Second, we have counted the frequency of folded rings at increasing concentrations of formamide. As in the preceding papers (Lee & Thomas, 1973; Pyeritz & Thomas, 1973), the folded rings are quite stable even at high formamide concentrations. Taking the stability and closure length information together indicates that a high degree of mismatching (about 2 to 3%) is very unlikely. Finally, electron microscope grids were prepared of folded rings in concentrations of formamide that partially denature the double helix. It a.ppears that some rings are closed by polynucleotide chains of sufficient length, composition and complementarity that they remain unopened even when nearby portions of the native double helix are partially denatured. Taken together, these studies set certain limits to the number of non-complementary nucleotides in the closure regions of folded rings.
2. Methods All procedures are similar to those described in the preceding papers of electron microscopy which will be described more fully here.
with
the exception
(a) Electron mksoscope grids of DNA in formamide The method of Davis et al. (1971) and Davis & Hyman (1971) was followed including the use of isodenaturing conditions during the mounting of DNA. This involves mixing the DNA sample with a certain concentration of formamide (e.g. 6Q% v/v) in 100 rnM-Tris (pH S), 10 mivr-EDTA, 0.01% cytochrome c, then spreading 50 ~1 of this solution on a hypophase that is 30% lower in formamide and IO-fold lower in electrolyte concentration (e.g. 30% v/v in 10 mM-Tris, 1 mM-EDTA). The simultaneous reduction of both formamide and salt content is thought to produce the same reduction in T, in both the spreading solution and hypophase. Therefore, the state of the molecules visualized on the electrou microscope grid is considered to be the same as those in the formamide spreading solution. Specimens were stained in uranyl acetate, rotary shadowed with platinum-palladium alloy and examined in a Hitachi HS-7S electron microscope. Molecules were photographed at 5000 to 10,000 x and projected and traced at a final magnification of 50,000~. Contour lengths of molecules were measured with a Hewlett-Packard calculator with an accessory digitizing board for contour length measurements.
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3. Results (a) Examination
of molecules in formamide
During the course of these studies with .iVecturws and Droso&la DNA fragmeni,s, more than 1000 folded rings have been directly examined on the electron microscope fluorescent screen following preparation of grids in various concentrations of formamide. Over 200 molecules have been photographed for closer scrutiny. Plate I shows what we believe to be representative examples of folded rings from Necturtis D fragments in 20, 60 and 80% formamide. Similarly, Plate II is a collection of Drosophila hydei salivary gland DNA rings mounted in 50 to 807& formamide. These micrographs are included to demonstrate that the vast majority of rings show no features that could be interpreted to indicate the presence of unpaired single-chain
II
I Tandem repetrtion .
.
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.
.
.
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intermiftent 1111
repetition ?mllEQc
Bra. 1. Competing models for ring formations. (I) Shows a g-region composed of tandemly-repeating sequences. No non-repetitious sequences exist within the g-region. The “closure length”, the number of nucleotide pairs in the duplex portion that is responsible for closing the ring, is a single unit ranging in length from a minimum of ho nucleotides (required to form a stable duplex) to a maximum of T nucleotides, the number of exposed nucleotides at each terminal. Note that either single-chain “gaps” (a or b) or single.. chain “whiskers” (b or c) bracket the closure length. (II) Shows a g-region composed of densely-clustered repetitious blocks (shaded regions) that are 9’ units long, and intervening non-repetitious DNA. Half or more of the nucleotides in the g-region are thought to be in g’-blocks, which are themselves non-repetitious. The maximum contiguous closure length cannot exceed g’, which may be bracketed by “gaps” (d) or “whiskers” (e)., Note also the predicted structure having multiple closure lengths (f). The important feature here is the unpaired region residing between two duplex portions. Assuming random fragmentation, this structure should be found in a certain proportion of such rings whenever the closure length exceeds (by 2bo) the length of the unpaired region.
regions that would support model II (Pig. 1 (f)). However, during this search, we have found five structures that might be taken to support model II. Plate III shows the five molecules demonstrating these features. These five are the on(y such molecules found amongst over 1000 carefully studied rings in the electron microscope. The finding of these five molecules indicates that if unpaired regions are a common characteristic of folded rings, they should not elude detection.
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(b) Measurement of closure length The structures diagrammed in Figure 1 show that some rings should be amenable to direct measurement of the closure length, namely, those molecules containing either “whiskers” or “gaps”. Unfortunately (for these purposes), most of the rings appear to be perfect (neither gaps nor whiskers) and extremely few gaps have been observed. This may be due to our inability to reliably identify gaps under the present spreading conditions. However, rings having two “whiskers” are not difficult to find (Fig. l(c) and (e)), although we have not measured their frequency. Plate IV shows some examples. Presuming that the closure region is the shortest length between two such whiskers, the closure length can be measured. Figure 2 shows a histogram of these lengths measured from Necturus and Drosophila folded and slipped rings. The closure lengths range from 100 to 1000 nucleotide pairs with nearly 80% of them falling between 100 and 600. Only two out of 21 folded rings had closures shorter than 100, and none of the additional 28 slipped rings displayed closures this short.
I
El Drosophila @
Necturus slipped
El
Drosophila slipped
n
Necturus folded
W
02 600
folded
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03 900
Mwons Nxleotide
pairs
Lengf h
FIG. 2. Histogram of closure lengths. The shortest distance between “whiskers” of the type shown in Plate IV is plotted as a composite histogram of both folded and slipped rings formed from Drosophila and Necturus DNA fragments. As one can see, only 2149 rings had closures of less than 100 nucleotides in length.
(c) Stability of rings in formamide T7 phage DNA rings were mixed with either Necturus or Droso@ilia folded rings, then adjusted to concentrations of formamide ranging from 0 to 90%. The fraction of rings of both types was obtained by direct counting on the electron microscope fluorescent screen. The normalized percentage of rings remaining is plotted in Figure 3(a) and (b). On the same graph, the “equivaIent temperature”, in so far as DNA denaturation is concerned, is calculated from the relation of McConaughy et aE. (1969) (1 o/0v/v formamide = 0.7 deg. C). Figure 3 shows three theoretical curves. The dashed
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a
Dhydei O077M-No’ r=800,i=l6/~m
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20
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80 A Equivalent temperature (“Cl
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Fra. 3. Stability of folded rings in formamide. (a) Necturus DXA fragments 3 to 4 pm in length were reseated to an average of 1200 nucleotides and annealed to produce 19% circular structures, mixed with intact T7 DXA folded rings, Ghen adjusted to increasing concentrations of formamide, held at 21% for 2 to 3 min and spread for electron microscopy at 21°C under isodenaturing conditions (see text). The normalized peroentage of rings of both types is plotted against the percentage (v/v) formamide, or equivalent temperature assuming 7 deg. C for each 10% increase in formamide (McConaughy et al., 1969). (4) T7 rings; (6) Necturus folded rings. The solid ourve is the theoretical expectation drawn from equation (A14) (Fig. A3) in Thomas & Dancis (Appendix to Lee & Thomas, 1.973), assuming that each terminal has been resected by 1200 nucleotides and that the T, of Necturus DNA is 75’6 (see arrow) as calculated from its presumed composition (42% G + C) and the salt concentration (0.039 II). The solid curve is a reasonable fit to the data. When this curve is shifted 8.2 deg. C lower to indicate the stability profile of rings closed by 100 nucleotides (dotted curve) we see a poor fit, and conclude that the closure lengths must be longer than 100 nucleotides. The expected Z’p,of T7 rings is 76”C, which is 820/260 or 3 deg. C lower than the T, of T7 DXA in this solvent, 79°C. The dashed line is drawn for CJ= 4 deg. C with a midpoint of 76%. The data agree with this expectation. (b) L). h$ei fragments I.5 pm in length that were resected to an average of 800 nucleotides and annealed to produce 13% circular structures, were mixed with T7 DXA folded rings, then adjusted to increasing concentrations of formamide in 0.077 x-salt, held at 23°C for 2 to 3 min and spread for electron microscopy at 23°C under isodenaturing conditions. The theoretical solid. and dotted lines are constructed in the same way as above assuming a uniform resection of 809 nuoleotides. The Tm of D. hydei DNA is assumed to be the same as that of 8. Melanogaster and D. viriEis DNAs (see Lee & Thomas, 1973) and corrected to the present salt concentration (0.077 x). Again, we see a good fit assuming complementary sequences that are longer than the resected length (as implied by equation (A14) referred to above}, and a poor fit assuming that &sure lengths are limited to 100 nuoleotides (dotted curve). The expected T, of T7 rings is 80°C which is 820/260 or 3 deg. C less than the T,,, of T7 DNA in this solvent, 83°C. The dashed line is drawn for cr = 4 deg. C with a midpoint of 80°C. The data are better represented by a T, that is 3 deg. C lower than this.
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line represents the expected transition from rings to linears if T7 rings were closed by 260 perfectly matched nucleotide pairs of a G + C content equal to the average of T7. This is calculated from equation (A22) of Thomas & Dancis (Appendix to Lee & Thomas, 1973). Here one sees fair agreement with the data in both Figure 3(a) and (b), although the points in (a) would be a better fit by a curve drawn 3 deg. C lower, The terminals of T7 are known to melt at slightly lower concentrations of formamide than do interior regions (Wolfson et al., 1972). The solid line is drawn from equation (A14) or Figure A3 of Thomas & Dancis (Appendix to Lee & Thomas, 1973), by substituting the measured average value of r, the number of nucleotides resected from each terminal, and the expected melting temperature T& . This expression assumes that the ring closures are uniformly distributed in length between some minimum value b, (taken as 33) and r, the maximum possible. Such would be the case if the repetitious blocks were much longer than the resected length or for a purely tandemly-repeating sequence. Within the precision of the experiment, we see that the data points are well represented by the theory. The dotted curve is the solid curve moved 8.2 deg. C to the left. If the closures were limited by the length of the intermittent repetitions (g’), and if the average value of these were 100 nucleotides, this curve should describe the stability of the rings. Similar thermal stability experiments have been reported in the preceding papers (Lee & Thomas, 1973; Fyeritz $ Thomas, 1973), with nearly the same results. From this we see that the folded fragment rings are clearly more stable than expected for closures limited to 100; they are probably more stable than expected from blocks 200 units long, and would be compatible with any number above 200. (d) Partial denaturation of rings and linear j’ragments In these experiments we sought to obtain direct evidence regarding the stability of folded rings by making electron micrographs from solutions of formamide that would partially denature native DNA. This is quite possible to do, and many rings can be found to survive formamide concentrations that partially denature nearby linear fragments. Most striking is the finding that some rings display partially-denatured regions, yet the closure region fails to denature and circularity is maintained. Some examples are shown in Plate V. We must attribute this to the fact that the closure region of these surviving rings is richer in G + C than is the region suffering partial denaturation. And further, that any destabilizing effect introduced by the finite length of the closure is more than over-balanced by the stability due to locally high G + C content. It might be argued that rings are preferentially made from more stable (richer in G + C) DNA and therefore are somehow different from the linear fragments. If this were the case, rings would contain few (or no) denatured regions while the lineara would contain many. This led to a study of the relative abundance of rings and linears showing one or more denatured regions. The results are shown in Figure 4. Here we see that the frequency of Necturus rings depends on length, as shown in the preceding paper (Pyeritz & Thomas, 1973). Longer fragments, or rings, are more frequently found to contain a denatured region. Therefore, any comparison must be made within size classes. When this is done, we see that rings and linears of comparable length are equally likely to contain one or more denatured regions. Thus, it appears that there is nothing special about rings, in so far as their sensitivity to partial denaturation by formamide is concerned.
3
PLAI.E
I (I?)
PI&LX I\.‘. Cloa~we lengths. Typical esampl~s o r moleculeb which wwc used for measuring thi: closure lox&h; the shortest distance between as plotted in Fig. 2. These molecules are derived from Xeclzsr’us and D. h,ytZei, either folded or slipped rings. I3ar ecluals 0.2 urn. Note that molecule e is tho same as c in Elate III. The closure length of this unusual ring is no difkrcnt from that of typical rings.
two whi*lte%
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% of mo!ecules blistered in each we class Rings
,5 ,4 ,7 ,2 25 _ -
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Ltnears
13) ,3 18 10 3s 37 3, 30 s2 37 55 70 43 100 - 50 -
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Length (pm)
FIG. 4. Partial denaturation The histogram shows the size distribution of linear DNA fragments and mounted for electron microscopy cules has been measured. The upper portion shows the per cent of molecules scale on the histogram) which contain one or more molecules containing denatured regions were present the region between 0.5 and 3.0 pm where the majority
of folded rings. and ring structures in 80% formamide.
formed from Necturus A total of 1196 mole-
within each size class (corresponding to ths3 denatured regions. In many cases too few (represented by dash). Of prime interest is of the rings resides.
4. Discussion Perhaps the most significant result from this work is a negative one: we sought to find rings with unusual closure regions and failed to find an appreciable number. The unusual closure regions that would be expected from the intermittent repetition model are those containing unpaired single chains (Fig. l(f)). The preparation of electron microscope grids in the presence of 20 to SOo/oformamide does reveal single chains when they exist, t,herefore, if non-complementary regions exist, they must be either too long to be contained within the closure region, or they are too short to be resolved by the method used. It was concluded in the previous paper (Fyeritz & Thomas, 1973) that t.he g-region must have at least 50% of its nucleotides in blocks of 8’ that are more than 200 units long. In order to account for the decreasing frequency of rings with decreasing fragment length, it was necessary to picture these g’-blocks as being even longer and internally non-repetitious; but let us not rely on this conclusion for the moment. The point is that if the y’-blocks are of the order of 200 units then the non-repetitive blocks must also be of the order of 200 units, which places them well above the limit of detection of a non-homologous region (currently estimat,ed to be about 50 nucleotides). Wlnat fraction of rings formed from fragments that have been resected by 16300
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nucleotides would be expected to contain an unpaired region 200 units long within the closure region? In the collection of 1000 rings studied, we would have expected ZOO,assuming regular spacing of such blocks. If the unpaired region were 600 units long, we should have found 100; if it were $00 units long, we should have found 50. We actually found five structures that could satisfy the requirements of Figure l(f). As a conservative conclusion, we presume that rings with unusual closures would not be produced or found if the unpaired single chains were of the order of 1000 or more nuoleotides in length. However, if the non-repetitious blocks are evading detection by being organized into relatively long blocks (1000 units or more) then the y’-blocks must be 2600 units long in order to provide a sufficient density of repetitious sequences (72%) to satisfy equation (4) of Pyeritz & Thomas (1973) and the observed frequency of rings (see Table 3 of Pyerit.z & Thomas (1973)). Again, one must remember that this is a “worst case” analysis, and if the g’-block were itself non-repetitious, then the required value of a would have to be higher. To summarize: the absence of unusual closures can be understood in terms of an intermittent-repetition model provided that the g’-blocks were long (microns long) and if the intervening non-repeating sequences were 1000 units or longer. What if the non-homologous regions within the closure region were too short to be recognized in the electron microscope? To approach this problem, we measured the thermal stability of Necturus and D. hydei rings in formamide and found (Fig. 3) that the rings must be closed by at least 200 perfectly matched nucleotides of average composition. If mismatching occurred, greater closure lengths would be required. In Pigure 2 the length of DNA between whiskers is shown. If these lengths are a sample of all the closure lengths, including those that cannot be measured, we conclude that 90% of the closure lengths are longer than 100 nucleotide pairs, and 80% fall between 100 and 600 nuoleotide pairs. At face value, this means that the thermal stability and measured closure length data agree. If one were to entertain the idea that mismatching occurs, then one must explain why the observed closure lengths are not greater. Numerically, the idea was developed earlier (Thomas & Dancis, Appendix to Lee & Thomas, 1973) that 3% mismatched nucleot,ides would lower the T, by 9 deg. C. Certainly this would be detected by a lowered ring stability unless the closure length were three to four times longer (or contained more G + C) to compensate. IS this possible? Probably not, because the maximum closure length cannot exceed r, the number of resected nucleotides, which in this case is an average of 1200 and 800 nucleotides. The average closure should be somewhat greater than r/2 or 400 to 600 units. The observed values fall in this range but the mean value is about 300 units. Thus the observed closure lengths are in reasonable accord with the average resections. Therefore, we do not think that substantial (>2 to 3%) mismatching can occur unless special conditions were placed on G + C content. This chain of speculation may be continued by using equation (A22) of Thomas & Dan&s (Appendix to Lee & Thomas, 1973). To summarize: the hypothesis that the closure regions contain non-resolvable unpaired single chains has been approached from the point of view of the formamide stability of the rings in conjunction with measured closure lengths and measured degrees of resection. Substantial (>2 to 3%) mismatching appears very unlikely, but none of our work can exclude a uniform 1 y0 mismatching or even visible (>50 nucleotides) mismatching provided it were infrequent enough to evade being included within the closure regions.
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5. Summary In this, the last paper in a group of three, it is reasonable to offer an over-all summary of these experiments and their interpretation, In the first paper (Lee & Thomas, 1973), it was demonstrated that folded rings could be made with good efficiency from polytene chromosome DNA, which is largely devoid of satellite and rapidly-associa,ting sequences as well as heterochromatic DNA. Further: alternative modes of fragmentation give the same frequency of folded rings a fact that suggests that breakage of the DNA at “special” points is not a pre-requisite for ring formation. The ring feequeney was seen to decrease as the fragment length increased. This must mean that the repetitious sequences of a given type are clustered into a relatively short, distinctive region of the chromatid. In the paper on Ring Theory (Thomas et al.: 197.3), this idea was expressed mathematically, and allows us to estimate the length of these regions. The average length of these regions (called g-regions) is about 5 pm in Duoiro$iEa or one-half the average amount of DNA in the ehromomere for this organism. Taking these numbers at face value suggests that about one-half of the chromosomal DNA resides in g-regions (y =05). From this we estimate that the efficiency E of ring formations is also about one-half. This estimate receives confirmation from the fact that if y = 0.5 and E = 05, then the number of g-regions turns out to be the same as the number of bands in the salivary gland chromosomes. This in turn suggests that one g-region is found in one chromomere. The second paper (Pyeritz & Thomas, 1973) deals with the organization of the g-regions. Here, two competing models were introduced and the merim of both discussed. The key observation here is the fact that fragments shorter than one to two microns cyclize with decreasing efficiency. It was argued that this observation demands a sycecing of the repetitive sequences within the g-region. This paper deals with the paradoxical demands of repetitive sequences that are clustered (yet spaced) within a g-region. At the same time they must be sufficiently dense to account for the observed ring frequency. Although all these observations are understandable in terms of g-regions composed of purely tandemly-repeating sequences, it would be possible to maintain the intermittent-repetition model provided each repetitious block of &-units was long enough to satisfy the thermal stability data (>200 units), abundant enough to satisfy the ring frequency (-50% of the g-region must be in g’-blocks) and noninternally-repetitious, so that the decrease in ring frequency seen with short fragments may be explained. This is tantamount to a tandemly-repeating sequence in which the repeating unit is composed of repeating and non-repeating portions. This model was called “fractional tandem”, although a suitably arranged “irregular disposition” of intermittent repetitions might suffice. The present study was undertaken with the hope of proving the existence or nonexistence of such non-repetitious DNA within the g-region. We have faiIed to find evidence for this non-repetitious DNA between the hypothetical repetitive blocks. Either the non-repetitive blocks are too long or much too short to be seen in the electron microscope, or they do not exist. If they are hypothesized to be too long, the model that emerges is a tandemly-repeating one, in which g’ replaces g. If they are too short the problem becomes one of determining the degree of mismatching. If our analysis is correct the mismatching must be less Lhan 2%. While most of the discussion has been concerned with variations of the intermittentrepetition model, we are impressed by the fact that every single observation is in
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accord with the idea that g-regions are composed of tandemly-repeating sequences. Indeed, many of the results were predicted theoretically before they were studied. Therefore, it seems very likely to us that a significant proportion of chromosomal DNA is organized into many thousands of regions, each containing tandemly-repeating sequences. One final note of caution: in all of these studies we have been concerned with only that port,ion of the DNA that cyclizes; or as we have come to speak of it, that portion residing within g-regions. This portion is crudely estimated to be about 5Oo/oof the total DNA. About the remainder, these experiments tell us nothing. We thank Drs David Dressler and Jon Wolfson for their help with formamide
electron
microscopy. We also thank our colleagues Drs C. S. Lee and R. E. Pyeritz for sharing their materials and ideas. We also benefited from many discussions with Dr B. M. Dancis. One of us (M.D.B.) is a Fellow of the Damon Runyon Memorial Fund, and another author (H.L.H.) holds a National Institute of Health Postdoctoral Fellowship. The research was supported by grants from the National Institutes of Health (no. AI 08186-03) and the
National Science Foundation (no. GB 31118X). We are pleased to thank these benefactors.
REFERENCES Davis, R. & Hyman, R. (1971). J. Mol. Biol. 62, 287. Davis, R., Simon, M. 8: Davidson, N. (1971). Methods in En,zynzoZogy, 21, 413. Lee, C. S. & Thomas, C. A., Jr (1973). J. Mol. Biol. 77, 25. McConaughy, B., Lair-d, C. & McCarthy, B. (1969). Biochenzistry, 8, 3289. Pyeritz, R. & Thomas, C. A., Jr (1973). J. Mol. Biol. 77, 57. Wolfson, J., Dressler, D. & Magazin, M. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 499.
tability and Fine Structure of Eukaryotic DNA Rings in Formamide M. D. BICK, H. L. HUANCJ AND C. A. THOMAS JE Harvard Medical School Depadment of Biological Chemistry Boston, Mass. 02115, U.X.A.
(Received 18 September 29’iZ) Folded rings formed from Drosophila and Necturus DNA fragments
were examined by electron microscopy in increasing concentrations of formamide, in an effort t,o identify regions of non-homology within the closure region. Unusual closure regions of this type were not found, in spite of an extensive search. If such regions exist, they must be too short to be detectable (<50 nucleotides), or longer than 1000 nucleotides. In this latter case, they could no% be contained within the overlap region of the ring. A study of the thermal (formamide) stability of these rings in relation to the observed closure lengths suggests that extensive (>2 to 3%) mismatching is not possible. At higher formamide conoentrations, some rings will partly denature, yet remain circular because the closure region remains intact.
1. Introduction IEvidence presented in the two preceding papers (Lee 8~ Thomas, 1973; Pyeritz 8r. Thomas, 1973) indicates that the ability to form rings seems to be a general feature of eukaryotic, but not of prokaryotic, DNA fragments. A study of the frequency of folded rings as a function of fragment length and degree of resection has led to the conclusions that repetitious sequences of a given type must be densely clustered into relatively short regions, called “g-regions”, that are about 5 to 10 pm (15,000 to 30,000 nucleotide pairs) in length. Paradoxically, fragments shorter than 1 pm cyclize less efficiently, suggesting that the repeating sequences are spaced at intervals of 600 to 6000 nucleotides, yet the absolute frequency of ring formation, together with the observed thermal stability of the rings, demands at least 50% of the g-region be represented in repeating blocks that are more than 200 units long. These observations can be explained by supposing that repetitious sequences are regularly, or nearly regularly, spaced at intervals ranging from 600 to 6000 nucleotide pairs. In other words, it, is suggested that the g-regions may be composed of approximately regularly spaced, intermittently repetitious sequences or tandemly-repeating sequences in which some portion, less than half, is non-repetitious. At this point, it appears that debate can profitably be focused on the following question: do non-repetitious blocks exist 76
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between the repetitious sequences within g-regions, or are g-regions composed of purely tandemly-repetitious sequences? If non-repetitious sequences were located between the repetitious blocks, the closure region of folded fragment rings might be expected to contain regions of double helix interrupted by regions of unassociated single polynucleotide chains. On the other hand, if the g-regions contained only repetitious sequences, the closure regions should not contain mismatched single-chain regions. The competing models and consequences are shown in Figure 1. In the experiments recounted here, we have examined folded rings (and some slipped rings) that have been mounted for electron microscopy in various concentrations of formamide. Under these conditions, the non-complementary single chains should be visible. Nothing we have found can be taken as evidence to support the view that g-regions contain non-repetitious sequences. It might plausibly be argued that the repetitious blocks, or the hypothetical nonrepetitious blocks, within the g-regions contain only somewhat different sequences. In this way, the closure region might contain very short mismatched chains that could be below the ability of formamide electron microscopy to detect. This limit is about 50 nucleotides. To approach this problem, we have attempted to measure the closure length in the electron microscope. Second, we have counted the frequency of folded rings at increasing concentrations of formamide. As in the preceding papers (Lee & Thomas, 1973; Pyeritz & Thomas, 1973), the folded rings are quite stable even at high formamide concentrations. Taking the stability and closure length information together indicates that a high degree of mismatching (about 2 to 3%) is very unlikely. Finally, electron microscope grids were prepared of folded rings in concentrations of formamide that partially denature the double helix. It a.ppears that some rings are closed by polynucleotide chains of sufficient length, composition and complementarity that they remain unopened even when nearby portions of the native double helix are partially denatured. Taken together, these studies set certain limits to the number of non-complementary nucleotides in the closure regions of folded rings.
2. Methods All procedures are similar to those described in the preceding papers of electron microscopy which will be described more fully here.
with
the exception
(a) Electron mksoscope grids of DNA in formamide The method of Davis et al. (1971) and Davis & Hyman (1971) was followed including the use of isodenaturing conditions during the mounting of DNA. This involves mixing the DNA sample with a certain concentration of formamide (e.g. 6Q% v/v) in 100 rnM-Tris (pH S), 10 mivr-EDTA, 0.01% cytochrome c, then spreading 50 ~1 of this solution on a hypophase that is 30% lower in formamide and IO-fold lower in electrolyte concentration (e.g. 30% v/v in 10 mM-Tris, 1 mM-EDTA). The simultaneous reduction of both formamide and salt content is thought to produce the same reduction in T, in both the spreading solution and hypophase. Therefore, the state of the molecules visualized on the electrou microscope grid is considered to be the same as those in the formamide spreading solution. Specimens were stained in uranyl acetate, rotary shadowed with platinum-palladium alloy and examined in a Hitachi HS-7S electron microscope. Molecules were photographed at 5000 to 10,000 x and projected and traced at a final magnification of 50,000~. Contour lengths of molecules were measured with a Hewlett-Packard calculator with an accessory digitizing board for contour length measurements.
STABILITY
AND
FINE
STRUCTURE
OF RIXGS
3. Results (a) Examination
of molecules in formamide
During the course of these studies with .iVecturws and Droso&la DNA fragmeni,s, more than 1000 folded rings have been directly examined on the electron microscope fluorescent screen following preparation of grids in various concentrations of formamide. Over 200 molecules have been photographed for closer scrutiny. Plate I shows what we believe to be representative examples of folded rings from Necturtis D fragments in 20, 60 and 80% formamide. Similarly, Plate II is a collection of Drosophila hydei salivary gland DNA rings mounted in 50 to 807& formamide. These micrographs are included to demonstrate that the vast majority of rings show no features that could be interpreted to indicate the presence of unpaired single-chain
II
I Tandem repetrtion .
.
.
.
.
.
.
.-
intermiftent 1111
repetition ?mllEQc
Bra. 1. Competing models for ring formations. (I) Shows a g-region composed of tandemly-repeating sequences. No non-repetitious sequences exist within the g-region. The “closure length”, the number of nucleotide pairs in the duplex portion that is responsible for closing the ring, is a single unit ranging in length from a minimum of ho nucleotides (required to form a stable duplex) to a maximum of T nucleotides, the number of exposed nucleotides at each terminal. Note that either single-chain “gaps” (a or b) or single.. chain “whiskers” (b or c) bracket the closure length. (II) Shows a g-region composed of densely-clustered repetitious blocks (shaded regions) that are 9’ units long, and intervening non-repetitious DNA. Half or more of the nucleotides in the g-region are thought to be in g’-blocks, which are themselves non-repetitious. The maximum contiguous closure length cannot exceed g’, which may be bracketed by “gaps” (d) or “whiskers” (e)., Note also the predicted structure having multiple closure lengths (f). The important feature here is the unpaired region residing between two duplex portions. Assuming random fragmentation, this structure should be found in a certain proportion of such rings whenever the closure length exceeds (by 2bo) the length of the unpaired region.
regions that would support model II (Pig. 1 (f)). However, during this search, we have found five structures that might be taken to support model II. Plate III shows the five molecules demonstrating these features. These five are the on(y such molecules found amongst over 1000 carefully studied rings in the electron microscope. The finding of these five molecules indicates that if unpaired regions are a common characteristic of folded rings, they should not elude detection.
78
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(b) Measurement of closure length The structures diagrammed in Figure 1 show that some rings should be amenable to direct measurement of the closure length, namely, those molecules containing either “whiskers” or “gaps”. Unfortunately (for these purposes), most of the rings appear to be perfect (neither gaps nor whiskers) and extremely few gaps have been observed. This may be due to our inability to reliably identify gaps under the present spreading conditions. However, rings having two “whiskers” are not difficult to find (Fig. l(c) and (e)), although we have not measured their frequency. Plate IV shows some examples. Presuming that the closure region is the shortest length between two such whiskers, the closure length can be measured. Figure 2 shows a histogram of these lengths measured from Necturus and Drosophila folded and slipped rings. The closure lengths range from 100 to 1000 nucleotide pairs with nearly 80% of them falling between 100 and 600. Only two out of 21 folded rings had closures shorter than 100, and none of the additional 28 slipped rings displayed closures this short.
I
El Drosophila @
Necturus slipped
El
Drosophila slipped
n
Necturus folded
W
02 600
folded
1
03 900
Mwons Nxleotide
pairs
Lengf h
FIG. 2. Histogram of closure lengths. The shortest distance between “whiskers” of the type shown in Plate IV is plotted as a composite histogram of both folded and slipped rings formed from Drosophila and Necturus DNA fragments. As one can see, only 2149 rings had closures of less than 100 nucleotides in length.
(c) Stability of rings in formamide T7 phage DNA rings were mixed with either Necturus or Droso@ilia folded rings, then adjusted to concentrations of formamide ranging from 0 to 90%. The fraction of rings of both types was obtained by direct counting on the electron microscope fluorescent screen. The normalized percentage of rings remaining is plotted in Figure 3(a) and (b). On the same graph, the “equivaIent temperature”, in so far as DNA denaturation is concerned, is calculated from the relation of McConaughy et aE. (1969) (1 o/0v/v formamide = 0.7 deg. C). Figure 3 shows three theoretical curves. The dashed
STABILITY
AND
FINE
STRUCTURE
OF RIXGS
79
a
Dhydei O077M-No’ r=800,i=l6/~m
I
20
L-
/
40
60
80 A Equivalent temperature (“Cl
0
Fra. 3. Stability of folded rings in formamide. (a) Necturus DXA fragments 3 to 4 pm in length were reseated to an average of 1200 nucleotides and annealed to produce 19% circular structures, mixed with intact T7 DXA folded rings, Ghen adjusted to increasing concentrations of formamide, held at 21% for 2 to 3 min and spread for electron microscopy at 21°C under isodenaturing conditions (see text). The normalized peroentage of rings of both types is plotted against the percentage (v/v) formamide, or equivalent temperature assuming 7 deg. C for each 10% increase in formamide (McConaughy et al., 1969). (4) T7 rings; (6) Necturus folded rings. The solid ourve is the theoretical expectation drawn from equation (A14) (Fig. A3) in Thomas & Dancis (Appendix to Lee & Thomas, 1.973), assuming that each terminal has been resected by 1200 nucleotides and that the T, of Necturus DNA is 75’6 (see arrow) as calculated from its presumed composition (42% G + C) and the salt concentration (0.039 II). The solid curve is a reasonable fit to the data. When this curve is shifted 8.2 deg. C lower to indicate the stability profile of rings closed by 100 nucleotides (dotted curve) we see a poor fit, and conclude that the closure lengths must be longer than 100 nucleotides. The expected Z’p,of T7 rings is 76”C, which is 820/260 or 3 deg. C lower than the T, of T7 DXA in this solvent, 79°C. The dashed line is drawn for CJ= 4 deg. C with a midpoint of 76%. The data agree with this expectation. (b) L). h$ei fragments I.5 pm in length that were resected to an average of 800 nucleotides and annealed to produce 13% circular structures, were mixed with T7 DXA folded rings, then adjusted to increasing concentrations of formamide in 0.077 x-salt, held at 23°C for 2 to 3 min and spread for electron microscopy at 23°C under isodenaturing conditions. The theoretical solid. and dotted lines are constructed in the same way as above assuming a uniform resection of 809 nuoleotides. The Tm of D. hydei DNA is assumed to be the same as that of 8. Melanogaster and D. viriEis DNAs (see Lee & Thomas, 1973) and corrected to the present salt concentration (0.077 x). Again, we see a good fit assuming complementary sequences that are longer than the resected length (as implied by equation (A14) referred to above}, and a poor fit assuming that &sure lengths are limited to 100 nuoleotides (dotted curve). The expected T, of T7 rings is 80°C which is 820/260 or 3 deg. C less than the T,,, of T7 DNA in this solvent, 83°C. The dashed line is drawn for cr = 4 deg. C with a midpoint of 80°C. The data are better represented by a T, that is 3 deg. C lower than this.
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line represents the expected transition from rings to linears if T7 rings were closed by 260 perfectly matched nucleotide pairs of a G + C content equal to the average of T7. This is calculated from equation (A22) of Thomas & Dancis (Appendix to Lee & Thomas, 1973). Here one sees fair agreement with the data in both Figure 3(a) and (b), although the points in (a) would be a better fit by a curve drawn 3 deg. C lower, The terminals of T7 are known to melt at slightly lower concentrations of formamide than do interior regions (Wolfson et al., 1972). The solid line is drawn from equation (A14) or Figure A3 of Thomas & Dancis (Appendix to Lee & Thomas, 1973), by substituting the measured average value of r, the number of nucleotides resected from each terminal, and the expected melting temperature T& . This expression assumes that the ring closures are uniformly distributed in length between some minimum value b, (taken as 33) and r, the maximum possible. Such would be the case if the repetitious blocks were much longer than the resected length or for a purely tandemly-repeating sequence. Within the precision of the experiment, we see that the data points are well represented by the theory. The dotted curve is the solid curve moved 8.2 deg. C to the left. If the closures were limited by the length of the intermittent repetitions (g’), and if the average value of these were 100 nucleotides, this curve should describe the stability of the rings. Similar thermal stability experiments have been reported in the preceding papers (Lee & Thomas, 1973; Fyeritz $ Thomas, 1973), with nearly the same results. From this we see that the folded fragment rings are clearly more stable than expected for closures limited to 100; they are probably more stable than expected from blocks 200 units long, and would be compatible with any number above 200. (d) Partial denaturation of rings and linear j’ragments In these experiments we sought to obtain direct evidence regarding the stability of folded rings by making electron micrographs from solutions of formamide that would partially denature native DNA. This is quite possible to do, and many rings can be found to survive formamide concentrations that partially denature nearby linear fragments. Most striking is the finding that some rings display partially-denatured regions, yet the closure region fails to denature and circularity is maintained. Some examples are shown in Plate V. We must attribute this to the fact that the closure region of these surviving rings is richer in G + C than is the region suffering partial denaturation. And further, that any destabilizing effect introduced by the finite length of the closure is more than over-balanced by the stability due to locally high G + C content. It might be argued that rings are preferentially made from more stable (richer in G + C) DNA and therefore are somehow different from the linear fragments. If this were the case, rings would contain few (or no) denatured regions while the lineara would contain many. This led to a study of the relative abundance of rings and linears showing one or more denatured regions. The results are shown in Figure 4. Here we see that the frequency of Necturus rings depends on length, as shown in the preceding paper (Pyeritz & Thomas, 1973). Longer fragments, or rings, are more frequently found to contain a denatured region. Therefore, any comparison must be made within size classes. When this is done, we see that rings and linears of comparable length are equally likely to contain one or more denatured regions. Thus, it appears that there is nothing special about rings, in so far as their sensitivity to partial denaturation by formamide is concerned.
3
PLAI.E
I (I?)
PI&LX I\.‘. Cloa~we lengths. Typical esampl~s o r moleculeb which wwc used for measuring thi: closure lox&h; the shortest distance between as plotted in Fig. 2. These molecules are derived from Xeclzsr’us and D. h,ytZei, either folded or slipped rings. I3ar ecluals 0.2 urn. Note that molecule e is tho same as c in Elate III. The closure length of this unusual ring is no difkrcnt from that of typical rings.
two whi*lte%
STABILITY
AND
FINE
STRUCTURE
OF RINGS
% of mo!ecules blistered in each we class Rings
,5 ,4 ,7 ,2 25 _ -
-
-
Ltnears
13) ,3 18 10 3s 37 3, 30 s2 37 55 70 43 100 - 50 -
-
-
-
-
-
-
-
-
-
-
Length (pm)
FIG. 4. Partial denaturation The histogram shows the size distribution of linear DNA fragments and mounted for electron microscopy cules has been measured. The upper portion shows the per cent of molecules scale on the histogram) which contain one or more molecules containing denatured regions were present the region between 0.5 and 3.0 pm where the majority
of folded rings. and ring structures in 80% formamide.
formed from Necturus A total of 1196 mole-
within each size class (corresponding to ths3 denatured regions. In many cases too few (represented by dash). Of prime interest is of the rings resides.
4. Discussion Perhaps the most significant result from this work is a negative one: we sought to find rings with unusual closure regions and failed to find an appreciable number. The unusual closure regions that would be expected from the intermittent repetition model are those containing unpaired single chains (Fig. l(f)). The preparation of electron microscope grids in the presence of 20 to SOo/oformamide does reveal single chains when they exist, t,herefore, if non-complementary regions exist, they must be either too long to be contained within the closure region, or they are too short to be resolved by the method used. It was concluded in the previous paper (Fyeritz & Thomas, 1973) that t.he g-region must have at least 50% of its nucleotides in blocks of 8’ that are more than 200 units long. In order to account for the decreasing frequency of rings with decreasing fragment length, it was necessary to picture these g’-blocks as being even longer and internally non-repetitious; but let us not rely on this conclusion for the moment. The point is that if the y’-blocks are of the order of 200 units then the non-repetitive blocks must also be of the order of 200 units, which places them well above the limit of detection of a non-homologous region (currently estimat,ed to be about 50 nucleotides). Wlnat fraction of rings formed from fragments that have been resected by 16300
82
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JR
nucleotides would be expected to contain an unpaired region 200 units long within the closure region? In the collection of 1000 rings studied, we would have expected ZOO,assuming regular spacing of such blocks. If the unpaired region were 600 units long, we should have found 100; if it were $00 units long, we should have found 50. We actually found five structures that could satisfy the requirements of Figure l(f). As a conservative conclusion, we presume that rings with unusual closures would not be produced or found if the unpaired single chains were of the order of 1000 or more nuoleotides in length. However, if the non-repetitious blocks are evading detection by being organized into relatively long blocks (1000 units or more) then the y’-blocks must be 2600 units long in order to provide a sufficient density of repetitious sequences (72%) to satisfy equation (4) of Pyeritz & Thomas (1973) and the observed frequency of rings (see Table 3 of Pyerit.z & Thomas (1973)). Again, one must remember that this is a “worst case” analysis, and if the g’-block were itself non-repetitious, then the required value of a would have to be higher. To summarize: the absence of unusual closures can be understood in terms of an intermittent-repetition model provided that the g’-blocks were long (microns long) and if the intervening non-repeating sequences were 1000 units or longer. What if the non-homologous regions within the closure region were too short to be recognized in the electron microscope? To approach this problem, we measured the thermal stability of Necturus and D. hydei rings in formamide and found (Fig. 3) that the rings must be closed by at least 200 perfectly matched nucleotides of average composition. If mismatching occurred, greater closure lengths would be required. In Pigure 2 the length of DNA between whiskers is shown. If these lengths are a sample of all the closure lengths, including those that cannot be measured, we conclude that 90% of the closure lengths are longer than 100 nucleotide pairs, and 80% fall between 100 and 600 nuoleotide pairs. At face value, this means that the thermal stability and measured closure length data agree. If one were to entertain the idea that mismatching occurs, then one must explain why the observed closure lengths are not greater. Numerically, the idea was developed earlier (Thomas & Dancis, Appendix to Lee & Thomas, 1973) that 3% mismatched nucleot,ides would lower the T, by 9 deg. C. Certainly this would be detected by a lowered ring stability unless the closure length were three to four times longer (or contained more G + C) to compensate. IS this possible? Probably not, because the maximum closure length cannot exceed r, the number of resected nucleotides, which in this case is an average of 1200 and 800 nucleotides. The average closure should be somewhat greater than r/2 or 400 to 600 units. The observed values fall in this range but the mean value is about 300 units. Thus the observed closure lengths are in reasonable accord with the average resections. Therefore, we do not think that substantial (>2 to 3%) mismatching can occur unless special conditions were placed on G + C content. This chain of speculation may be continued by using equation (A22) of Thomas & Dan&s (Appendix to Lee & Thomas, 1973). To summarize: the hypothesis that the closure regions contain non-resolvable unpaired single chains has been approached from the point of view of the formamide stability of the rings in conjunction with measured closure lengths and measured degrees of resection. Substantial (>2 to 3%) mismatching appears very unlikely, but none of our work can exclude a uniform 1 y0 mismatching or even visible (>50 nucleotides) mismatching provided it were infrequent enough to evade being included within the closure regions.
STABILITY
AXD
FINE
STRUCTURE
OF RIKGS
5. Summary In this, the last paper in a group of three, it is reasonable to offer an over-all summary of these experiments and their interpretation, In the first paper (Lee & Thomas, 1973), it was demonstrated that folded rings could be made with good efficiency from polytene chromosome DNA, which is largely devoid of satellite and rapidly-associa,ting sequences as well as heterochromatic DNA. Further: alternative modes of fragmentation give the same frequency of folded rings a fact that suggests that breakage of the DNA at “special” points is not a pre-requisite for ring formation. The ring feequeney was seen to decrease as the fragment length increased. This must mean that the repetitious sequences of a given type are clustered into a relatively short, distinctive region of the chromatid. In the paper on Ring Theory (Thomas et al.: 197.3), this idea was expressed mathematically, and allows us to estimate the length of these regions. The average length of these regions (called g-regions) is about 5 pm in Duoiro$iEa or one-half the average amount of DNA in the ehromomere for this organism. Taking these numbers at face value suggests that about one-half of the chromosomal DNA resides in g-regions (y =05). From this we estimate that the efficiency E of ring formations is also about one-half. This estimate receives confirmation from the fact that if y = 0.5 and E = 05, then the number of g-regions turns out to be the same as the number of bands in the salivary gland chromosomes. This in turn suggests that one g-region is found in one chromomere. The second paper (Pyeritz & Thomas, 1973) deals with the organization of the g-regions. Here, two competing models were introduced and the merim of both discussed. The key observation here is the fact that fragments shorter than one to two microns cyclize with decreasing efficiency. It was argued that this observation demands a sycecing of the repetitive sequences within the g-region. This paper deals with the paradoxical demands of repetitive sequences that are clustered (yet spaced) within a g-region. At the same time they must be sufficiently dense to account for the observed ring frequency. Although all these observations are understandable in terms of g-regions composed of purely tandemly-repeating sequences, it would be possible to maintain the intermittent-repetition model provided each repetitious block of &-units was long enough to satisfy the thermal stability data (>200 units), abundant enough to satisfy the ring frequency (-50% of the g-region must be in g’-blocks) and noninternally-repetitious, so that the decrease in ring frequency seen with short fragments may be explained. This is tantamount to a tandemly-repeating sequence in which the repeating unit is composed of repeating and non-repeating portions. This model was called “fractional tandem”, although a suitably arranged “irregular disposition” of intermittent repetitions might suffice. The present study was undertaken with the hope of proving the existence or nonexistence of such non-repetitious DNA within the g-region. We have faiIed to find evidence for this non-repetitious DNA between the hypothetical repetitive blocks. Either the non-repetitive blocks are too long or much too short to be seen in the electron microscope, or they do not exist. If they are hypothesized to be too long, the model that emerges is a tandemly-repeating one, in which g’ replaces g. If they are too short the problem becomes one of determining the degree of mismatching. If our analysis is correct the mismatching must be less Lhan 2%. While most of the discussion has been concerned with variations of the intermittentrepetition model, we are impressed by the fact that every single observation is in
84
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JR
accord with the idea that g-regions are composed of tandemly-repeating sequences. Indeed, many of the results were predicted theoretically before they were studied. Therefore, it seems very likely to us that a significant proportion of chromosomal DNA is organized into many thousands of regions, each containing tandemly-repeating sequences. One final note of caution: in all of these studies we have been concerned with only that port,ion of the DNA that cyclizes; or as we have come to speak of it, that portion residing within g-regions. This portion is crudely estimated to be about 5Oo/oof the total DNA. About the remainder, these experiments tell us nothing. We thank Drs David Dressler and Jon Wolfson for their help with formamide
electron
microscopy. We also thank our colleagues Drs C. S. Lee and R. E. Pyeritz for sharing their materials and ideas. We also benefited from many discussions with Dr B. M. Dancis. One of us (M.D.B.) is a Fellow of the Damon Runyon Memorial Fund, and another author (H.L.H.) holds a National Institute of Health Postdoctoral Fellowship. The research was supported by grants from the National Institutes of Health (no. AI 08186-03) and the
National Science Foundation (no. GB 31118X). We are pleased to thank these benefactors.
REFERENCES Davis, R. & Hyman, R. (1971). J. Mol. Biol. 62, 287. Davis, R., Simon, M. 8: Davidson, N. (1971). Methods in En,zynzoZogy, 21, 413. Lee, C. S. & Thomas, C. A., Jr (1973). J. Mol. Biol. 77, 25. McConaughy, B., Lair-d, C. & McCarthy, B. (1969). Biochenzistry, 8, 3289. Pyeritz, R. & Thomas, C. A., Jr (1973). J. Mol. Biol. 77, 57. Wolfson, J., Dressler, D. & Magazin, M. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 499.