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Residual activity of denatured transforming DNA of Haemophilus influenzae: A naturally occurring cross-linked DNA
J. Mol. Biol. (1968) 82, 423435
Residual Activity of Denatured Transforming DNA of Haemophilus inzuenzae: A Naturally Occurring Cross-linked DNA CAREL
MULDER~
AND PAUL DOTY
Department of Chemistry, Harvard University Cambridge,Massachusetts, U.S.A. (Received13 June 1967) The residual activity of denatured transforming DNA of Haemophilusinfluenzae is accounted for by the presence of cross-linked molecules in all DNA preparations, whereas single-stranded DNA has no detectible transforming activity. The specific transforming activity of the cross-linked molecules is at least 90% that of native DNA. This conclusion is reached from the observation that the residual activity sediments l-3 to 1.4 times faster than the bulk of the denatured DNA through an alkaline sucrose gradient, almost identical to the rate of an artifloially cross-linked DNA. Similarly, the residual aotivity is eluted from a Sepharose column 1.5 times faster than the bulk of the denatured DNA and ahnost identical to the elution pattern of added native DNA of the same homogeneous size. Moreover, the buoyant density in CsCl of the molecules carrying the residual activity was found to be close to that of native DNA and considerably lighter than that of denatured DNA; with denatured biological hybrid (15NaH/14N1H) DNA, this fraction was found only at a buoyant density slightly heavier than native hybrid DNA; no activity was found near the densities of native or denatured heavy DNA or native light DNA. The intrinsic residual activity is equal for all melting conditions tried and for nine different markers studied. It was found to be 1 1*5o/o for a DNA preparation with an average molecular weight of 30 x lo6 daltons, and it is linearly related to the molecular weight of the DNA. Therefore, it is estimated that about three cross-links are present per bacterial genome if they pre-exist in tivo.
1. Introduction Transforming DNA losesmost of its activity after denaturation by heat, alkali or a variety of other conditions. The residual activity, which is usually a few per cent of the original activity, has three possibleorigins (Rownd, Lanyi & Doty, 1961): (1) a small fraction of the molecules is resistant to strand separation; (2) a small degree of renaturation inevitably occurs on removal of the melting conditions; and (3) singlestranded DNA can be taken up by recipient cells to a small extent and initiate transformation. The second possibility was ruled out by Ginoza & Zimm (1961) and by Rownd (1963), who showed that the level of residual activity is independent of the DNA concentration and of the ionic environment at denaturation. The residual activity of Bacillus subtilis DNA was found to originate from molecules with native buoyant density in CsCl (Rownd, 1963; Rownd, Green, Sternglans $ f Present
St. Louis,
address: Institute for Missouri 63110, U.S.A.
Molecular
Virology, 423
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Medical
School,
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Doty, 1968), thus indicating a fraction resistant to denaturati0n.i Recently, it was shown (Alberts, 1965; Alberts & Doty, 1968) that these molecules with surviving activity are a “naturally occurring” cross-linked fraction in B. subtilis DNA. A similarly cross-linked fraction was shown to exist in DNA preparations from other bacterial, viral and mammalian sources. On the other hand, the third possibility, a small intrinsic activity of denatured DNA, was preferred for Diplowccus pneumoniue by Guild (1961) and by Guild & Robison (1963) from studies of the buoyant density of residually active DNA in CsCland by Ginoza & Zimm (1961) from experiments on the kinetics of heat inactivation of DNA. These results have recently been challenged, however (Mulder & Fox, manuscript in preparation). The residual activity of thermally denatured DNA of Haemophilw injluenzae was similarly found in one report at a buoyant density of denatured DNA (Rownd et al. 1961). However, later findings indicated that the activity has properties unlike denatured DNA: its resistanceto degradation by exonucleaseI (Barnhardt $ Herriott, 1963) and the similarity of its elution pattern from hydroxyapatite columns to that of native DNA (Chevallier & Bernardi, 1965) suggesteda bihelical configuration. In an intensive study, Barnhardt (1965) suggestedintrastrand hydrogen bonding of separated single strands as configuration of the residually active molecules. Recently, Postel6 Goodgal (1966) showed that under their experimental conditions the residual activity of denatured DNA had a buoyant density in between that of native and of denatured DNA. In view of these conflicting observations, it was of interest to examine the nature of the residual activity of H. in&ensue DNA in more detail. In this study it has been shown that the transforming activity of denatured H. injthenzae DNA is due to a small fraction of naturally cross-linked molecules which zipper up upon cooling. When isolated, these molecules show almost the samespecific activity as native DNA and a bihelical conflguration; by contrast, single-stranded DNA separated from these crosslinked moleculesdoes not show any biological activity. Some of the results presented here were briefly referred to in a recent abstract (Mulder, 1967).
2. Materials and Methods (a) Strains anu!DNA H. influenzae Rd obtained from Dr S. H. Goodgal (originally from Alexander BELeidy (1963)) was usedthroughout. It was brought into competency, and transformation assays carried out according to standard techniques (Goodgal & Herriott, 1961; Spencer L Herriott, 1906).All transformation assayswere done in the linear nx~ponse range of DNA, 6 mm/ml. or below. Strains bearing erythromycin (Er = Ee6) resistance or the linked streptomycin (Sr = 52000) and cathomycin (novobiocin) (Cr = Caas) resistancemarkerswere obtained by transforming Rd cells with Sr, Cr or Erf DNA provided by Dr J. Lanyi. Temperatnresensitive mutanta of strain Rd were induced with nitrosognanidine as described below. For isolation of DNA, the organisms were grown in Difco brain-heart infusion broth t The terminology of Kohn, Spears & Doty (1966) is used to describe the various con&gurations of DNA and the transformations among them : however, the widely used term “reversibly denaturable DNA” is retsined to describe cross-linked DNA, although “reversibly meltable” would be more consistent with this terminology. $ Abbreviations used: Sr, streptomycin-reaice ; Cr, cethomycin-resistance ; Er, erythromycinresistance; SSC, 0.16 ar-N&l-O-O16 ar-trieodiom citrate (pH 7-O); pH 9.2 buffer, O-02 x-KIHPOI (pH 9.2).
NATURALLY
CROSS-LINKED
H. influenzoe
425
DNA
supplemented with hemin (Eastman-Kodak, 10 &ml.) and NAD chemicals Corp., 2.6 pg/ml.) (medium HD) until late logarithmic phase. resistant strains, the medium was supplemented with the antibiotic. sensitive strains were grown at 3O”C, the permissive temperature. DNA was isolated as described by Alberts & Doty (1968), except that of organisms was about 1.6 x lOlo during the lysis of the cells.
(Nutritional BioFor the antibioticThe temperaturethe concentration
(b) Isolation, melting, CEenaturation and shear-induced brcukuge of DNA The experimental methods involved in the isolation of DNA by phenol have been described in the preceding paper (Alberta & Doty, 1908). The sedimentation rates @‘!&,) of these DNA preparations varied from 31 to 36.8 s, which corresponds to an average molecular weight of 20.4 to 31 x lo8 daltons. When a preparation with a value of fc0.w of 34 s (mol. wt of 25*6 x 10e) was sedimented in alkali according to Studier (1905), the melted DNA had a value of S&,, = 38 s, which corresponds to a mol. wt of 13.8 X 10’ ; therefore, this preparation was free of single-strand breaks. DNA samples were stored at 3°C in SSC (0.15 M-NaCl-0.016 ma-t&odium citrate, pH 7.0) or in pH 9.2 buffer (0.02 MKzHP04, pH 9.2). The melting and subsequent denaturation of DNA and its shear-induced breakage were performed as described by Alberta t Doty (1968). (c) cioss-h&king of DNA with nitrow acid DNA was cross-linked with nitrous acid by the method of Becker, Zimmerman & Geiduschek (1964) as described by Alberta & Doty (1968). However, the pH used was lowered to pH 4.6, as it was found that the extent of cross-linking of H. irzfluenzae DNA at pH 6.0 was only 10% of that found for B. mbtilk DNA. (d) C&l and sucrose gradient sedinzeaWim The experimental techniques used for equilibrium banding in analytical and preparative CsCl gradients and those for sedimentation through neutral and alkaline sucrose gradients are described by Albert8 & Doty (1968). (e) Biological hybrid DNA Density-hybrid DNA was isolated from Sr-cells grown for many generations in the medium described by Spencer & Herriott (1966) made up in aHaO (Bio-Rad, 96%), the NH&I being replaced by 16NH&1 (40 m&f) (Bio-Rad, 99.7%). The phosphate buffer was replaced by 3aP-labeled potassium phosphate (10 mM, pH 7) (Cambridge Nuclear Company) with a specific activity of 0.2 me/m-mole. After washing, the organisms were transferred to medium HD without radioactive label and collected after one generation of growth. The DNA extracted from these organisms was banded in a preparative C&l gradient, and the DNA in the peak fractions of the hybrid density band used for the experiments. The density of this DNA was 1.705 g/cm3 calibrated against dAT = 1.678 g/cm3. After denaturation, this DNA yielded two equal bands with buoyant densities of 1.714 and l-727 g/cm3, respectively. The density of [‘Hl’N]DNA of H. injZunzae wss found to be I.698 g/cm3 for native and 1.714 g/cm3 for denatured DNA. (f) Filtration through Sepharos Sepharose 2B (lot 3616) and 4B (lot 3270), and a Sephadex laboratory column (type K 16/90) were a gift from Pharmacia Fine Chemicals, Inc. Columns (1.6 cm x 86 cm) were washed with 600 ml. buffer S (20 mM-K,HP04-4 mar-EDTA, pH 8.8) before applying the sample. Samplee (60 to 100 pg denatured DNA in 1 to 2 ml. buffer S) were eluted with buffer S at a rate of 6 ml&r. The operating water pressure during the whole procedure was 60 cm measured from the outlet of the effluent tubing to the buffer reservoir. The DNA samples were sheared to a more uniform size before heat-denaturation. (g) Radtiotope determination Fractions from C&l gradients were counted for 3zP in Bray’s (1960) Cab-O&l in a Packard Tricarb liquid-scintillation spectrometer (model 314EX).
solution
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(h) Nitrosoguunidine-induced mutation Rd cells in early logarithmic growth in HD medium were incubated with N-methylN’-nitro-N-nitrosoguanidine (50 pg/ml.) in medium HD at 37°C for 10 min. After dilution, the organisms were incubated on HD-agar at 30°C. After replication, 15 colonies were found to be unable to grow at 41°C.
3. Results (a) Residual transforming activity The residual transforming activity of H. in$henzae DNA surviving exposure to melting (denaturing) conditions for various lengths of time followed by rapid quenching showed a course very similar to that of B. subtilis DNA (Alberta & Doty, 1968). The curves in Fig. 1 show a sharp break point between zero and two minutes. The activity decreasesrapidly during the first minute or two and then much more slowly. The initial rapid loss of activity is thought to be caused by the collapse of the helical structure and the consequent strand separation of most molecules. The slope of this Time
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of heat denaturation on the transforming eotivity of H. injluenzae DNA. 7 ml.) of Sr DNA (0.1 pg/ml.; mol. wt 21 x IO6 daltons) in potassium phosphate (0.02 M, pH 9.2 (-O--O--) or pH 7.0 (. . . 0 . . .O . . .)) in thin-walled tubes were heated in a Fro.
Samples
1. Effeot (0.2
boiling water bath. samples in potassium 15,30 and 45 min, a of these samples are by comparison with taneously.
After 2, 4, 6, 8 and 10 phosphate (0.1 M, pH tube was transferred to plotted as percentages l/l00 and l/10 dilutions
min, 11.7 a 0% of the of
a tube was transferred to a 0°C bath. Identical (- A -. . .- A -)) were heated at 40°C. After bath and neutralized. The tmnsforming activities activity of the unheated sample, as determined the original native DNA sample plated aimul-
NATURALLY
CROSS-LINKED
H. iltfluenzae DNA
427
initial decrease is of the same order of magnitude for all methods of melting and denaturation used. The slope of the second part of the curve, after two minutes, is strongly dependent on the melting conditions. This further decrease of activity is probably due to depurinization and single-chain scission(Eigner, Boedtker BEMichaels, 1961; Greer & Zamenhof, 1962). Heating at 100°C in SSC at pH 7, a widely used method of melting DNA, causesa relatively rapid lossof activity in this secondregion, considerably more than at 100°C at pH 9-2, for example. A very mild procedure for melting DNA, heating at 40°C at pH 11.7, produces hardly any further inactivation (Fig. 1). Back extrapolation of this secondpart of the curve gives the intrinsic residual activity : this is found to be independent of the melting conditions used, being 11.5% for a DNA preparation of 30 x 106 daltons. The size of this fraction is found to be directly proportional to the molecular weight ; the samepreparation when reduced by shear to 5 x lo6 daltons displayed only 2% residual activity. The intermediate molecular weight used in Fig. 1 showed 8% intrinsic residual activity. All genetic markers described for H. injluenme are for antibiotic resistance and could therefore be located on a specific region of the genome. In order to examine whether the residual activity was of the sameorder of magnitude for different regions of the genome, some temperature-sensitive mutants of H. injluenme were isolated and the residual activity of denatured DNA carrying these markers was examined. Temperature-sensitive markers are expected to be randomly distributed over the genome; accordingly, no genetic linkage was found between any of these markers or between these and the antibiotic resistance markers. Figure 1 showsonly data for the streptomycin-resistance (Saooo)marker, but identical zero-time extrapolations were obtained for the residual activity of eight other markers investigated for this sample (resistance to two antibiotics (Ws and E6’6) and six temperature-sensitive markers (ts 2, 4, 5, 7, 12 and 14)). Very similar inactivation curves were obtained when DNA artificially cross-linked by nitrous acid was subjected to the samemelting conditions, except that, due to the cross-linkings, the size of the residually active fraction was considerably larger (17 to 91%). Studies to determine the critical temperatures for inactivation of the three antibiotic markers and five temperature-sensitive markers gave identical results, namely 865°C in standard saline-citrate solution (SK!). This contrasts with similar examination of D. pneumoniae DNA, where differences up to 4°C in the critical inactivation temperature have been observed (Roger & Hotchkiss, 1961). (b) Alkaline m.mosegradients Since the sedimentation rate of melted DNA in alkali dependsin a known way on its molecular weight (Studier, 1965), the sedimentation rate of the residual activity through an alkaline sucrosegradient was examined and compared with that of DNA with artificially induced cross-links. The DNA was sheared to a more uniform molecular weight to avoid the broad sedimentation pattern of molecules of heterogeneous sizes as found in a normal DNA preparation. Cross-linked molecules should move quickly in such sedimentations, since their molecular weights should be double that of the separated single strands. The sedimentation pattern of the bulk-denaturedDNA from the gradient was obtained by absorbance measurement, that of the residual activity by transformation of suitably diluted samples. As can be seenfrom Fig. 2, this activity sediments 1.3 to l-4 times faster than does denatured DNA. Under the
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I
I
Fraction
no.
I
---r--ri,6
FIG. 2. Sediment&ion of the residual activity and of arti&&lly cross-linked DNA through an &al& zucroze gradient. A mixture of Er DNA (4 pg/ml.), crass- linked with nitrous acid to 21% (as measured by ito residual tranzforming aotivity after den&x&ion) and untreated Sr DNA (60 a/ml.) wa8 sheered to a more uniform size in aVirtie homogenizer ret 7700 rev./min for 30 min at 0% (avemge mol. wt 6.6 x lo* d&onz). To 8 nnmple (O-18 ml.) of this sheared DNA mixture, N&l (12 mg) and NaOH (0.02 ml., M) were added at 4%. This melted sample wae layered on top of & discontinuous sucrose gradient formed from 0.86~ml. layers of zucroze solutions (20, 16, 12, 8 end 4% sucrose in N&l (0.9 M), N&OH (0.1 M)), cooled in the Polyellomer tubes uzed. After 6.6 hr at 38,000 rev./min in s Spinco SW39 rotor at 4’C, fractions (about 0.16 ml.) were collected from the bottom of the tube onto 8 pad of Trie buffer (0.1 ml., M, pH 7.4). The absorbance 8t 260 rnp (-a-. . .- a---) and the transforming activity (using 6 4. of each fraction) were measured for all f&&ions for the Sr (-- A --A --) and the Er (- 0 - 0 -) ma.rkers.
conditions used, the sedimentation rate increases 1.32 times for a doubling in singlestranded molecular weight (Studier, 1965). This would mean that the molecular weight of the residually active molecules is about twice that of denatured DNA, strongly suggesting that the residual activity is due to a naturally occurring crosslinked fraction, rather than to an intrinsic activity of the separated single strand. Notice that, as a control, the transforming activity of artificially cross-linked Er DNA sediments at about the same rate as the naturally occurring residual activity. In neutral sucrose gradients the transforming activities of the denatured Sr and Er DNA samples were found to band together in the place expected for native DNA. The position of the bulk of the DNA could not be determined in these gradients, as the amount of native DNA required for absorbance readings would have interfered with the sediment&ion. No radioisotopes were used in this study (except for the densityhybrid experiments, see below) in order to avoid any possible oross-linking of DNA by emitted B-rays. (c) i3ep?mroee column Jiltration Sepharose, a beaded form from agarose for gel titration, fractionates in the range of 3 x lo6 to 3 x lo6 (type 4B) and 2 up to 20 to 30 x IO8 daltons (type 2B) according to
NATURALLY
CROSS-LINKED
H.
influenrae
429
DNA
the manufacturer. Therefore, denatured DNA was filtered through agarose oolumns of either type and the elution pattern of denatured DNA, obtained by absorbance measurements, was compared with that of its residual activity, with DNA containing artificially induced cross-links and with native DNA, all measured by transformation of suitably diluted samples. The DNA was sheared to a more uniform molecular weight to avoid the expected broad elution pattern of molecules of heterogeneous sizes 8s found in a normal DNA preparation. Cross-linked DNA molecules should move more rapidly through a Sepharose column than denatured DNA because of their higher molecular weight and bihelical structure. Figure 3 shows such an elution pattern from Sepharose 2B; as can be seen,
7
17
25
33 Fraction
41
49
57
no.
FIG. 3. Filtration of native, denatured and arti&ally cross-linked DNA through an egaroee oohmn. A mixture (1.6 ml.) of Sr DNA (46 pg) and Er DNA (10 H, oroae-linked with nitrous acid for 21%) was sheared to a mol. wt of 6.5 x 10” d&one ae described in Fig. 2, and wee denatured by heating at 100% for 2 min. After admixture with native Cr DNA (1 ccp;, 6epelMely she-d to the same extent), the sample ws8 applied to a Sepharose 2B column (86 om X 1.6 cm) and eluted with buffer S. Fractions (2.2 ml.) were aaeayed for absorbance at 260 rnp (-0 -. . . -0 -) and the transforming motivity for the Sr (. . A. . . A. . ), Er (- O0 -) and Cr (-- A --A --) markers.
the residual activity is eluted in a sha,rp peak, identical to that of artificially crosslinked Er DNA and very similar to that of added native ti DNA. The denatured molecules are eluted in a broad band: its peak is eight fmctions (18 ml.) removed from the residual activity. The broad elution pattern is probably intrinsic to denetured DNA which is expected to have some tendency to stick to the polysacoharides of the gel at the low ionio strength of the eluent. A molecular weight heterogeneity of the single strands contributes probably only little to the broadness of the peak, since the low molecular weight and the mild den&m&ion conditions (two minutes at lOO”C, pH 9.2) will have prevented an appreciable chain soission. The small difference in elution pattern between native DNA s,nd the residual a&ivities of the denatured
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and artificially cross-linked DNA’s is probably caused by a small difference in molecular weight : the native DNA was separately sheared at a lower DNA concentration and may, therefore, be slightly smaller in average size. On a similar column of Sepharose4B, the peak of the residual activity and of the absorbance differed only one fraction. This is not unexpected, as even the singlestrand molecular weight (about 2.8 x 106)is closeto the excluded size (3 x 106). These results confirm that the molecules bearing the residual activity have a substantially higher molecular weight than denatured molecules; in fact they are eluted at the position where the original native moleculeswould have been eluted. This again suggeststhat the residual activity is due to a naturally occurring cross-linked fraction. A native-like configuration is also suggestedby the sharpnessof the elution pattern of the residual activity, in contrast to the broad pattern of denatured DNA. (d) Cesiurnchloride density-gradients In view of the evidence from alkaline sedimentation and Sepharosefiltration that the residually active molecules are not identical to denatured DNA, the buoyant density of these moleculesin CsClwas re-examined to test the previous claim (Rownd et al., 1961) that the buoyant density of these molecules is that of denatured DNA. As a control, the residual activity of the denatured normal molecules was again compared with that of an admixture of differently genetically marked, artificially crosslinked molecules. Figure 4 shows the results of such a preparative CsCl gradient.
0
0 30
35 Fraction
FIG.
4. C&l
density-gradient
of native,
denatured
40
45
no.
and cross-linked
H. injkmwae
DNA.
A mixture of Er DNA (1.6 pg, cross-linked by nitrous acid to the extent of 21%) and Cr DNA (8.4 pg) in 1 ml. KpHPOd (0.02 I, pH 9.2) was denatured by hating et 1OO’C for 3 min followed by rapid cooling in a 0% bath. Native Sr DNA (0.2 pg in 1 ml. pH 9.2 buffer) and CsCl (2.867 g) were added [TV = 1*4003]. Equilibrium was established by centrifugation for 3 days in an SW39 rotor in a Spinco model L at 38,000 rev./min at 19°C. Fractions (about (O-046 ml.) were collected in 0.2 ml. pH 9.2 buffer from the bottom of the tube, and assayed for the absorbance at 260 rnp .- l -) and the transforming activity for the Sr (-- A --A --), Er (- 0 - 0 -) (-•-.. and Cr (--A--A--) markers.
NATURALLY
CROSS-LINKED
H.
influenzoe
431
DNA
Clearly, under these conditions the naturally occurring residual activity (Cr marker) separates well from the bulk of the denatured DNA, as represented by its absorbance profile. Its density is in fact almost indentical to that of the residual activity of the nitrous acid cross-linked (Er) DNA, which is in turn about 0*003 g/cm3 higher than that of added native (Sr) DNA. Since this result is in conflict with previous variations observations in this laboratory (Rownd et al., 1961), experimental were tried in an attempt to reproduce the earlier result. Varying the melting conditions from three minutes at pH 9.2 to those of Rownd et al. (10 minutes at pH 7) did not change the result substantially, although the density at which the residual activity bands is now about 0.004 to 0$@5 g/cm3 heavier than that of native DNA, probably as a result of a larger number of strand scissionsoccurring in the residually active molecules. However, at the higher concentration of DNA previously used (71.5 pg instead of 6.5 pg/3 ml.), a different result was sometimesobtained. The residual activity was now found both in the previous position (slightly heavier than native DNA) and in the position of true denatured DNA. It can be shown, however, that this result is an artifact caused by the high concentration of denatured DNA in the gradient; when the peak fraction in this denatured DNA band was recentrifuged in a density gradient, the residual activity it contained now bands closeto added native DNA, well separated from its previous denatured DNA position. It cannot adequately be explained why molecules with an apparent native-like density sometimesband with the denatured molecules,nor why this apparent aggregation is not always observed under seemingly identical conditions : in four other experimer1t.susing high concentrations of DNA (60 and 70 pg), t’he residual activity was found in one band near the native density only. 1
TABLE
tSpeci$ctransforming activity of purified moleculescarrying
DNA
A native B denatured in CsCl C purified
Concentration during transformation (in mpg/ml.) 4.0 4.0 1.9
No.
residual activity
of transformed colonies (X 10-4)
Per cent of A
17.7 1.4 7.9
100 8 94
Cr DNA (30 pg) was denatured by incubation at 40% (pH 11.7) for 10 min. After cooling to 0% and adjusting the pH to 9.2, the DNA was diluted to 2.1 ml. and mixed with &Cl (2.87 g). Equilibrium eentrifugation and fractionation were performed as described in Fig. 4. The peak fraction of the residual activity was used as purified “residually active molecules”; its AZsO value was 0.076.
The specific activity of residually active molecules purified in CsCl gradients after short heating times was found to be about 909/othat of native (Table l), indicating that if all damage to the melted strands could be avoided, the activity of the naturally cross-linked DNA could well be identical to that of native DNA. When this purified fraction is subjected to a renewed thermal denaturation, the residual activity decreases only gradually and fits completely the extrapolated secondpart of the curves in Fig. I. This indicates that this gradual decreaseis causedby thermal degradation (depurinization and chain breakage) not by strand separation. 28
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(e) Biological hybrid molecules Perhaps the most conclusive means to establish the nature and origin of the residually active molecules is the investigation of the behavior in a CsCl gradient of denatured DNA, derived from density-hybrid DNA isolated from bacteria grown for many generations in heavy-isotope medium followed by one generation in light medium. Were the transforming activity due to single strands of DNA, it would form separate bands at the denatured heavy and denatured light positions; if due to renaturation between separated strands, three peaks would be expected (native heavy, native hybrid and native light) ; and if caused by a cross-linked fraction of the A26O
42
45
A26O
50
55 Fraction
FIU.
6. C&l
density-gradient
60
,
-24
1 65
A0
no.
of denatured
biological
hybrid
DNA.
Biological hybrid (1sNaH3aP/14N1H31P) Sr DNA (p = l-705) (6.7 pg) in 2 ml. pH 9.2 but& was denatured by heating at 1OO’C for 3 min followed by rapid cooling in ice water. Native (light) Cr DNA (0.2 pg) in 0.8 ml. pH 9.2 buffer and CsCl (3.86 g) were added [vn = 1*4009]. Further details as in Fig. 4. The fractions were assayed for radioactivity (- q - 0 -), transforming activity of markers and absorbance at 260 rnp; the the Sr (--A--r--) and Cr (--A-----A--) absorbance curve is omitted, only the two peak fractions being indicated by an arrow.
DNA, the activity should be found solely at native hybrid density. Figure 5 shows the result of such a study : the transforming activity of denatured hybrid DNA bands in a single peak closeto the native hybrid position. Although this position is also close to the density of light denatured DNA, no biological activity was found near the band of the 3aP-labeled denatured heavy molecules. Therefore, it can be concluded that the active speciesmust arise from a true cross-linked fraction. 4. Discussion The evidence presented in this paper attributes the residual activity of denatured DNA of H. injluenxae to a reversibly denaturable fraction in this DNA, similar to the “naturally occurring” cross-linked fraction previously described in B. subtilis DNA by Rownd (1963) and characterized by Alberts & Doty (1968). No differences have been
NATURALLY
CROSS-LINKED
H.
influenzae
DNA
I?? *.
detected between artificially (nitrous acid) cross-linked DNA and the residually active molecules. The increase of the sedimentation rate in alkaline sucrosegradients by a factor of 1.3 to 1.4 over the single-strands (Fig. 2) indicates that the mean molecular weight of the residually active moleculesis about twice that of the denatured molecules(Studier, 1966), a value to be expected for cross-linked molecules when the two strandsthough melted-are held together by the cross-linkage. Similarly, the elution pattern from a Sepharose2B column (Fig. 3) showsthat the moleculeswith residual activity have nearly the samemolecular weight and configuration as the original native molecules. This, again, is expected for reversibly denaturable (cross-linked) molecules where the two strands have zippered up after cooling. The buoyant density in CsClpoints to a native-like, bihelical structure. The slight shift towards heavier density is probably caused by some denatured regions in the molecules, most likely as a result of strand scissionof one of the strands in the melted state. Barnbardt (1965) stated that the residual activity of heat-denatured H. injhenme DNA had clearly a greater buoyant density than native DNA. However, the buoyant density of his residually active moleculesappears not to be substantially different from the data presented in this paper, after recalculating his data by correcting for the inhibiting effect of the large excess of native DNA present in his gradient. The conflicting data of Rownd et al. (1961), who found residual activity at, a density identical to denatured DNA, is most probably explained by aggregation at, the high concentration of DNA used. Barnhardt (1965), in a study of the residual activity of heat-denatured Ei. ijljhenzae DNA, claimed that this activity is clearly different from that of native DNA. However, his arguments against a native structure are all consistent with a crosslinked native structure, with the exception of his report that the residually active molecules are less sensitive to ultraviolet light than native molecules. This claim, however, could not be confirmed (Colarusso& Mulder, unpublished data). The explanation that the residual activity originates from naturally occurring crosslinked molecules is in agreement with the data of Chevallier & Bernardi (1965), who showed that the residual activity is eluted from a hydroxyapatite column well after the bulk of the denatured DNA and at a salt concentration that normally elutes native molecules. The evidence given in the present paper indicates that denatured molecules cannot transform, probably becausecompetent cells are unable to absorb irreversibly these single strands. Recently, however, Postel & Goodgal (1966) showedthat in exceptional circumstances (exposure of the cells to a low pH in the presenceof EDTA) competent cells of H. ir@uenzuecan absorb and be transformed by denatured DNA. In B. subtilis only fully native moleculeshave substantial activity in transformation and partially denatured moleculeshave no activity (Alberts & Doty, 1968) or somehut very little activity (Rownd et al., 1968), whereasin H. injluenzae these moleculeswit’h bihelical configuration and an unmatched, denatured, extension of one of t,he strands at either or both endsappear to be active too. Shen & Mulder (unpublished data) have found that the residual activity resides in both phasesof the polyethylene glycoldextran phase partition system of Albertsson (1962) as modified by Alberts (1967), whereas the residual activity of B. subtilis DNA is concentrated in the top phase, nearly completely separated from the denatured and partially helical DNA in the bottom phase. In this partition system only perfectly native molecules move into
434
C. MULDER
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the top phase. Another indication that partially denatured molecules may have activity is that the average buoyant density of residually active molecules is 0.003 to O-005 g/cm3 heavier than perfectly native DNA. It is of interest to note that the residual activity of denatured DNA of D. pneumoniae also has an average buoyant density about halfway between those of native and denatured DNA (Mulder & Fox, results to be published), whereas the residual activity of B. subtilis was found by Alberta & Doty (1968) to be exclusively at the density of native DNA. So far the origin and the chemical structure of the naturally occurring cross-links remain unsolved. In an extensive study of the naturally occurring cross-linked fraction in B. subtilis DNA, Alberts (1968) failed to find any conclusive evidence that these cross-links arise in vivo. He observed that nitrous acid-induced cross-linked DNA invariably sediments somewhat faster than the naturally occurring cross-linked molecules, and he concluded that this could be an indication that the naturally occurring oross-links are terminal. From this and other evidence it was suggested that the cross-links are induced by shear at the Grst few breaking points of a whole genome upon isolation of the DNA. These observations in alkaline sucrose gradients could not be confirmed for H. inJluenzue DNA (Fig. 2), although such an effect could possibly have been masked in the latter case by the probable activity of partially helical DNA molecules in the H. in&ensue transformation system; also the approximately tenfold higher resistance of H. in$uenzue DNA to cross-linking by nitrous acid means that the treatment inflicts more damage on H. injluenzae DNA than on B. subtilis DNA, possibly causing single-strand breaks which would selectively reduce the sedimentation rate in alkali of the cross-linked species by nitrous acid. The residual activity of a DNA preparation with an average molecular weight of 21 x lo6 daltons was found to be 8%, extrapolated to zero-time melting (Fig. 1). Since the specific activity of the cross-linked molecules responsible for the activity is almost normal, this would mean that one cross-linkage occurs for about 260 x 10e daltons of DNA. This is about three per genome, assuming the genome of H. inJluenzae to be on the order of 8 x 10s daltons (Berns & Thomas, 1965) ; since an equal activity was found for all markers tested, these cross-links must be randomly distributed on the genome. It is not excluded, however, that the cross-links, if they pre-exist in viva, ase unequally distributed over the bacteria: a fraction of the organisms may have a large number of cross-links in their genome, whereas the majority of the organisms are free of cross-links. In conclusion, the present data support the conception that DNA preparations of H. injuenzae contain a naturally cross-linked fraction of unknown origin and nature, distributed apparently at random at an average frequency of about one cross-linkage per 260 x lo6 daltons of DNA. This fraction is solely responsible for the residual activity of denatured DNA. Single-stranded DNA displays no detectible transforming activity for H. in$uenzae under normal conditions, although it is active when incubated with competent cells at pH 4 (Postel & Goodgal, 1966). Partially denatured molecules which are inactive with B. subtilis apparently have some activity with H. inJluenzae. We are indebted to Drs sions, and to Mi-s Wei Liu completion of the present
B. M. Alberta, Shen and Mr
manuscript,
M. S. Fox, L. Colarusso we learned
and R. Sternglanz for helpful discusfor expert technical assistance. After from Dr G. Bernardi that Dr M.-R.
NATURALLY
CROSS-LINKED
H. inffluenzue
DNA
435
Chevallier and he had continued their previous work snd had reached via different ways essentially the same conclusions as expressed in this paper. Their account is also published in this issue, Chevallier & Bernardi (1968). The work was supported by U.S. National Institutes of Health grant HD 01229. REFERENCES Alberta, B. M. (1966). Ph.D. thesis, Harvard University. Alberta, B. M. (1967). Biochemktry, 6, 2627. Alberts, B. M. (1968). J. Mol. Biol. 32, 465. Alberts, B. M. & Doty, P. (1968). J. Mol. Biol. 32, 379. Albertason, P. A. (1962). Arch. Biochem. Biophy&, Suppl. 1, 264. Alexander, H. E. BE Leidy, G. (1953). J. Exp. Med. 97, 17. Bamhardt, B. J. (1966). J. Bact. 89, 1271. Barnhardt, B. J. & Herriott, R. M. (1963). Biochim. whys. Actu, 76, 25. Becker, E. F., Zimmerman, B. K. & Geiduschek, E. P. (1964). J. Mol. BioZ. 8, 377. Berns, K. I. & Thomss, C. A., Jr. (1965). J. Mol. BioZ. 11, 476. Bray, G. A. (1960). Andyt. Biochem. 1, 279. Chevallier, M.-R. & Bernardi, G. (1965). J. Mol. BioZ. 11, 668. Chevallier, M.-R. & Bernardi, G. (1968). J. Mol. B&Z. 32, 437. Eigner, J., Boedtker, H. & Michaels, G. (1961). Biochim. biophys. Acta, 51, 165. Ginoza, W. & Zimm, H. (1961). Proc. Nat. Acad. Sci., Wash. 47, 639. Goodgal, S. H. BE Herriott, R. M. (1961). J. Qen. Physiol. 44, 1201. Greer, S. L Zamenhof, S. (1962). J. Mol. Biol. 4, 123. Guild, W. R. (1961). Proc. Nat. Acud. Sci., Wash. 47, 1660. Guild, W. R. t Robison, M. (1963). Proc. Nat. Acud. Sci., Wmh. 50, 106. Kohn, K., Spears, C. & Doty, P. (1966). J. Mol. BioZ. 19, 266. Mulder, C. (1967). Fed. Proc. 26, 396. Postel, E. H. & Goodgal, S. H. (1966). J. Mol. BioZ. 16, 317. Roger, M. & Hotchkiss, R. D. (1961). Proc. Nat. Acad. Sci., Wash. 47, 6El. Rownd, R. (1963). Ph.D. thesis, Harvard University. Rownd, R., Green, D. M.. Sternglanz, R. I% Doty, P. (1968). J. Mol. BioZ. 32, 369. Rownd, R., Lanyi, J. & Doty, P. (1961). B&him. biophys. Actu, 53, 225. Spencer, H. T. t Herriott, R. M. (1965). J. Bad. 90, 911. Studier, F. W. (1966). J. Mol. BioZ. 11, 373.
Residual Activity of Denatured Transforming DNA of Haemophilus inzuenzae: A Naturally Occurring Cross-linked DNA CAREL
MULDER~
AND PAUL DOTY
Department of Chemistry, Harvard University Cambridge,Massachusetts, U.S.A. (Received13 June 1967) The residual activity of denatured transforming DNA of Haemophilusinfluenzae is accounted for by the presence of cross-linked molecules in all DNA preparations, whereas single-stranded DNA has no detectible transforming activity. The specific transforming activity of the cross-linked molecules is at least 90% that of native DNA. This conclusion is reached from the observation that the residual activity sediments l-3 to 1.4 times faster than the bulk of the denatured DNA through an alkaline sucrose gradient, almost identical to the rate of an artifloially cross-linked DNA. Similarly, the residual aotivity is eluted from a Sepharose column 1.5 times faster than the bulk of the denatured DNA and ahnost identical to the elution pattern of added native DNA of the same homogeneous size. Moreover, the buoyant density in CsCl of the molecules carrying the residual activity was found to be close to that of native DNA and considerably lighter than that of denatured DNA; with denatured biological hybrid (15NaH/14N1H) DNA, this fraction was found only at a buoyant density slightly heavier than native hybrid DNA; no activity was found near the densities of native or denatured heavy DNA or native light DNA. The intrinsic residual activity is equal for all melting conditions tried and for nine different markers studied. It was found to be 1 1*5o/o for a DNA preparation with an average molecular weight of 30 x lo6 daltons, and it is linearly related to the molecular weight of the DNA. Therefore, it is estimated that about three cross-links are present per bacterial genome if they pre-exist in tivo.
1. Introduction Transforming DNA losesmost of its activity after denaturation by heat, alkali or a variety of other conditions. The residual activity, which is usually a few per cent of the original activity, has three possibleorigins (Rownd, Lanyi & Doty, 1961): (1) a small fraction of the molecules is resistant to strand separation; (2) a small degree of renaturation inevitably occurs on removal of the melting conditions; and (3) singlestranded DNA can be taken up by recipient cells to a small extent and initiate transformation. The second possibility was ruled out by Ginoza & Zimm (1961) and by Rownd (1963), who showed that the level of residual activity is independent of the DNA concentration and of the ionic environment at denaturation. The residual activity of Bacillus subtilis DNA was found to originate from molecules with native buoyant density in CsCl (Rownd, 1963; Rownd, Green, Sternglans $ f Present
St. Louis,
address: Institute for Missouri 63110, U.S.A.
Molecular
Virology, 423
St.
Louis
University
Medical
School,
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C. MULDER
AND
P. DOTY
Doty, 1968), thus indicating a fraction resistant to denaturati0n.i Recently, it was shown (Alberts, 1965; Alberts & Doty, 1968) that these molecules with surviving activity are a “naturally occurring” cross-linked fraction in B. subtilis DNA. A similarly cross-linked fraction was shown to exist in DNA preparations from other bacterial, viral and mammalian sources. On the other hand, the third possibility, a small intrinsic activity of denatured DNA, was preferred for Diplowccus pneumoniue by Guild (1961) and by Guild & Robison (1963) from studies of the buoyant density of residually active DNA in CsCland by Ginoza & Zimm (1961) from experiments on the kinetics of heat inactivation of DNA. These results have recently been challenged, however (Mulder & Fox, manuscript in preparation). The residual activity of thermally denatured DNA of Haemophilw injluenzae was similarly found in one report at a buoyant density of denatured DNA (Rownd et al. 1961). However, later findings indicated that the activity has properties unlike denatured DNA: its resistanceto degradation by exonucleaseI (Barnhardt $ Herriott, 1963) and the similarity of its elution pattern from hydroxyapatite columns to that of native DNA (Chevallier & Bernardi, 1965) suggesteda bihelical configuration. In an intensive study, Barnhardt (1965) suggestedintrastrand hydrogen bonding of separated single strands as configuration of the residually active molecules. Recently, Postel6 Goodgal (1966) showed that under their experimental conditions the residual activity of denatured DNA had a buoyant density in between that of native and of denatured DNA. In view of these conflicting observations, it was of interest to examine the nature of the residual activity of H. in&ensue DNA in more detail. In this study it has been shown that the transforming activity of denatured H. injthenzae DNA is due to a small fraction of naturally cross-linked molecules which zipper up upon cooling. When isolated, these molecules show almost the samespecific activity as native DNA and a bihelical conflguration; by contrast, single-stranded DNA separated from these crosslinked moleculesdoes not show any biological activity. Some of the results presented here were briefly referred to in a recent abstract (Mulder, 1967).
2. Materials and Methods (a) Strains anu!DNA H. influenzae Rd obtained from Dr S. H. Goodgal (originally from Alexander BELeidy (1963)) was usedthroughout. It was brought into competency, and transformation assays carried out according to standard techniques (Goodgal & Herriott, 1961; Spencer L Herriott, 1906).All transformation assayswere done in the linear nx~ponse range of DNA, 6 mm/ml. or below. Strains bearing erythromycin (Er = Ee6) resistance or the linked streptomycin (Sr = 52000) and cathomycin (novobiocin) (Cr = Caas) resistancemarkerswere obtained by transforming Rd cells with Sr, Cr or Erf DNA provided by Dr J. Lanyi. Temperatnresensitive mutanta of strain Rd were induced with nitrosognanidine as described below. For isolation of DNA, the organisms were grown in Difco brain-heart infusion broth t The terminology of Kohn, Spears & Doty (1966) is used to describe the various con&gurations of DNA and the transformations among them : however, the widely used term “reversibly denaturable DNA” is retsined to describe cross-linked DNA, although “reversibly meltable” would be more consistent with this terminology. $ Abbreviations used: Sr, streptomycin-reaice ; Cr, cethomycin-resistance ; Er, erythromycinresistance; SSC, 0.16 ar-N&l-O-O16 ar-trieodiom citrate (pH 7-O); pH 9.2 buffer, O-02 x-KIHPOI (pH 9.2).
NATURALLY
CROSS-LINKED
H. influenzoe
425
DNA
supplemented with hemin (Eastman-Kodak, 10 &ml.) and NAD chemicals Corp., 2.6 pg/ml.) (medium HD) until late logarithmic phase. resistant strains, the medium was supplemented with the antibiotic. sensitive strains were grown at 3O”C, the permissive temperature. DNA was isolated as described by Alberts & Doty (1968), except that of organisms was about 1.6 x lOlo during the lysis of the cells.
(Nutritional BioFor the antibioticThe temperaturethe concentration
(b) Isolation, melting, CEenaturation and shear-induced brcukuge of DNA The experimental methods involved in the isolation of DNA by phenol have been described in the preceding paper (Alberta & Doty, 1908). The sedimentation rates @‘!&,) of these DNA preparations varied from 31 to 36.8 s, which corresponds to an average molecular weight of 20.4 to 31 x lo8 daltons. When a preparation with a value of fc0.w of 34 s (mol. wt of 25*6 x 10e) was sedimented in alkali according to Studier (1905), the melted DNA had a value of S&,, = 38 s, which corresponds to a mol. wt of 13.8 X 10’ ; therefore, this preparation was free of single-strand breaks. DNA samples were stored at 3°C in SSC (0.15 M-NaCl-0.016 ma-t&odium citrate, pH 7.0) or in pH 9.2 buffer (0.02 MKzHP04, pH 9.2). The melting and subsequent denaturation of DNA and its shear-induced breakage were performed as described by Alberta t Doty (1968). (c) cioss-h&king of DNA with nitrow acid DNA was cross-linked with nitrous acid by the method of Becker, Zimmerman & Geiduschek (1964) as described by Alberta & Doty (1968). However, the pH used was lowered to pH 4.6, as it was found that the extent of cross-linking of H. irzfluenzae DNA at pH 6.0 was only 10% of that found for B. mbtilk DNA. (d) C&l and sucrose gradient sedinzeaWim The experimental techniques used for equilibrium banding in analytical and preparative CsCl gradients and those for sedimentation through neutral and alkaline sucrose gradients are described by Albert8 & Doty (1968). (e) Biological hybrid DNA Density-hybrid DNA was isolated from Sr-cells grown for many generations in the medium described by Spencer & Herriott (1966) made up in aHaO (Bio-Rad, 96%), the NH&I being replaced by 16NH&1 (40 m&f) (Bio-Rad, 99.7%). The phosphate buffer was replaced by 3aP-labeled potassium phosphate (10 mM, pH 7) (Cambridge Nuclear Company) with a specific activity of 0.2 me/m-mole. After washing, the organisms were transferred to medium HD without radioactive label and collected after one generation of growth. The DNA extracted from these organisms was banded in a preparative C&l gradient, and the DNA in the peak fractions of the hybrid density band used for the experiments. The density of this DNA was 1.705 g/cm3 calibrated against dAT = 1.678 g/cm3. After denaturation, this DNA yielded two equal bands with buoyant densities of 1.714 and l-727 g/cm3, respectively. The density of [‘Hl’N]DNA of H. injZunzae wss found to be I.698 g/cm3 for native and 1.714 g/cm3 for denatured DNA. (f) Filtration through Sepharos Sepharose 2B (lot 3616) and 4B (lot 3270), and a Sephadex laboratory column (type K 16/90) were a gift from Pharmacia Fine Chemicals, Inc. Columns (1.6 cm x 86 cm) were washed with 600 ml. buffer S (20 mM-K,HP04-4 mar-EDTA, pH 8.8) before applying the sample. Samplee (60 to 100 pg denatured DNA in 1 to 2 ml. buffer S) were eluted with buffer S at a rate of 6 ml&r. The operating water pressure during the whole procedure was 60 cm measured from the outlet of the effluent tubing to the buffer reservoir. The DNA samples were sheared to a more uniform size before heat-denaturation. (g) Radtiotope determination Fractions from C&l gradients were counted for 3zP in Bray’s (1960) Cab-O&l in a Packard Tricarb liquid-scintillation spectrometer (model 314EX).
solution
426
C. MULDER
AND
P. DOTY
(h) Nitrosoguunidine-induced mutation Rd cells in early logarithmic growth in HD medium were incubated with N-methylN’-nitro-N-nitrosoguanidine (50 pg/ml.) in medium HD at 37°C for 10 min. After dilution, the organisms were incubated on HD-agar at 30°C. After replication, 15 colonies were found to be unable to grow at 41°C.
3. Results (a) Residual transforming activity The residual transforming activity of H. in$henzae DNA surviving exposure to melting (denaturing) conditions for various lengths of time followed by rapid quenching showed a course very similar to that of B. subtilis DNA (Alberta & Doty, 1968). The curves in Fig. 1 show a sharp break point between zero and two minutes. The activity decreasesrapidly during the first minute or two and then much more slowly. The initial rapid loss of activity is thought to be caused by the collapse of the helical structure and the consequent strand separation of most molecules. The slope of this Time
at 4O’C
(min)
15
30
'.._.( ._ l
.E
‘..,
.g
2E
0 ‘k,
ly-
‘.__ ‘.._ “k,. l
04 0
, 2
I 4 Time
1 6 at 100°C
I 6
‘.... ‘.., .‘....,
"..7.0 _ a
I IO
(min)
of heat denaturation on the transforming eotivity of H. injluenzae DNA. 7 ml.) of Sr DNA (0.1 pg/ml.; mol. wt 21 x IO6 daltons) in potassium phosphate (0.02 M, pH 9.2 (-O--O--) or pH 7.0 (. . . 0 . . .O . . .)) in thin-walled tubes were heated in a Fro.
Samples
1. Effeot (0.2
boiling water bath. samples in potassium 15,30 and 45 min, a of these samples are by comparison with taneously.
After 2, 4, 6, 8 and 10 phosphate (0.1 M, pH tube was transferred to plotted as percentages l/l00 and l/10 dilutions
min, 11.7 a 0% of the of
a tube was transferred to a 0°C bath. Identical (- A -. . .- A -)) were heated at 40°C. After bath and neutralized. The tmnsforming activities activity of the unheated sample, as determined the original native DNA sample plated aimul-
NATURALLY
CROSS-LINKED
H. iltfluenzae DNA
427
initial decrease is of the same order of magnitude for all methods of melting and denaturation used. The slope of the second part of the curve, after two minutes, is strongly dependent on the melting conditions. This further decrease of activity is probably due to depurinization and single-chain scission(Eigner, Boedtker BEMichaels, 1961; Greer & Zamenhof, 1962). Heating at 100°C in SSC at pH 7, a widely used method of melting DNA, causesa relatively rapid lossof activity in this secondregion, considerably more than at 100°C at pH 9-2, for example. A very mild procedure for melting DNA, heating at 40°C at pH 11.7, produces hardly any further inactivation (Fig. 1). Back extrapolation of this secondpart of the curve gives the intrinsic residual activity : this is found to be independent of the melting conditions used, being 11.5% for a DNA preparation of 30 x 106 daltons. The size of this fraction is found to be directly proportional to the molecular weight ; the samepreparation when reduced by shear to 5 x lo6 daltons displayed only 2% residual activity. The intermediate molecular weight used in Fig. 1 showed 8% intrinsic residual activity. All genetic markers described for H. injluenme are for antibiotic resistance and could therefore be located on a specific region of the genome. In order to examine whether the residual activity was of the sameorder of magnitude for different regions of the genome, some temperature-sensitive mutants of H. injluenme were isolated and the residual activity of denatured DNA carrying these markers was examined. Temperature-sensitive markers are expected to be randomly distributed over the genome; accordingly, no genetic linkage was found between any of these markers or between these and the antibiotic resistance markers. Figure 1 showsonly data for the streptomycin-resistance (Saooo)marker, but identical zero-time extrapolations were obtained for the residual activity of eight other markers investigated for this sample (resistance to two antibiotics (Ws and E6’6) and six temperature-sensitive markers (ts 2, 4, 5, 7, 12 and 14)). Very similar inactivation curves were obtained when DNA artificially cross-linked by nitrous acid was subjected to the samemelting conditions, except that, due to the cross-linkings, the size of the residually active fraction was considerably larger (17 to 91%). Studies to determine the critical temperatures for inactivation of the three antibiotic markers and five temperature-sensitive markers gave identical results, namely 865°C in standard saline-citrate solution (SK!). This contrasts with similar examination of D. pneumoniae DNA, where differences up to 4°C in the critical inactivation temperature have been observed (Roger & Hotchkiss, 1961). (b) Alkaline m.mosegradients Since the sedimentation rate of melted DNA in alkali dependsin a known way on its molecular weight (Studier, 1965), the sedimentation rate of the residual activity through an alkaline sucrosegradient was examined and compared with that of DNA with artificially induced cross-links. The DNA was sheared to a more uniform molecular weight to avoid the broad sedimentation pattern of molecules of heterogeneous sizes as found in a normal DNA preparation. Cross-linked molecules should move quickly in such sedimentations, since their molecular weights should be double that of the separated single strands. The sedimentation pattern of the bulk-denaturedDNA from the gradient was obtained by absorbance measurement, that of the residual activity by transformation of suitably diluted samples. As can be seenfrom Fig. 2, this activity sediments 1.3 to l-4 times faster than does denatured DNA. Under the
428
C. MULDER
AND
P. DOTY
I
I
Fraction
no.
I
---r--ri,6
FIG. 2. Sediment&ion of the residual activity and of arti&&lly cross-linked DNA through an &al& zucroze gradient. A mixture of Er DNA (4 pg/ml.), crass- linked with nitrous acid to 21% (as measured by ito residual tranzforming aotivity after den&x&ion) and untreated Sr DNA (60 a/ml.) wa8 sheered to a more uniform size in aVirtie homogenizer ret 7700 rev./min for 30 min at 0% (avemge mol. wt 6.6 x lo* d&onz). To 8 nnmple (O-18 ml.) of this sheared DNA mixture, N&l (12 mg) and NaOH (0.02 ml., M) were added at 4%. This melted sample wae layered on top of & discontinuous sucrose gradient formed from 0.86~ml. layers of zucroze solutions (20, 16, 12, 8 end 4% sucrose in N&l (0.9 M), N&OH (0.1 M)), cooled in the Polyellomer tubes uzed. After 6.6 hr at 38,000 rev./min in s Spinco SW39 rotor at 4’C, fractions (about 0.16 ml.) were collected from the bottom of the tube onto 8 pad of Trie buffer (0.1 ml., M, pH 7.4). The absorbance 8t 260 rnp (-a-. . .- a---) and the transforming activity (using 6 4. of each fraction) were measured for all f&&ions for the Sr (-- A --A --) and the Er (- 0 - 0 -) ma.rkers.
conditions used, the sedimentation rate increases 1.32 times for a doubling in singlestranded molecular weight (Studier, 1965). This would mean that the molecular weight of the residually active molecules is about twice that of denatured DNA, strongly suggesting that the residual activity is due to a naturally occurring crosslinked fraction, rather than to an intrinsic activity of the separated single strand. Notice that, as a control, the transforming activity of artificially cross-linked Er DNA sediments at about the same rate as the naturally occurring residual activity. In neutral sucrose gradients the transforming activities of the denatured Sr and Er DNA samples were found to band together in the place expected for native DNA. The position of the bulk of the DNA could not be determined in these gradients, as the amount of native DNA required for absorbance readings would have interfered with the sediment&ion. No radioisotopes were used in this study (except for the densityhybrid experiments, see below) in order to avoid any possible oross-linking of DNA by emitted B-rays. (c) i3ep?mroee column Jiltration Sepharose, a beaded form from agarose for gel titration, fractionates in the range of 3 x lo6 to 3 x lo6 (type 4B) and 2 up to 20 to 30 x IO8 daltons (type 2B) according to
NATURALLY
CROSS-LINKED
H.
influenrae
429
DNA
the manufacturer. Therefore, denatured DNA was filtered through agarose oolumns of either type and the elution pattern of denatured DNA, obtained by absorbance measurements, was compared with that of its residual activity, with DNA containing artificially induced cross-links and with native DNA, all measured by transformation of suitably diluted samples. The DNA was sheared to a more uniform molecular weight to avoid the expected broad elution pattern of molecules of heterogeneous sizes 8s found in a normal DNA preparation. Cross-linked DNA molecules should move more rapidly through a Sepharose column than denatured DNA because of their higher molecular weight and bihelical structure. Figure 3 shows such an elution pattern from Sepharose 2B; as can be seen,
7
17
25
33 Fraction
41
49
57
no.
FIG. 3. Filtration of native, denatured and arti&ally cross-linked DNA through an egaroee oohmn. A mixture (1.6 ml.) of Sr DNA (46 pg) and Er DNA (10 H, oroae-linked with nitrous acid for 21%) was sheared to a mol. wt of 6.5 x 10” d&one ae described in Fig. 2, and wee denatured by heating at 100% for 2 min. After admixture with native Cr DNA (1 ccp;, 6epelMely she-d to the same extent), the sample ws8 applied to a Sepharose 2B column (86 om X 1.6 cm) and eluted with buffer S. Fractions (2.2 ml.) were aaeayed for absorbance at 260 rnp (-0 -. . . -0 -) and the transforming motivity for the Sr (. . A. . . A. . ), Er (- O0 -) and Cr (-- A --A --) markers.
the residual activity is eluted in a sha,rp peak, identical to that of artificially crosslinked Er DNA and very similar to that of added native ti DNA. The denatured molecules are eluted in a broad band: its peak is eight fmctions (18 ml.) removed from the residual activity. The broad elution pattern is probably intrinsic to denetured DNA which is expected to have some tendency to stick to the polysacoharides of the gel at the low ionio strength of the eluent. A molecular weight heterogeneity of the single strands contributes probably only little to the broadness of the peak, since the low molecular weight and the mild den&m&ion conditions (two minutes at lOO”C, pH 9.2) will have prevented an appreciable chain soission. The small difference in elution pattern between native DNA s,nd the residual a&ivities of the denatured
430
C. MULDER
AND
P. DOTY
and artificially cross-linked DNA’s is probably caused by a small difference in molecular weight : the native DNA was separately sheared at a lower DNA concentration and may, therefore, be slightly smaller in average size. On a similar column of Sepharose4B, the peak of the residual activity and of the absorbance differed only one fraction. This is not unexpected, as even the singlestrand molecular weight (about 2.8 x 106)is closeto the excluded size (3 x 106). These results confirm that the molecules bearing the residual activity have a substantially higher molecular weight than denatured molecules; in fact they are eluted at the position where the original native moleculeswould have been eluted. This again suggeststhat the residual activity is due to a naturally occurring cross-linked fraction. A native-like configuration is also suggestedby the sharpnessof the elution pattern of the residual activity, in contrast to the broad pattern of denatured DNA. (d) Cesiurnchloride density-gradients In view of the evidence from alkaline sedimentation and Sepharosefiltration that the residually active molecules are not identical to denatured DNA, the buoyant density of these moleculesin CsClwas re-examined to test the previous claim (Rownd et al., 1961) that the buoyant density of these molecules is that of denatured DNA. As a control, the residual activity of the denatured normal molecules was again compared with that of an admixture of differently genetically marked, artificially crosslinked molecules. Figure 4 shows the results of such a preparative CsCl gradient.
0
0 30
35 Fraction
FIG.
4. C&l
density-gradient
of native,
denatured
40
45
no.
and cross-linked
H. injkmwae
DNA.
A mixture of Er DNA (1.6 pg, cross-linked by nitrous acid to the extent of 21%) and Cr DNA (8.4 pg) in 1 ml. KpHPOd (0.02 I, pH 9.2) was denatured by hating et 1OO’C for 3 min followed by rapid cooling in a 0% bath. Native Sr DNA (0.2 pg in 1 ml. pH 9.2 buffer) and CsCl (2.867 g) were added [TV = 1*4003]. Equilibrium was established by centrifugation for 3 days in an SW39 rotor in a Spinco model L at 38,000 rev./min at 19°C. Fractions (about (O-046 ml.) were collected in 0.2 ml. pH 9.2 buffer from the bottom of the tube, and assayed for the absorbance at 260 rnp .- l -) and the transforming activity for the Sr (-- A --A --), Er (- 0 - 0 -) (-•-.. and Cr (--A--A--) markers.
NATURALLY
CROSS-LINKED
H.
influenzoe
431
DNA
Clearly, under these conditions the naturally occurring residual activity (Cr marker) separates well from the bulk of the denatured DNA, as represented by its absorbance profile. Its density is in fact almost indentical to that of the residual activity of the nitrous acid cross-linked (Er) DNA, which is in turn about 0*003 g/cm3 higher than that of added native (Sr) DNA. Since this result is in conflict with previous variations observations in this laboratory (Rownd et al., 1961), experimental were tried in an attempt to reproduce the earlier result. Varying the melting conditions from three minutes at pH 9.2 to those of Rownd et al. (10 minutes at pH 7) did not change the result substantially, although the density at which the residual activity bands is now about 0.004 to 0$@5 g/cm3 heavier than that of native DNA, probably as a result of a larger number of strand scissionsoccurring in the residually active molecules. However, at the higher concentration of DNA previously used (71.5 pg instead of 6.5 pg/3 ml.), a different result was sometimesobtained. The residual activity was now found both in the previous position (slightly heavier than native DNA) and in the position of true denatured DNA. It can be shown, however, that this result is an artifact caused by the high concentration of denatured DNA in the gradient; when the peak fraction in this denatured DNA band was recentrifuged in a density gradient, the residual activity it contained now bands closeto added native DNA, well separated from its previous denatured DNA position. It cannot adequately be explained why molecules with an apparent native-like density sometimesband with the denatured molecules,nor why this apparent aggregation is not always observed under seemingly identical conditions : in four other experimer1t.susing high concentrations of DNA (60 and 70 pg), t’he residual activity was found in one band near the native density only. 1
TABLE
tSpeci$ctransforming activity of purified moleculescarrying
DNA
A native B denatured in CsCl C purified
Concentration during transformation (in mpg/ml.) 4.0 4.0 1.9
No.
residual activity
of transformed colonies (X 10-4)
Per cent of A
17.7 1.4 7.9
100 8 94
Cr DNA (30 pg) was denatured by incubation at 40% (pH 11.7) for 10 min. After cooling to 0% and adjusting the pH to 9.2, the DNA was diluted to 2.1 ml. and mixed with &Cl (2.87 g). Equilibrium eentrifugation and fractionation were performed as described in Fig. 4. The peak fraction of the residual activity was used as purified “residually active molecules”; its AZsO value was 0.076.
The specific activity of residually active molecules purified in CsCl gradients after short heating times was found to be about 909/othat of native (Table l), indicating that if all damage to the melted strands could be avoided, the activity of the naturally cross-linked DNA could well be identical to that of native DNA. When this purified fraction is subjected to a renewed thermal denaturation, the residual activity decreases only gradually and fits completely the extrapolated secondpart of the curves in Fig. I. This indicates that this gradual decreaseis causedby thermal degradation (depurinization and chain breakage) not by strand separation. 28
432
C. MULDER
AND
P. DOTY
(e) Biological hybrid molecules Perhaps the most conclusive means to establish the nature and origin of the residually active molecules is the investigation of the behavior in a CsCl gradient of denatured DNA, derived from density-hybrid DNA isolated from bacteria grown for many generations in heavy-isotope medium followed by one generation in light medium. Were the transforming activity due to single strands of DNA, it would form separate bands at the denatured heavy and denatured light positions; if due to renaturation between separated strands, three peaks would be expected (native heavy, native hybrid and native light) ; and if caused by a cross-linked fraction of the A26O
42
45
A26O
50
55 Fraction
FIU.
6. C&l
density-gradient
60
,
-24
1 65
A0
no.
of denatured
biological
hybrid
DNA.
Biological hybrid (1sNaH3aP/14N1H31P) Sr DNA (p = l-705) (6.7 pg) in 2 ml. pH 9.2 but& was denatured by heating at 1OO’C for 3 min followed by rapid cooling in ice water. Native (light) Cr DNA (0.2 pg) in 0.8 ml. pH 9.2 buffer and CsCl (3.86 g) were added [vn = 1*4009]. Further details as in Fig. 4. The fractions were assayed for radioactivity (- q - 0 -), transforming activity of markers and absorbance at 260 rnp; the the Sr (--A--r--) and Cr (--A-----A--) absorbance curve is omitted, only the two peak fractions being indicated by an arrow.
DNA, the activity should be found solely at native hybrid density. Figure 5 shows the result of such a study : the transforming activity of denatured hybrid DNA bands in a single peak closeto the native hybrid position. Although this position is also close to the density of light denatured DNA, no biological activity was found near the band of the 3aP-labeled denatured heavy molecules. Therefore, it can be concluded that the active speciesmust arise from a true cross-linked fraction. 4. Discussion The evidence presented in this paper attributes the residual activity of denatured DNA of H. injluenxae to a reversibly denaturable fraction in this DNA, similar to the “naturally occurring” cross-linked fraction previously described in B. subtilis DNA by Rownd (1963) and characterized by Alberts & Doty (1968). No differences have been
NATURALLY
CROSS-LINKED
H.
influenzae
DNA
I?? *.
detected between artificially (nitrous acid) cross-linked DNA and the residually active molecules. The increase of the sedimentation rate in alkaline sucrosegradients by a factor of 1.3 to 1.4 over the single-strands (Fig. 2) indicates that the mean molecular weight of the residually active moleculesis about twice that of the denatured molecules(Studier, 1966), a value to be expected for cross-linked molecules when the two strandsthough melted-are held together by the cross-linkage. Similarly, the elution pattern from a Sepharose2B column (Fig. 3) showsthat the moleculeswith residual activity have nearly the samemolecular weight and configuration as the original native molecules. This, again, is expected for reversibly denaturable (cross-linked) molecules where the two strands have zippered up after cooling. The buoyant density in CsClpoints to a native-like, bihelical structure. The slight shift towards heavier density is probably caused by some denatured regions in the molecules, most likely as a result of strand scissionof one of the strands in the melted state. Barnbardt (1965) stated that the residual activity of heat-denatured H. injhenme DNA had clearly a greater buoyant density than native DNA. However, the buoyant density of his residually active moleculesappears not to be substantially different from the data presented in this paper, after recalculating his data by correcting for the inhibiting effect of the large excess of native DNA present in his gradient. The conflicting data of Rownd et al. (1961), who found residual activity at, a density identical to denatured DNA, is most probably explained by aggregation at, the high concentration of DNA used. Barnhardt (1965), in a study of the residual activity of heat-denatured Ei. ijljhenzae DNA, claimed that this activity is clearly different from that of native DNA. However, his arguments against a native structure are all consistent with a crosslinked native structure, with the exception of his report that the residually active molecules are less sensitive to ultraviolet light than native molecules. This claim, however, could not be confirmed (Colarusso& Mulder, unpublished data). The explanation that the residual activity originates from naturally occurring crosslinked molecules is in agreement with the data of Chevallier & Bernardi (1965), who showed that the residual activity is eluted from a hydroxyapatite column well after the bulk of the denatured DNA and at a salt concentration that normally elutes native molecules. The evidence given in the present paper indicates that denatured molecules cannot transform, probably becausecompetent cells are unable to absorb irreversibly these single strands. Recently, however, Postel & Goodgal (1966) showedthat in exceptional circumstances (exposure of the cells to a low pH in the presenceof EDTA) competent cells of H. ir@uenzuecan absorb and be transformed by denatured DNA. In B. subtilis only fully native moleculeshave substantial activity in transformation and partially denatured moleculeshave no activity (Alberts & Doty, 1968) or somehut very little activity (Rownd et al., 1968), whereasin H. injluenzae these moleculeswit’h bihelical configuration and an unmatched, denatured, extension of one of t,he strands at either or both endsappear to be active too. Shen & Mulder (unpublished data) have found that the residual activity resides in both phasesof the polyethylene glycoldextran phase partition system of Albertsson (1962) as modified by Alberts (1967), whereas the residual activity of B. subtilis DNA is concentrated in the top phase, nearly completely separated from the denatured and partially helical DNA in the bottom phase. In this partition system only perfectly native molecules move into
434
C. MULDER
AND
P. DOTY
the top phase. Another indication that partially denatured molecules may have activity is that the average buoyant density of residually active molecules is 0.003 to O-005 g/cm3 heavier than perfectly native DNA. It is of interest to note that the residual activity of denatured DNA of D. pneumoniae also has an average buoyant density about halfway between those of native and denatured DNA (Mulder & Fox, results to be published), whereas the residual activity of B. subtilis was found by Alberta & Doty (1968) to be exclusively at the density of native DNA. So far the origin and the chemical structure of the naturally occurring cross-links remain unsolved. In an extensive study of the naturally occurring cross-linked fraction in B. subtilis DNA, Alberts (1968) failed to find any conclusive evidence that these cross-links arise in vivo. He observed that nitrous acid-induced cross-linked DNA invariably sediments somewhat faster than the naturally occurring cross-linked molecules, and he concluded that this could be an indication that the naturally occurring oross-links are terminal. From this and other evidence it was suggested that the cross-links are induced by shear at the Grst few breaking points of a whole genome upon isolation of the DNA. These observations in alkaline sucrose gradients could not be confirmed for H. inJluenzue DNA (Fig. 2), although such an effect could possibly have been masked in the latter case by the probable activity of partially helical DNA molecules in the H. in&ensue transformation system; also the approximately tenfold higher resistance of H. in$uenzue DNA to cross-linking by nitrous acid means that the treatment inflicts more damage on H. injluenzae DNA than on B. subtilis DNA, possibly causing single-strand breaks which would selectively reduce the sedimentation rate in alkali of the cross-linked species by nitrous acid. The residual activity of a DNA preparation with an average molecular weight of 21 x lo6 daltons was found to be 8%, extrapolated to zero-time melting (Fig. 1). Since the specific activity of the cross-linked molecules responsible for the activity is almost normal, this would mean that one cross-linkage occurs for about 260 x 10e daltons of DNA. This is about three per genome, assuming the genome of H. inJluenzae to be on the order of 8 x 10s daltons (Berns & Thomas, 1965) ; since an equal activity was found for all markers tested, these cross-links must be randomly distributed on the genome. It is not excluded, however, that the cross-links, if they pre-exist in viva, ase unequally distributed over the bacteria: a fraction of the organisms may have a large number of cross-links in their genome, whereas the majority of the organisms are free of cross-links. In conclusion, the present data support the conception that DNA preparations of H. injuenzae contain a naturally cross-linked fraction of unknown origin and nature, distributed apparently at random at an average frequency of about one cross-linkage per 260 x lo6 daltons of DNA. This fraction is solely responsible for the residual activity of denatured DNA. Single-stranded DNA displays no detectible transforming activity for H. in$uenzae under normal conditions, although it is active when incubated with competent cells at pH 4 (Postel & Goodgal, 1966). Partially denatured molecules which are inactive with B. subtilis apparently have some activity with H. inJluenzae. We are indebted to Drs sions, and to Mi-s Wei Liu completion of the present
B. M. Alberta, Shen and Mr
manuscript,
M. S. Fox, L. Colarusso we learned
and R. Sternglanz for helpful discusfor expert technical assistance. After from Dr G. Bernardi that Dr M.-R.
NATURALLY
CROSS-LINKED
H. inffluenzue
DNA
435
Chevallier and he had continued their previous work snd had reached via different ways essentially the same conclusions as expressed in this paper. Their account is also published in this issue, Chevallier & Bernardi (1968). The work was supported by U.S. National Institutes of Health grant HD 01229. REFERENCES Alberta, B. M. (1966). Ph.D. thesis, Harvard University. Alberta, B. M. (1967). Biochemktry, 6, 2627. Alberts, B. M. (1968). J. Mol. Biol. 32, 465. Alberts, B. M. & Doty, P. (1968). J. Mol. Biol. 32, 379. Albertason, P. A. (1962). Arch. Biochem. Biophy&, Suppl. 1, 264. Alexander, H. E. BE Leidy, G. (1953). J. Exp. Med. 97, 17. Bamhardt, B. J. (1966). J. Bact. 89, 1271. Barnhardt, B. J. & Herriott, R. M. (1963). Biochim. whys. Actu, 76, 25. Becker, E. F., Zimmerman, B. K. & Geiduschek, E. P. (1964). J. Mol. BioZ. 8, 377. Berns, K. I. & Thomss, C. A., Jr. (1965). J. Mol. BioZ. 11, 476. Bray, G. A. (1960). Andyt. Biochem. 1, 279. Chevallier, M.-R. & Bernardi, G. (1965). J. Mol. BioZ. 11, 668. Chevallier, M.-R. & Bernardi, G. (1968). J. Mol. B&Z. 32, 437. Eigner, J., Boedtker, H. & Michaels, G. (1961). Biochim. biophys. Acta, 51, 165. Ginoza, W. & Zimm, H. (1961). Proc. Nat. Acad. Sci., Wash. 47, 639. Goodgal, S. H. BE Herriott, R. M. (1961). J. Qen. Physiol. 44, 1201. Greer, S. L Zamenhof, S. (1962). J. Mol. Biol. 4, 123. Guild, W. R. (1961). Proc. Nat. Acud. Sci., Wash. 47, 1660. Guild, W. R. t Robison, M. (1963). Proc. Nat. Acud. Sci., Wmh. 50, 106. Kohn, K., Spears, C. & Doty, P. (1966). J. Mol. BioZ. 19, 266. Mulder, C. (1967). Fed. Proc. 26, 396. Postel, E. H. & Goodgal, S. H. (1966). J. Mol. BioZ. 16, 317. Roger, M. & Hotchkiss, R. D. (1961). Proc. Nat. Acad. Sci., Wash. 47, 6El. Rownd, R. (1963). Ph.D. thesis, Harvard University. Rownd, R., Green, D. M.. Sternglanz, R. I% Doty, P. (1968). J. Mol. BioZ. 32, 369. Rownd, R., Lanyi, J. & Doty, P. (1961). B&him. biophys. Actu, 53, 225. Spencer, H. T. t Herriott, R. M. (1965). J. Bad. 90, 911. Studier, F. W. (1966). J. Mol. BioZ. 11, 373.