The relation of single-stranded regions in bacteriophage PM2 supercoiled DNA to the early melting sequences

J. Mol. Bid. (1975) 96, 693-702

The Relation of Single-Stranded Regions in Bacteriophage PM2 Supercoiled DNA to the Early Melting Sequences CHRISTINE BRACK, THOMAS A. BICKLE AXD ROBERT YUAN

Abteikng Mikrobiologie, Biozentrwn der Universitd Klingelbergstrasse 70, CH-4056 Ba-sel, XwitzerEand (Received 3 Murch 1975) Bacteriophage PM2 supercoiled DNA contains one to three small single-stranded regions that can be detected in the electron microscope after various treatments. The relative positions of these regions were mapped against the unique cleavage site for the restriction endonuclease R*HapII on PM2 DNA. Any of eight sharply defined regions of the genome may be single-stmnded in supercoiled molecules. They are found in all possible combinations of three or less and at approximately the same frequency. A comparison of this map of supercoiled DNA with the alkaline denaturation pattern of nicked circular or linear PM2 DNA showed that these same regions were also the earliest melting regions in non-supercoiled DNA.

1. Introduction Single-stranded regions in small, supercoiled DNA molecules (form I DNA) have been demonstrated by titration with formaldehyde or methyl mercury (Dean & Lebowitz, 1971; Beerman & Lebowitz, 1973), by their susceptibility to nucleaaes specific for single-stranded DNA (Beard et al., 1973; Germond et al., 1974), or by their ability to form complexes with single-stranded DNA binding proteins such as the gene 32 product of bacteriophage T4 (Delius et al., 1972; Morrow & Berg, 1972). Small, supercoiled DNA molecules such as that of simian virus 40 often show one such single-stranded region which can be mapped in a specific part of the genome (Morrow & Berg, 1972). It has been suggested that these single-stranded regions represent the most A+T-rich segments of the DNA (Beerman & Lebowitz, 1973; Jacob et al., 1974) although no direct evidence has yet been presented in support of this idea. The DNA from phage PM2 is the most tightly supercoiled DNA known with approximately 50 supernumerary turns per molecule of molecular weight 6.3 x lo6 (Espejo et al., 1969). It was of interest to determine whether such a relatively large and tightly supercoiled molecule also contained single-stranded regions and, if ao, whether these regions mapped in specific places on the DNA. The results presented in this paper demonstrate the existence of such regions by different electron microscopic techniques. Their positions on the DNA are mapped against the unique cleavage site on this DNA for the restriction endonuclease R.HapII and this map is compared with the map of the early melting sequences in nicked circular and linear DNA. 693

694

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2. Materials and Methods (a) Materiaz8 Purified (Franklin, 1974) bacteriophage PM2 was a gift from Dr 12. Schafer. Form I DNA was isolated by sucrose gradient centrifugation after disruption of the phngo with sodium dodecyl sulphate (Schafer & Franklin, 1975). Preparations contained between 0.5 and 3% contamination with nicked or broken molecules. Nicked circular DNA was prepared in a similar way from an aged preparation of phage that contained about 5076 nicked DNA molecules. The average number of nicks per molecule was thus estimated to be between 1 and 2. Bacteriophage T4 gene 32 protein (P32) was purified as described by Alberts & Frey (1970) from the overproducing mutant T4 55- (amBL292) (Epstein et al., 1963; Krisch et al., 1974). The sample used for the present denaturation study contained a mixture of P32 and P32* (Hosoda et aZ., 1974). The restriction endonuclease from Haemophilus uphrophilus (Takanami, 1973) was a kind gift from Dr B. Allet (b) Alkaline denaturation Partial alkaline denaturation of PM2 DNA was carried out as described by Chattoraj & Inman (1972). PM2 DNA (2 to 4 pg/ml) was incubated with a buffer containing 12% formaldehyde at 25°C and a given pH. Progressive denaturation was obtained by increasing the denaturation time from 10 to 120 min. The DNA samples were spread in a mixture containing 50% formamide (Fluka, Puriss.) and O*Olo/o cytochrome c on a hypophaso of distilled water (Inman & SchnSs, 1970), picked up on carbon-coated grids, dried in 95% ethanol and rotary shadowed with platinum at an angle of 6 to 8”. Form I DNA molecules were not denatured under the conditions in which nicked and linear molecules showed extensive denaturation (15 to 20 loops). More severe conditions were used, by increasing the formamide concentration to 63%, or increasing the temparature to 40 to 50°C. (c) Denaturation by T4 gene 32 protein Complexes between PM2 form I DNA and P32 were made by incubating the DNA (20 pg/ml) with P32 (180 rg/ml) at 37°C for 10 min in a buffer containing 20 mM-TriseHCl (pH 8.5) and 1 mM-NasEDTA (final vol. 20 to 30 ~1.) (Delius et al., 1972). Complexes were fixed with O.Olo/o glutaraldehyde for 10 min at 37°C and purified by gel filtration (Biogel A 1.5 M), eluting with 1 mM-EDTA. The purified complexes were spread for electron microscopy by adsorption to positively charged carbon films (Dubochet et al., 1971), stained for 2 to 3 s with a 2% aqueous solution of uranyl acetate, dried by blotting on filter paper and rotary shadowed with platinum. Nicked and linear molecules were not denatured under these conditions. It was found that under conditions of very low ionic strength (1 mar-EDTA) progressive denaturation of nicked and linear DNA could be obtained with relatively low amounts of protein (DNA 20 rg/ml, P32 120 pg/ml).

Native PM2 form I DNA cleaved with the restriction (pH 7.4), 60 mM-NaC1 and purified on Biogel A (1.5 M)

(d) Enzyme treatment and purified complexes of PM2 form I DNA with P32 were endonuclease R *Hap11 in a buffer containing 10 mna-Tris *HCl 7 mM-MgCl,. After the reaction, the digested molecules were as described in section (c), above.

(e) EZectron microscopy Micrographs were recorded on 70-mm roll films (Kodalith LR2572) on a Philips 301 electron microscope at magnifications of 25,000 or 32,000. Magnifications were calibrat,ed with a carbon grating replica (Pelco, 54,000 L/in). (f) Length memrements and mapping Negatives were enlarged 10 times with a projector and the molecules traced on translucent paper. Length measurements were made with a map measurer. Mapping of singlestranded regions or P32-loops were made by measuring the distances between the centres

DENATURATION

MAPPING

OF PHAGE

PM2

696

DNA

of the loops and expressing them per cent of the total genome length. The endo R .HapII cleavage site was taken as the zero point and the denatured site nearest to the endo R *Hap11 cut (16%) was arbitrarily taken as site I.

3. Results (a) A comparison

of the different

denatwation

methods

The contour length measurements of PM2 DNA molecules prepared by the two different electron microscopic techniques described in Materials and Methods are shown in Table 1. The contour length after cytochrome c spreading from formamide is about loo/” longer and more subject to variation than after adsorption to charged carbon films. With increasing denaturation, the contour length of formamide spread molecules increases to a maximum of approximately 115:/, of that of the native molecule. TABLE 1 Length measurements Alkaline denaturation, formamide spreading

1’32 denaturation-adsorption

on PM2

DNA

molecules

Double-stranded

under different

nicked circle

co~nditions

3.55&0.29

pm

Double-stranded “relaxed” form I DNA with 1 to 3 singlestranded regions

3.49f0.25

pm

Partly denatured nicked circle (10 to 12 single-stranded regions)

4.09 & 0.40 pm

Single-stranded circle (denatured nicked molecule)

3.62 10.25

Form I DN4

3.25 + 0.04 pm

with

1 to 3 loops

pm

Denaturation with P32 offers the following advantages. First, complex formation proceeds under mild conditions of pH and ionic strength. Second, the complexed regions of the DNA are far more visible in the electron microscope than the singlestranded regions after alkaline denaturation or formaldehyde treatment (compare Plates I and II). Lastly, the P32 complexes can be locked in place by glutaraldehyde fixation, the complexes purified and used in further studies.

(b) The number and extent of single-stranded

regions in PM2

supercoils

PM2 supercoiled DNA was prepared for electron microscopy after either alkaline denaturation, treatment with formaldehyde and spreading from formamide, or after complex formation with P32. Formaldehyde-treated molecules showing one or two single-stranded regions are presented in Plate I. The complex of form I DNA with P32 is shown in Plate II. All three methods showed the existence of single-stranded regions in the DNA ranging in number from one to three with an occasional molecule (less than 2% of the total number) displaying four or more such regions. A summary of the results obtained with the different techniques employed in this study on both

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supercoiled and nicked DNA is given in Table 2. The extent of the denatured regions varied between 1 and 4% of the total genome length with either of the first two methods and was slightly larger, 2 to 8% (mean 5.6%) after denaturation with P32. This last result was expected. It has been shown that single-stranded DNA coated with P32 is up to 50% longer than the naked DNA (Delius et al., 1972). TABLE

2

Comparison between different denaturation methods and results Number Form I DNA

Denaturation conditions

of denatured sites Nicked circle

pH 7.0 formaldehyde 26°C

+ 60% formamide,

l-3

0

pH 8.0 formaldehyde 25°C

+ 50% formamide,

l-3

2-3

pH 9.86 formaldehyde 26%

+ 60% formamide,

l-3

lo-16

pH 9.86 formaldehyde + 63% formamide, 26°C or 60% formamide. 60°C P32:DNA = 9:l 20 rnM-Tris/EDTA P32:DNA = 6:l 1 mm-EDTA

2-3 or completely single-stranded

extensively

l-3 l-3

denatured

0 l-16 or completely

(c) The position of the single-stranded regions An attempt was made to map the positions of the single-stranded regions relative to each other. The distances between loops on a large number of molecules were measured and the molecules were aligned by rotation and inversion to give the maximum correspondence between sites. It soon became evident that this method would give at best highly ambiguous results due to the circular permutation of the system and, in hindsight, the relatively large number of possible sites. It was therefore decided to map the sites against the unique cleavage site on PM2 DNA for the restriction endonuclease R *Hap11 (Wang, 1974). Complexes between PM2 form I DNA and P32 were fixed with glutaraldehyde, dialysed and digested with endo R+HapII. The DNA-protein complexes were separated from free protein by gel filtration and the samples were spread for electron microscopy. Plate III shows molecules prepared in this way. It was now possible to measure the positions of the denatured regions relative to the ends of the linear molecules and to attempt to order them. This was done in the following way. Many molecules were found with P32 binding sites clustered at distances of 16% and 20% from one end of the molecule. The finding of a number of molecules with denaturation loops at 16% from one end and 20% from the other allowed the assignment of these regions to opposite ends of the molecule. Molecules containing either of these sites and any other site could now be used to map the positions of all the other P32 binding sites.

hATIS I. PM2 form I DNA with single-stranded regions (+). Cytochrome e spreading fro111 fi)rmaldehycle-formamide pH 7.0. Magnification 60,000 x . The bar on all the F’lat~es wpwwnt~ lb.5 pm.

/ /r,<~iirr,,‘, +:or;

DENATURATION

MAPPING

OF

PHAGE

PM2

DNA

697

Figure l(a) shows a histogram of the results of this study. Seven sharply defined regions of the PM2 genome were found to be single-stranded in this experiment. Any three of the seven could be single-stranded on the same DNA molecule. No one of these regions had a much higher probability of being single-stranded than any other (a maximum t,hree-fold differences between sites III and V). On different DNA molecules, any combination of two different single-stranded regions can be found. In addition, it was suspected that another possible single-stranded region was situated at, or near, the endo R-Hap11 cutting site because a proportion of the P32-complexed supercoils were resistant to digestion by the enzyme. This expectation was confirmed by the finding among the resistant molecules of distances between dena(tured regions that could only be accounted for by the existence of a P32 binding site near the endo R *Hap11 cleavage point. The single-stranded regions on form I DNA prepared by formamide spreading of formaldehyde-treated molecules (Plate I) were measured and were aligned using as a basis the map shown in Figure l(a). A small proportion of the molecules (8%) gave ambiguous results and were not included in the analysis. The result is shown in Figure 1(b). The same pattern of single-stranded regions was detected with formaldehyde as with P32, although the relative frequency at which the different sites were found was somewhat different. In addition, the eighth site, close to the endo ReHupII cleavage site was now detected at about the same frequency as the others and could be assigned to a position at 990/ of the genome length on the arbitrary scale used in Figme I.. (d) The denaturation map of nicked or linear PJf2 DNA Randomly nicked circular or endo R *HapII-cleaved linear PM2 DNA was partially denatured as described in Materials and Methods with either alkali or with P32 at low ionic strength. Circular molecules prepared with both methods and endo R. HapIIproduced linear molecules treated with P32 are shown in Plates IV to VI. The pattern of denaturation was essentially the same with either technique. At low levels of denaturation a few isolated loops are seen. With increasingly severe conditions more loops appear until at advanced stages of denaturation from 15 to 20 small denatured regions are seen distributed fairly evenly along the DNA. At no stage is a particularly distinctive arrangement of denatured and native DNA apparent and the individual denatured regions do not merge with each other to form large denatured areas of DNA. There are, therefore, no distinctive features of extensively denatured DNA as there are, for example, in coliphage A DNA (Inman & Schnos, 1970). It’ was of interest to determine whether the early melting regions of non-supercoiled DNA were the same as the single-stranded regions in form I DNA. To this end, alkali-denatured, nicked circular molecules with two or three denatured regions were measured. It was then determined whether the distances between the denatured regions could be correlated with the map of single-stranded regions in form I DNA shown in Figure 1. The histogram in Figure 2 shows the distribution of the denatured regions aligned as in Figure 1. Again, eight sharply defined peaks are found. Table 3 presents a comparison of the positions of the single-stranded regions in form I DNA (both after P32 complex formation and after formaldehyde treatment) with the positions of the early melting regions in nicked circles. Agreement between

C. BRACK,

698

T. L4. BICKLE

4ND

R.

YUAS

(a)

(b)

20 -

> 15 : 2 $ Ii IO -

5-

% of total genome length

FIG. 1. The positions of single-stranded DNA in PM2 form I DNA. (a) The complex between PM2 form I DNA and P32 protein w&s prepared and digested with endo R-Hap11 8s described in Materials and Methods. The preparation was spread for electron microscopy and individual linear molecules bearing P32 complexes were photographed, enlarged and traced at a final megnification of 300,000. Tne positions of the centres of the complexed regions from the ends of the molecules were measured and the molecules were inverted if necessary to align the regions as described in Results. The positions of the sites are expressed as a percentage of the total genome length. (b) Formaldehyde-denatured supercoils prepared as described in Materials and Methods were spread for electron microscopy and the positions of the centres of the denatured regions relative to each other was measured. The molecules were rotated and if necessary inverted using s,s a guide the results shown in Fig. l(a).

DENATURATION

MAPPING

OF PHAGE

PM2

699

DNA

the three sets of results is excellent (within one standard deviation) and it is concluded t,hat the early melting regions are also those which have the tendency t)o become single-stranded in supercoils. P32-treated endo ReHapII-produced linear molecules also showed the same pattern of denaturation (results not shown). In addition, many of these molecules were denatured at one extremity and displayed a P32-coated “split end” (Plate VI) generated by denaturation of site VIII (Table 3). 25

% of total genome length

FIG. 2. The early melting regions in PM2 DNA. Randomly nicked PM2 DNA was alkalidenatured at pH 8.0 and 26°C and prepared for electron microscopy aa described in Materials and Methods. The relative positions of the centres of the denatured regions in molecules containing 2 or 3 such regions were measured. The moleoules were then aligned by rotation and inversion using as a guide the positions of the single-stranded regions in form I DNA shown in Fig. 1.

TABLE

3

Position, of single-stranded regions in y. genome length from the endo R *HapII site. Comparison of different methods

Sit,o

Form I DNA-P32 complex cleaved with endo R *Hap11

Form I DNA single-stranded regions formaldehyde-formamide

cleavage

Nicked molecule alkaline donaturation

-1 II III 1V V VI VII VlII

16.0610.44 24.63 * 0.62 28.26kO.67 31.93*1*10 64*76* 1.68 76.68kO.24 79.43+0.88 endo R *Hap11

16.28hO.9 24.37*0.19 28.64f0.23 32.03&0.16 64*20* 1.27 76.61+0.71 79.41 kO.33 99*68&0.16

16~41*0~10 24.04&0*64 28.20&0.48 31.86kO.76 64.17,t 1.46 74.96& 1.39 79.63-&0.23 99.98hO.14

700

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T. A. BICKLE

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R. YUAN

An attempt to align more extensively denatured nicked molecules with the map was also made. However, molecules with four or more denaturation loops often contained denatured regions that could not be reconciled with the map. It would appear therefore that all of the eight sites on the map need not be denatured before new sites of denaturation appear.

4. Discussion Single-stranded regions have been detected in supercoiled DNA by a number of techniques: titration of the unpaired bases (Dean & Lebowitz, 1971; Beerman & Lebowitz, 1973), digestion of supercoils with single-strand-specific nucleases (Beard et al., 1973; Germond et al., 1974) and reaction with proteins that form complexes with single-stranded DNA (Delius et al., 1972; Morrow b Berg, 1972). In this paper we present yet another method, spreading of aupercoils from formamide after reaction with formaldehyde. The method depends on hydroxymethylation by formaldehyde of non-base-paired amino groups in single-stranded regions of the DNA. Such structures are incapable of re-hybridizing during spreading for electron microscopy and can then be visualized as stable, single-stranded regions. Up to 4% of the supercoiled DNA is found to be single-stranded in these experiments after formaldehyde treatment and spreading from formamide at either neutral or alkaline pH. This is very similar to the 3.7% of bases in PM2 form I DNA that Beerman t Lebowitz (1973) found to be reactive with methyl mercury. Interestingly, it did not prove possible to further denature the covalently closed DNA beyond this point by increasing the severity of the alkali denaturation conditions (higher formamide concentrations, increasing the temperature, the time of incubation or the pH). Under conditions in which nicked DNA is almost completely denatured (see Table 2), form I DNA still shows the same pattern of denaturation as at neutral pH. Under even more severe conditions some of the supercoiled DNA molecules melt to become completely single-stranded, with no detectable intermediate denatured more than the 4% found at neutral pH (Plate VII). Any melting of covalently closed, unstrained DNA would require the introduction of positive supernumerary turns. It is quite clear from the results described above that the thermodynamic barrier against such positive supercoiling is high. The physical map of the PM2 genome determined in this study is shown in Figure 3. It shows the eight readily denaturable regions and the endo R+ Hap11 cleavage site. The orientation of the map is arbitrary, the site closest to the endo R *Hap11 cleavage site at 16% of the total genome length was called I. Such maps are useful for many purposes, especially for organisms such as PM2 where no genetic markers are available. For example, the map has been used to localize the cleavage sites on PM2 DNA for the restriction enzyme from Escherichia wli strain K (Brack et al., unpublished results). A map of the single-stranded regions in PM2 form I DNA has recently been published that does not agree with the one presented here (Jacob et al., 1974). This map, however, was obtained by aligning the alkali-denatured regions in circular molecules. This does not allow the distinction to be made between, for example, the intervals between sites VIII and II or VIII and VII (both 24 to 25% of the genome length), or between any of the pairs, II-III, III-IV and VI-VII (all 3 to 4%). Under those circumstances, it is difficult to decide on the number of sites, a decision that

DENATURATION

MAPPING

OF PHAGE

PM2

DNA

701

79% 24% 75%

f

\

lY

32%

FIG. 3. A physical map of PM2 DNA. The reletive positions of the early PM2 DNA relative to the endo R*HapII cleavage site are shown.

melting

regions in

has to be made before the molecules can be aligned. In the present study only two intervals could be confused with each other, those between the endo R *Hap11 cutting site and regions II and VI, both about 24 to 25%. This ambiguity, however, was resolved whenever sites II or VI occurred in combination with any other site. The results show that the single-stranded regions in form I DNA are the same aa the earliest melting sequences in linear or nicked circular DNA and that each of these regions has about the same probability of being single-stranded in form I DNA as of melting early during alkali or P32 denaturation. This is hardly surprising and had been previously suggested by several authors (Beerman & Lebowitz, 1973; Beard et al., 1973; Jacob et al., 1974). This study is, however, the first in which the correlation has been demonstrated. sites were found to be singleAll possible combinations of the eight denaturable stranded in form I DNA but no more than three were found on any given molecule. This suggests that form I DNA might be in a state of dynamic equilibrium in which all of the potentially single-stranded regions become single-stranded at different times. Such a resonating molecule might have a lower free energy than a static one. Other explanations are, of course, possible. The A+T-rich regions of DNA have often been implicated in the binding of RNA polymerase and the initiation of RNA synthesis (Le Talaer & Jeanteur, 1971; Shishido & Ikeda, 1971; Heyden et al., 1972; Le Talaer et aE., 1973). Brack & Delain (1975) have demonstrated

that

in kinetoplast

DNA

from

Trypanosoma

cruzi,

the

four early melting regions of the minicircular DNA are also binding sites for E. coli RNA polymerase. Studies have been initiated to determine whether this is also true for PM2 DNA. Wo would like to thank Dr B. Allet and R. Schiifer for gifts of materials used in this study and Drs W. Arber and V. Pirrotta for helpful criticisms of the manuscript. P32 protein W&B prepared in collaboration with Dr J. Hosoda, who was supported in part by the Roche Research Foundation for Scientific Exchange and Biomedical Collaboration with Switzerland. One of us (T. A. B.) held an EMBO long term fellowship during part of this work. This research was partially supported by grant no. 3.0640.73 from the Swiss National Foundation for Scientific Research.

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REFERENCES Alberts, B. & Frey, L. (1970). Nature (London), 227, 1313-1318. Beard, P., Morrow, J. & Berg, P. (1973). J. Birol. 12, 1303-1313. Beerman, T. A. & Lebowitz, J. (1973). J. Mol. Biol. 79, 451-470. Brack, Ch. & Delain, E. (1975). J. Cell Sci. 17, 287-306. Chattoraj, D. K. & Inman, R. B. (1972). J. Mol. BioZ. 66, 423-434. Dean, W. W. & Lebowitz, J. (1971). Nature New BioZ. 231, 5-8. Delius, H., Mantell, N. J. & Alberts, B. (1972). J. Mol. BioZ. 67, 341-350. Dubochet, J., Ducommun, M., Zollinger, M. & Kellenberger, E. (1971). J. UZtrastruct. Res. 35, 147-167. Epstein, R. H., Bolle, A., Steinberg, Ch. M., Kellenberger, E., Boy de la Tour, E., Chevalley, R., Edgar, R. S., Sussman, M., Denhardt, G. H. & Lielausis, A. (1963). Cold Spring Harbor

Symp.

Quunt.

BioZ. 28, 375-392.

Espejo, R. T., Canelo, E. S. & Sinsheimer, R. C. (1969). Proc. Nut. Acud. Sci., U.S.A. 1164-1168. Franklin, R. M. (1974). Current Topics Microbial. Immunol. 68, 107-160. Germond, J. E., Vogt, V. M. & Hirt, B. (1974). Eur. J. Biochem. 43, 591-600. Heyden, B., Nuesslein, C. & Schaller, H. (1972). Nature New BioZ. 244, 257-260. Hosoda, J., Takacs, B. & Brack, Ch. (1974). FEBS Letters, 47, 33&343. Inman, R. B. & Schniis, M. (1970). J. MoZ. BioZ. 49, 93-98. Jacob, R. L., Lebowitz, J. & Kleinschmidt, A. K. (1974). J. ViroZ. 13, 11761185. Krisoh, H. M., Bolle, A. & Epstein, R. H. (1974). J. Mol. BioZ. 88, 89-104. Le Talaer, J-Y. & Jeanteur, P. (1971). FEBS Lettera, 12, 253. Le Talaer, J-Y., Kermici, M. & Jeanteur, P. (1973). Proc. Nut. Acud. Sci., U.S.A. 2911-2915. Morrow, J. & Berg, P. (1972). Proc. Nut. Acad. Sk, U.S.A. 69, 3365-3369. Schafer, R. & Franklin, R. M. (1975). J. Mol. Biol. In the press. Shishido, K. & Ikeda, Y. (1971). Biochem. Biophys. Res. Commun. 44, 1429-1428. Takanami, M. (1973). FEBS Letters, 34, 31&322. Wang, J. C. (1974). J. Mol. BioZ. 87, 797-816.

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