Distribution of intercalative dye binding sites in chromatin

PAWt F. LWR

Aetinomyein D (AMD) to chromatii isolated and weak process anal tion of the dye intercalation low r-values (dye bound per dye released from chromatin an AMD dissociation profiles cl denaturation profiles; whereas cated 3 major transitions main corrponent correspon DNA. The DNA-like component w chromatin but could be generated of residual non acid-soluble protems treatment or dissociation and reconstitution sion to the DNA-like component with 1 indicates that more than o __.__ ” ______ _._.” * l_-_l-... _ .^__

of chrom

INTRODUCTION

The cytological application of DNA ink metry techniques offers considerable structure of individu nucl%ti.(review N.R.C.C. No. 16028. a Present address: Centre d’Etude da I’Ener (Belgique). b To whom reprint requests may be directed. Abbreviations: AMD, atinomycin D; EB, ethidium bromide. *

tion is marred somewhat by the lack of control over the amount of dye that is actually interacting with the DNA. For a dye such as acridine orange identification of the types complexes formed has been difficult to resolve spectro~opi~~ly f2]. Since several properties of DNA are known to be affected by variation in the dye-DNA ratio [3-81 further characterization of chromatindye complexes is needed in order to determine the exact nature of the binding sites. Xn this paper we have examined the tiinding properties of EB with chromatin isolated from various avian tissues. The binding parameters of EB were compared to those of AMD using partially dehistonized erythrocyte chromatin. The complexes were further characterized by studying the dissociation patterns of strongly bound dye as a function of DNA and chromatinDNA secondary disruption. MATERIALS AND METHDDS

Poly d(A-T).d(A-T) and poly dG.dC were obtained from Miles Laboratories and single-stranded poly dT from P-L Biochemic~s. EB was purchased from B.D.H. Ltd. AMD (A-grade) and bovine serum albumin were obtained from Calbiochem. Highly polymerized calf thymus DNA was purchased from Worthington Biochemical Corp. tion of chrornatin and nucki All tissues were obtained from 1-2-year-old domestic ganders. Mature erythrocyte chromatin was prepared from nuclei as previously described f9). Immatu~ e~throcy~ c~omatin was prepared from nuclei isolated from an erythroblast=enriched cell fraction (approximately 78% immature cells, includes both large and small basophilic erythroblasts and polychromatic erythrocytes) obtained by isopycnic centrifugation [IO]. Chromatin from liver and kidney were prepared according to a previously described procedure [ll].The imclei were washed in distilled water before dialysis against 1 mM Tris-HCl pH 8 for 6 h.

Pmparu

of ~i~t~~te-di~§~ci~~ed c~ro~a~in and DNA Partially and fully dehistonined chromatin was prepared by a stepwise acid extraction method [9] . Certain aspects of the low pH extraction me~od should be emph~ized: histone extraction is more easily facili~~d using nuclei than prepared chromatin, under the present conditions the nuclei remain intact [ 121 and recovery of residual nuclei is rapid; proper neutralization of the residual nuclei is important particularly for the pH 1.0 extracted nuclei. Acidextracted nuclei usually require two washes in 0.1 M Tris-HCI, pH 8.0 (10 vol.) before a uniform viscous chromatin is obtained. After neutralization the fibers were 20-100 A [12]. While we are aware of the po~n~i~ hazard of acid depurination [9,13 J it should be noted that total low pH exposure time, including neutralization, was 15 min per preparation at 2-6”C; these conditions are well within the limits before changes in template properties are detected [ 9 ] . This technique was chosen because

~~par~t~~n

28

of the high selectivity, rapidity and reproducibility that is achieved in the preparation of histone I and histone V depleted chromatin, and dehistonized chromatin. DNA was prepared from mature erythrocyte chromatin dehistonized by salt or acid [9,14] following pretreatment with trypsin (1 : X5, trypsin : chromatin) for 4 h and incubation with autodigested pronase (1 : 10) at room temperature for 16 h. The erythrocyte DNA concentration was baded on calf thymus DNA as a standard [15].

Urea and high salt treatment of chromatin Approx. 2 ml of 1.5 - lo-’ M DNA-P mature erythrocyte chromatin was rapidly homogenized in 9 vol. of 6.6 M urea or 2.2 M NaCl-6.6 M urea in 1 mM Tris buffer. The urea was exchanged with 1 mM Tris buffer using an Amicon ultrafiltration apparatus with an UM-2 membrane at a maximum rate of minus 0.3 M urea/h. Chromatin in the salt-urea mixture was exchanged with Tris buffer generated first as a negative salt gradient (2.0 M NaCl to 0.1 M NaCl), followed by a second gradient, 6.0 M urea in 0.1 M NaCl. Control chromatin in each case was homogenized and exchanged with 0.1 M NaCl-1 mM Tris buffer only. Following dialysis, 15 h for each, the chromatin was pelleted at 20 000 g 40 min. The supematant was concentrated by ultrafiltration. The pH 1.0 soluble proteins of both the pellet and supematant were analyzed by disc gel electrophoresis at pH 2.7

[W. Determination of nucleic acid and protein Chromatin was fractionated into nucleic acids and protein components [9]. DNA concentrations were determined colorimetricaily using calf thymus DNA as a standard assuming an extinction coefficient per mole DNA-phosphate at 260 nm (e$‘j’) = 6500 [15]. Ultraviolet absorption spectra were determined on a Cary 14R or a Gilford spectrophotometer. Protein contents were determined by the microbiuret method and dissociated histones and histones remaining with chromatin after various treatments were identified by polyacrylamide gel electrophoresis [16]. For disc electrophoresis extracts were precipitated with 9 vol. of acetone, redissolved in 6 M urea-O.9 N acetic acid and electrophoresed at pH 2.7. The gels were stained with 0.5% Amido Black-40% ethanol-7% acetic acid for 20 min and destained in a solution of 7% acetic acid-40% ethanol.

Dye binding studies The binding of EB and AMD to DNA and to chromat,in was estimated by measuring the spectral changes that those dyes undergo during complex formation with nucleic acids. To ensure reproducible binding results all chromatin samples were lightly sonicated (two lo-set pulses, 54 W output Model W-185-C Sonifier Cell Disruptor, Heat Systems Co., Melville, N.Y.). The features of the spectrophotometric analysis and Scatchard plot calculations have been extensively discussed [ 2+?,17 ] .

29

The binding of EB was followed by measuring the absorbance at 460 nm at 20°C of a series of dilutions containing DNA or chromatin-DNA ranging from 0 to 18 - 10B4 M. EB concentration was determined by measuring the absorbance at 480 nm of a solution of free EB using the value e,480= 5600 [4 ] . The binding of AMD was monitored at 425 nm in a solution containing 0 to 1.2 * 10 -4 M DNA-P. The concentration of free dye was determined from the absorbance at 440 nm using c,,40= 2480 [18]. The non-specific absorption of each chromatin sample was measured and subtracted from the total & zs and Aa so as to measure the AMD and EB contributions only. Thermal denaturation and dye dissociation

Thermal denaturation studies on chromatin and DNA were performed using a modified Gilford 240 spectrophotometer equipped with a model 2427 therm0 programmer or a modified Beckman DB-G spectrophotometer with a Moseley Autograph 7001 AM X-Y recorder. For the latter tie temperature was recorded by a calibrated thermistor couple and bridge circuit that gave a linear voltage response from 0 to 105°C. The thermistor was located in the sample cuvette just above the optical pathway. .,\ Haake temperaturecontrolled circulating ethylene glycol (85%) bath was uscrd to provide a heating rate of 0.4” to OS”C/min. The Gilford 2427 therm0 programmer electrically heats the sealed four-chambered cuvette assembly at preselected rates of 0.22”, 0.45” or 0.85”C/min. Absorbance studies at 260 nm were made on samples containing 1.0 to 1.4 - 10V4 M DNA-P. The release or dissociation of the bound dyes upon melting of DNA samples was measured using either EB at 460 nm or AMD at 425 nm and DNA concentrations of 6.3 * 10s4 to 24.0 - 10e4 M DNA-P. The concentration of the dye required for each study was arrived at after the study of Scatchard plots. Absorbance changes due to scattering were corrected by having a sample in the reference cuvette without the addition of dye. Both sample and reference solutions were degassed and overlaid with mineral oil to avoid evaporation. In a series of control experiments the effects of heating and cooling on free AMD and EB spectral properties were examined. With temperatures above 60°C the absorption maximum ,of free EB was shifted from 480 nm to 490 nm at 90°C. The temperature-related absorption shift was insignificant at 460 nm in comparison with the absorbance decrease due to liquid expansion. The absorption spectrum of free AMD remained unperturbed at high temperatures. Correcting for the light scattering contribution to A,,, for chromatin the hyperchromic effect was 33 to 36%, which was comparable to that for erythrocyte DNA. Maximum hyperchromicity for both dye release and DNA were normalized to that obtained for deproteinized DNA. The degree of dissociation of dye and helicity of DNA were determined from the following formula A$,,, -A” A&, -A&

’ loo

where X is 260 nm, 425 nm or 460 nm.

A conductivity meter (Radiometer) was used to estimate the ionic strength of each DNA-dye sample. All denaturation studies were done within the ionic strength (I) range, 0.005 to 0.009. Data from the recorders were converted to numerical form using an Autotrol-Digitalizer. These data were processed by IBM 360 computer using a program similar to that described by Ansevin and Brown 1191 to give computerdriven plots of hyperchromicity vs. temperature, the temperature derivative of hyperchromicity vs. temperature and an analysis of residuals to compare observed with expected data. RESULTS

Dye binding to chromatin and DNA Spectral differences between free and bound dye can be used to determine the extent of dye binding to polynucleotides provided free and bound dye have a common isasbestic point [ 51. Fig. 1 shows that EB with mature erythrocyte chromatin has an isosbestic point about 510 nm. The same result was obtained for immature erythrocyb, liver and kidney chromatin. This suggests only bound and free dye coexist in solution, each species characterized by a given molar extinction coefficient. Scatchard plots made from the spectrophotometric titrations of various types of chromatin (see METHODS and Fig. 2) similarly displayed characteristics of more than one type of binding site, similar to those of purified DNA. The data shown in Fig. 2 also indi-

2.5

Fig. 1 Spectra of free EB and EB bound to mature erythrocyte chromatin. Total molar EB concentrations (M DNA-P), curve A = 0, B = 0.9 concentration is lo+; chromatin-DNA - 10w4, C = 1.8 * 10w4, D = 3.6 * lo4 and E = 4.6 - lo+. Fig. 2. Scatchard plots for the binding of EB to DNA and various types of avian chromatin. -z-o-, mature erythrocyte chromatin; -e-o-, spleen chromatin; -A-A-, kidney chromablood; tin; -A-A-, liver chromatin; -O-O-, chromatin prepared from erythroblast-enriched -a-~-, erythroryte DNA. Further calculations we summarized in Table I.

31

TABLE

I

PARAMETERS Chromatin

OF ETHIDIUM

BROMIDE

Protein/ DNA

source

______~_____

Mature erythrocyte Immature erythrocyte Liver Kidney Erythrocyte DNA -.

0.97 1.26 1.89 1.41 0.006

BINDING

TO DNA AND CHROMATIN

Sites/ nucleotide n .lO”

nucleotides/ site

33 70 58 47 192

30.3 14.3 17.2 21.3 5.2

i/n

.._._____

K * lo-’

% DNA

I/mole ___

____

16.0 34.8 29.0 23.5 100

54.0 64.1 65.3 66.2 27.0

cate that erythrocyte DNA binds EB to the same extent as DNA from other sources [4--81. All types of chromatin studied had fewer EB primary binding sites than deproteinized DNA (Table I and Fig. 2). The number of EB primary binding sites for mature erythrocyte chromatin was less than half the number of sites calculated for chromatin from a blood cell fraction highly enriched for erythroblasts. These observations are consistent with those of cytological studies on hen erythrocytes [20,21] and fully supports our observations of increased nuclear activity and in vitro transcription of chromatin isolated from very immature cells fractionated by isopycnic centrifugation [lo] . Effect of histone removal on chromatin-dye binding Removal of histones and residual proteins from chromatin resulted in a progressive increase in EB primary binding sites [3,22-251. Fig. 3 shows the histone composition of five types of erythrocyte chromatin used for this study. Analysis of the acid extracts indicated that the low pH method for histone removal was very selective for histories I and V and to a lesser extent for IIb (cf. Fig. 3b, d and f). The residuai nonhistone proteins (acid insoluble) of this chromatin have been extensively described 1261. By this extracTABLE

II

PARAMETERS CHRGMATIN

OF

ACTINOMYCIN

----_-.--Chromatin

AND

----_-_-___ status

-.--..~~~..~-“---_---

Mature erythrocyte Less histone 1 Less histones I and V Less histones I, V and IIb Less total histones DNA

32

D

BROMIDE

AMD EB l_l_l___--33 37 90 105 157 192

BINDING

_____~__

_--I------____

Sites/ nucleotide

16.5 17.0 19.5 22.0 40.5 70.5

ETHIDIUM

TO

___._

__

10-Y

Nucleotidesl site __---_ --

‘70DNA --.~-

l/mole - __~----

AMD

AMD EB _ --__

AMD __-

EB

24.4 24.2 27.7 31.2 57.4 100

1.52 1.64 2.03 1.84 1.76 2.62

54.0 53.5 45.0 38.5 30.0 27.0

60.6 58.8 51.3 45.5 24.7

EB ---_-.--.30.3 27.0 11.1 9.5 6.4

h’

16 18 47 55 82 100

.

.

e

ul ‘

e

a

tion method differences in EB primary binding values (n) were obtained for each type of chromatin (Table II). While increments of n may reflect the degree of histone interaction with DNA, such as in the case of the removal of histone I and V and possibly III and IV, no particular histone class preferentially effects the association constant (K,,,,.). The KBssoc. was found to decrease with removal of protein (Table II). The actual energy involved in the binding process for chromatin was found to be in good agreement with the generally accepted values for a primary or process I binding, AGc = -6 to -9 k&/mole ]4,5,27]. Our calculated values ranged from -6 to -6.5 kcal/mole for all chromatin and DNA studied. The significance of the Kaggw.differences, if any, is discussed below. Binding of AMD to calf thymus native [28,29] and partially deproteinized [ 281 chromatin has been studied. The presence of chromosomal proteins was found to markedly reduce the number of strongly bound dye molecules [ 281. The same phenomenon is shown to occur in erythrocyte chromatin’ (Fig. 4). The computed value of n is approximately the same as that for hen erythrocyte chromatin [ 291 but smaller than that reported for calf thymus [28,29] . Specific removal of chromosomtll proteins also promoted an in-

LL-L--Ili

Tempwoture,

“C

Fig. 4. Scatchard plots for the binding of AMD to native and partially deproteinized erythrocyte chromatin and DNA. The molar concentration of free actinomycin D is CF and r is the moles of AMD bound/mole DNA nucleotide. Details are described in text and Table II. -O-O-, native erytbrocyte chromatin; -a-+, erythrocyte chromatin with histones I and V removed; -fU-, fully dehistonized erythrocyte chromatin; -A-A-, erythrocyte DNA. Fig. 5. Thermal disruption of DNA-ethidium bromide complexes: correlation of erythrocyte denaturation DNA with dissociations of DNA-bound ER. Conditions and equipment was monitored at 260 nm for hyare described in the text. DNA denaturation (--) perchromicity using a solution containing 1.7 * 10-4 M DNA-P and 0.54 * lo-’ M EB. EB dissociation (-) was monitored at 460 nm for increased free EB concentration using a solution containing 10 times the amount of DNA and dye. Reference samples contained either DNA or dye at complementary concentrations. Right hand figure shows the corresponding thermal derivative prof;?es of DNA and dye.

34

crease in the number of AMD primary binding sites (Table II) and also the Kassoc.~ but not as great as &.,,. for EB. The K values given in Table II are in good agreement with those obtained for calf thymus chromatin [28]. Moreover, removal of histone I and V did not promote as large an AMD binding increment as observed for EB. On the other hand, removai of all histones, particularly the arginine-rich histones, and also the residual acid insoluble proteins sharply increased AMD binding. Characterization of dye complexes by thermal dissociation The Tm (temperature giving 50% hyperchromicity) of DNA with AMD [ 181 or with proflavin [ 301 (2,8diaminoacridine) is related to the amount of dye bound per nucleotide. The increase is considered due to the nature of the complexes, the intercalated dye [30] holding together base pairs at a temperature at which the hydrogen bonds would normally be broken. This interpretation has gained support from DNA renaturation studies using EB-DNA complexes [6]. In general low r values below saturation of primary binding sites, would not be expected to appreciably increase the Tm of DNA nor its rate of renaturation. In turn, nucleic acid-intercalative dye complexes can be characterized by quantitating dye release or dissociation upon heating the complex and subsequent destruction of the secondary structure [ 18,30331. Since EB could affect the melting temperature of DNA it was important to first establish the degree of coincidence of the dye dissociation from DNA and DNA denaturation, as functions of increasing temperature. Dye absorption in the 260 nm region of the DNA absorption spectrum is non-contributory due to the reduction of the dye-DNA complex concentration necessary to monitor DNA denaturation. Fig. 5 shows that there is a near complete overlap of these two interrelated functions (dye dissociation and DNA denaturation). Similar results were also obtained using cells with a l/lOth cm path length for reduction of DNA concentration instead of dilution of the complex concentration. For four separate experiments maximum perturbance of Tm was 6°C higher than the Tm of DNA without dye present. It should be stressed that only P, the actual EB bound per nucleotide, needed to be GO.05, and not total EB/nucleotide. Once Scatchard plots are obtained (Table I and Fig. 2), r can be computed [34] . The results in Fig. 5 indicate the possibility that the sites for EB primary binding are regularly distributed with respect to DNA base sequence. A similar study using a 1 : 1 mixture of commercial duplex polydeoxyribonucleotides (d(A-T) and dG.dC) further indicated that no major base compositional binding preference at low ionic strengths (I < 0.01) occurs. However, when I is greater than 0.01 the primary binding value (n) decreases. Under these conditions a larger proportion of EB was releated with the lower melting region of the DNA profile. These results suggest some binding preference for low melting DNA at higher ionic strengths or possibly an enhance8 *restabilization of the DNA by the bound dye, With the same conditions the dissociation of EB bound to single-stranded poly dT was different than any type of duplex DNA used in both temper-

35

E:B was released (40 f 5°C at I = 0.005) and amount of dye bound (<8% of that obtained for duplex DNA).

atures at which

Comparison of EB and AMD binding patterns

Fig. 6 shows the results of a complcmentry dye-DNA thermal dissociation study using mature erythrocyte chromatin. In contrast to the EB-DNA complex, the EB dissociation and erythrocyte chromatin-DNA melting profiles were not coincident but became so only after removal of certain proteins. Most of the EB is released from chromatin at a temperature which is even lower than that observed for deproteinized DNA. The release of nearly 50% of the bound EB corresponds to the denaturation of no more than 3% of the total DNA. The temperature derivative of dye release vs. temperature shows two major transitions. Although n increases by about 5 to 8 EB per 1000 r-----T--

T-‘-’

1

‘00 @

:f

80

60

I I I

40 20

: :

tr

_/i t--c-------+I

c-/

_-

‘00 @

80.

:

80 40 20

:

:

,,:

_a--

L-

30

50

m

so

Temperature,

‘C

6. Correlation of chromatin-DNA thermal denaturation in the presence ofethidium bromide measured at 260 nm (- - - ) with thermal dissociation of chromatin-bound ethidium bromide measured at 460 nm (). Experimental procedure is described in Fig. 6. (a) Native mature erythrocyte chromatin; (b) ch,romatin with histones I and V removed (refer Fig. 3); (c) fully dehistonized chromatin. Right hand figure shows the corresponding thermal derivative profiles. Fig.

36

nucleotide pairs with the removal of histone I no significant change in the overall EB release pattern was observed. Since n nearly doubles after the removal of I and V the appearance of an additional dye dissociation derivative component must reflect the formation of new EB binding sites. The pattern of dye release resembled that obtained for dehistonized chromatin (Fig. 6c) and deproteinized DNA. Full coincidence of the EB release and DNA denaturation profiles was not obtained until after deproteinization, especially removal of all the histones. These observations suggest that dye binding in chromatin is confined to specific regions of the DNA and that more than one type of intercalation-like binding site exists. Thermal melting profiles of AMD-chromatin complexes were examined by monitoring the appearance of unbound AMD upon heating the complexes at 425 nm [X3]. Differences between the AMD dissociation curves of the various types of chromatin after chromosomal proteins were removed from chromatin were noted (Fig. 7). The AMD-native chromatin complex, as EB, also exhibited a biphasic dissociation profile as a function of chromatin-DNL1 melting. The prominence of the first phase of the dissociation curve was

2.5

4.0

.2.5

il.0

t

__’

30

90 30 50 Temperature, ‘C

70

90

Fig. 7. Dissociation of actinomycin D from erythrocyte chromatin and DNA complexes as a function of temperature. Release of free actinomycin D (9.1 * low6 M) was monitored at 425 nm. DNA samples which were the same ones used in Fig. 6. (a) Native chroma-); (b) fully dehistonized and histone I and V depleted chromatin ( tin ( -) -). Right hand figures show the corand erythrocyte DNA ( chromatin ( -----) responding derivative profiles.

35

much smaller than that obtained for EB (Fig. 6a). Even though removal of histone I and V from the chromatin increased the AMD binding sites by about 3 per 1000 base pairs the binding pattern of the mature chromatin was significantly altered. From the 425 nm and the 260 nm measurements it is also apparent that DNA denaturation and AMD release are more closely associated than for EB and DNA in chromatin. In all observations the bulk of the AMD released occurred at higher melting temperatures than for EB. This suggests that the intercalation sites for EB and AMD in chromatin are different and that the latter possess a resistance to dissociation by heat much closer to that of the bulk chromatin. These results agree with those of Gellert and coworkers [ !$I who showed that more AMD was dissociated from DNA at a temperature above the Tm for calf thymus DNA. Comparison of EB binding patterns

in chromatin

from different sources

To determine whether the distribution of EB binding sites in mature erythrocyte chromatin was typical of other types of gander chromatin a study of the binding patterns of chromatin with different n values was made. Fig. 8 shows the EBdissociation temperature derivative profiles for various chromatin types. The results contrast those shown in Fig. 6a but are themselves quite uniform. In each case three main transitions were obtained which were similar to those for dehistonized erythrocyte chromatin (Fig. 6~). These results suggest that mature erythrocyte chromatin lacks a distinct DNA-like EB dissociation pattern which is observed in other chromatin types and when proteins are removed. Since immature chromatin does have this binding pat-

Temperature,

‘C

Fig. 8. Comparisons of the ethidium bromide binding patterns for various types of chromatin. Derivative thermal dissociation profiles of ethidium bromide bound to (a) mature erythrocyte chromatin () and immature erythrocyte chromatin (-), and (b) goose liver (-) chromatin. Conditions for forming the ) and kidney ( complexes were obtained from Table I and for thermal denaturation at 460 nm from Fig. 5.

38

Fig, 9, C~m~ari~cn3 ctf the a~idi~rn ~rcm~de ~~~d~~~ patterns in urea ~~~~~~~and salt>e~n~t~t~ted mature er~thr~cyte c~~arnati~, ~er~~~t~ve thermal EB dj~c~ati~r~ broker and cuntrol c~r~rn~t~~ (--- -- ----I and ia in (a) are for 6 M urea t~~atrne~t (-----I and contra1 chromatj~ (--- - -). C~~djtj~n~. for urea {b) far rec~~~tit~ted C--) treatment, salt di~cj~t~o~ and ~cconst~t~t~on of ch~cmat.~n are described in M~~T~C~~. Conditions for thermal denaturatian are the Same as in Fig. 5.

39

tin, Senshu 1361 has shown how erythrocyte chromatin when extracted 0.15 M to 0.2 M NaCl in 6 M urea releases both IIb histones.

with

DISCUSSION

Aside from in situ cytological evidence, little is known about the distribution of dye binding sites in chromatin. From the studies of Sobell and COworkers [3’7] and Hyman and Davidson [ 381, AMD would be expected to favour binding to G-C rich DNA. Similar evidence now exists for an A-T base preference for the binding of acridines (proflavine and quinacrine). Evidence of a base preference for the binding of EB is still not clear [ 39-411. However, studies by Richardson [8] indicate two modes of template restriction by EB, as well as differences in binding modes for DNA molecules that have very similar EB-binding values. We have demonstrated that avian chromatin not only has fewer EB primary binding sites (n) than DNA .(Table I), but also n appears to be directly proportional to the amount of in vitro template activity associated with the chromatin [ 3,421. Recently these observations have been confirmed by Nicolini and Baserga 1431 using a completely unrelated chromatin system. On the other hand, AMD-chromatin binding values, which exhibit a much narrower range (
40

tative analysis of protein-DNA complexes but both the technique and interpretations are far from definitive. Under conditions where most, if not all, the EB or AMD was bound to the DNA at a relatively low ionic strength (
41

However at the very low r-values used in our studies redistribution would not be expected to be favoured. We see no alteration in the qualitative dissociation (multiphasic) profiles. Rebinding of dissociated dye would be reflected by a negative derivative profile and would not be expected to be closely associated with the DNA helix-coil transition (Fig, 6). On the other hand, the dye dissociation transitions observed for the various tylees of ehromatin (Figs. 6-9) may be accounted for in part by postulating at le tribution of bound dye. Nevertheless, the transitions are ve of the type of chromatin used (Fig. 8). Based upon previous studies on the nature of the helix-coil transitions in nucleoproteins [ 19,461 and from our observations on EB binding patterns of chromatin treated with urea, reconstituted chromatin and histone depleted chromatin (Figs. 6-9) we suggest that the presence of the third (highest temperature) dye transition may also reflect dye-DNA interactions involving a limited number of highly restricted binding sites which for the most part are determined by DNA interactions with proteins. The DNA melting stability of mature erythrocyte chromatin has been shown to be much higher than for liver chromatin [ 141. From Fig. 6 it appears that about of the EB dissociated from mature erythrocyte chromatin can be cl fied with the third dye dissociation transition of more template-active chromatin types (Fig. 8) and may be the result of reassociation of previously dissociated dye as discussed in the preceding paragraph. Alternatively, the results suggest that for either transcriptionally active or inactive chromatin templates the more tightly bound proteins are responsible for preventing EB binding into the AT (75 to 86”) component of ehromatin. These observations are consistent with chromatin and EB circular dichroic data [3] as well as with polarization studies [ 23,261. Angerer and co-workers [ 231 have suggested that intercalated EB molecules are more clustered in chromatin than DNA. This signifies a clustering along the DNA of specific proteins or of a specific higher order of nucleoprotein structures which inhibit dye binding. Such structures may be similar to those described by Qlins and Olins [47: and van Holde and co-workers [48] , For mature erythrocyte chromatin progressive removal of histones is associated with the generation of a corresponding DNA-like EB dissociation profile. In particular, histone I and V, but not I alone, may be partially responsible for the major perturbations observed for mature erythrocyte chromatin to the other types of chromatin we studied, their removal generates an EB.binding patTern similar to that obtained for the more template-active types such as liver, kidney or immature erythroeyte. We have previously ested that’ the decreased binding may be due to marked conformati changes in the chromatin-DNA [S]. In goose [49] and chicken [12] erythrocytje only the highly compact ehromatin (>lOO”A fibers) was found to be disrupted by the removal of histone I and V with very little disruption o the chromatin subunit-like structure. Whether the increased EB bindi is associated with the availability of “spacer” DNA or DNA within the subunit structure [47,48] remains to be estab42

1 P.F. Lurquin, The

ture, them-Bid. la % E. Fredwicq and C a 4

&br

$ 3.8. Hutton *n

8 3.P. Richardson, Mechankm of ethidium bram J. Mol. Bid., 78 (1979) 703-714.

9 V.L. Seligy and J.M. Neelin, Transcription properties of stepwise acid-extracted chicken erythrocyte chromatin, Biochim. Biophys. Acta, 213 (1970) 380-390. 10 V-L, Se]&, G.M.H. Dams and J.M. Neelin, The role of histones in avian erythropoiesis, in J.K. Pollak and J. Wilson Lee (Eds.), The Biochemistry of Gene Expression in Higher Organisms, Australia and New Zealand Book Co., Sydney, 1973, pp. 117-190. Y. Moule and C. Rouiller, Isolation of pure and unaltered liver nuclei 11 J, Chdveau, morphology and biochemical comp~iti(~n. Exptl. Cell Res., 11 (1956) 317-321. 12 K. Brasch, G. Setterfield and J.M. Neelin, Effects of ~quentiai extraction of histone proteins on structural organization of avian erythrocyto and liver nuclei, Exptl. Cell Res., 74 (1972) 2’7-41. I.3 S. Banni, and H. Tereyama, Template activity of chromatins isolated from regenerating rat liver, J. Biochem., 66 (1969) 289-297. 14 V.L. Seligy and M. Miyagi, Comparison of template-property changes after salt extraction of avian’erythrocyte and liver chromatin, Eur. J. Biochem., 46 (1974) 259-269. 15 H.R. Mahler, B. Kline and B.D. Mehrota, Some observations on the hypochrom~m of DNA, J. Mol. Biol,, 9 (1964) 801-811. 16 S. Panyim and R. Chalkley, The heterogeneity of histone, I. A quantitative analysis of calf thymus histones in very long polyacrylamide gels, Biochemistry, 8 (1969) 39723979. 17 D.G. Dalgleish, G. Dingsoyr and A.R. Peacocke, A comparison of the interactions of proflavine with DNA and with deoxyribonucleohistone using circular dichroism spectroscopy, Biopolymers, 12 (1973) 445-457. ia M, Gellert, CE. Smith, D. Neville and Gary Felsenfeld, Actinomycin binding to DNA: M~hanism and specificity, J. Mol. Biol., 11 (1965) 445-457. 19 A.T. Ansevin and B.W. Brown, Specificity in the association of bistones with deoxyribonucleic acid, Evidence from derivative thermal denaturation profiles, Biochemistry, 10 (1971) 1133-1142. 20 A.M. Kernell, L. Bolund and N.R. Ringertz, Chromatin changes during erythropoiesis, Exptl. Cell Res., 65 (1971) l-6. 21 G. Campbell and B.L. Gledhill, Chromatin of primitive erythroid cells from the chick embryo, Chromosoma (Berl.), 41 (1973) 385-394. 22 L.M. Angerer and E.N. Moudrianakis, interaction of ethidium bromide with whole and selectively deproteinized deoxyribonucleoproteins from calf thymus~ J. Mol. Biol., 63 (1972) 505-521. 23 L.M. Angerer, S. Georghiou and E.N. Moudrianakis, Studies on the structure of deoxyribonucleoproteins. Spectroscopic characterization of the ethidium bromide binding sites, Biochemistry, 13 (1974) 1075-1082. 24 P.F. Lurquin and V.L. Seligy, Binding of ethidium bromide to avian erythrocyte chromatin, Biochem. Biophys. Res. Commun,, 46 (1972) 1339-1404. 26 J. Lawrence and M. Lewis, Kthidium bromide as a probe of chromatin structure, FEBS Letters, 40 (1974) 9-12. 26 K.R. Shelton and J.M. Neelin, Nuclear residual proteins from goose erythroid cells and liver, Biochemistry, 10 (1971) 2342-2348. 27 J.B. Lepecq and C. Paoletti, A fluorescent complex between ethidium bromide and nucleic acids, 3. Mol. Biol., 27 (1967) 87-106. 28 L. Kleiman and RX. Huang, Binding of actinomycin-D to calf thymus chromatin, J. Mol. Biol., 56 (1971) 503-521. 29 N. Ringertz and L. Bolund, Actinomycin binding capacity of deoxyribonucleoprotein, Biochim. Biophys. Acta, 174 (1969) 147-154. 30 J. Chambron, M. Daune and C. Sadron, Etude thermodynamique de l’interaction de la Proflavine avec l’aeide deoxyribonucl~ique, II, Biochim, Biophys. Acta, 123 (1966) 319-328, 31 H. Grosjean, J. Werenne and H. Chantrenne, The binding of proflavine to transfer ribonucleic acid: Dependence on secondary structure, Biochim. Biophys. Acta, 166 11968) 616-620.

32 P.F. Lurquin and P. Buehet-Math& Biological activity of ethidium bromide-transfer RNA complexes, FEBS Letters, 12 (1971) 244-249. 33 C. Urbanke, R. Romer and G. Maass, The binding of ethidium bromide to different conformations of t.RNA. Unfolding of tertiary structure, Eur. d. Biochem., 33 (1973) 511-516. 34 A.R. Peacocke and J.N.H. Skerett, The interaction of aminogcridin acids, Trans. Faraday Sot., 52 (1956) 261-267. 35 Y.V. Ilyin, A.Y. Varshavsky, U.N. Mickel r and G.P. Georgiev, Stu ribonucleoprotein structure, Eur. J. Bioche 22 (1971) 235-345. 36 T. Senshu, Selective extractron of histones with sodium chloride and Biophys. Acta, 236 (1971) 349-352. 37 H.M. Sobell, S.C. Jain, T.D. Sakore and C.E. Nordman, Stereochem tin DNA binding, Nature New Biology, 231 (1971) 200-205. 38 R.W. Hyman and N. Davidson, Kkletics of the in vitro inhibition of t Actinomycin D, J. Mol. Biol., 50 (: 970) 421-438. 39 C. Tsai, S.C. Jain and H.M. Sobeil, X-Ray crystallographic visualixation cleic acid intercalative binding: structure of an ethidiumdinucleoside mon crystalline complex, ethidium: b-iodouridylyl (3’.5’)adenosine, Proc. Natl. Acrd. Sci. USA, 72 (1975) 628-632. 40 R.E. Williams and V.L. Seligy, The interaction of ethidium bromide with sy polydeoxyribonucleic acids. Effect of base composition and sequence on the i circular dichroism spectra, Can. J. Biochem., 52 (1974) 281-287. 41 S. Aktipis, W.W. Martz and A. Kindelis, Thermal denaturation of the DNAethidium complex. Redistribution cf the intercalated dye during melting, Biochemistry, 14 (1975) 326-331. 42 V.L. Seligy and P.F. Lurquin, Relationship between dye binding and template activity of isolated avian chromatir., Nature New Biology, 231 (1973) 20-21. 43 C. Nicolini and R. Baserge, Circular dichroism and ethidium bromide binding studies of chromatin from WI-38 fibroblasts stimulated to proliferate, Chem:Biol. Interact., 11(1975) 101. 44 L. Bolund and E.W. JohnA. Chick-erythrocyte chromatin as a template for RNA synthesis in vitro, Eur. J. Bicchem., 40 (1973) 591-598. 45 R.S. Tobin and V.L. Seligy, Characterization of chromatinbound erythrocyte h V(fSc), J. Biol. Chem., 250 (1975) 358-364. 46 H.J. Li, C. Chang and M. Weiskopf, Helix-coil transition in nucleoprotein-chromatin structure, Biochemistry, 12 (1973) 1763-1771. 47 D.E. Olins and A.L. Olins, Spheroid chromatin units (v) bodies, Science, 183 (1974) 330-332. 48 K.E. van Holde, C.G. Sahasrabuddhe and B. Shaw, A model for particulate structure in chromatin, Nucleic Acid Res., 1 (1974) 1579-1585. 49 V.L. Seligy, M.J. Dove and H. Poon, Biochemical and morphological study of chrotnatin and sub-components from nuclei obtained from avian erythroblas erythrocytes, Proc. Can. Fed. Biol., 18 (1975) 472. 50 R.F. Itxhaki, Studies on the accessibility of deoxyribonucleic acid and deoxyribonucieoprotein to cationic molecules, Biochem. J., 122 (1971) 583-394. , J. Mol. 51 R.F. Itzhaki and H.K. Cooper, Similarity of chromatin from different ti Biol., 76 (1973) 119-128. 52 R.J. Clark and G. Felsenfeld, Structure of chromatin, Nature NEWk-i] (1971) 101-106.

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