Studies on RNA synthesis primed by damaged templates

126

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 96389

STUDIES ON RNA SYNTHESIS P R I M E D BY DAMAGED T E M P L A T F S I. DNA TEMPLATES DAMAGED BY D E O X Y R I B O N U C L E A S E T R E A T M E N T AND BY ?,-RADIATION j . P. G O D D A R D , J. J. W E I S S AND (7..~,I. W t l E E I . E R

Laboratory o] Radiation Chemistry, University o/ Newcastle upon Tyne, Newcastle upon Tyne, (Great Britain) (Received A u g u s t x t t h , lq6q)

SUMMARY

I. The template activity of native DNA in RNA synthesis in vitro has been studicd after damage by deox)~ibonuclease I, deoxyribonuclease I [ and y-radiation under different conditions. 2. I:rom the results obtained with deoxyribonuclease-danlaged templates the model proposed earlier in which damaged sites on native DNA are considered to bc capable of binding RNA polymerase, but fail to initiate synthesis of RNA, was used to calculate n, the number of nueleotide ]lairs per binding site on native DNA, for which a value of about 50o was found. This is in reasonable agreement with values obtained bv other workers using different experimental techniques. 3- 7 -irradiation of aqueous solutions of DNA under aerobic conditions was found to lead to an increased binding capacity for RNA polymerase but a lowering of its ilriming ability for RNA synthesis in vitro. 4. These observations can be explained by the radiation-produced formation of new binding sites, i.e. 2.I sites per IOO eV of radiation energy absorbed, of which however only o.6 site per IOO eV was active in the RNA synthesis. 5. DNA in aqueous systems irradiated under conditions where (rely radiationproduced hydrated electrons can react with the DNA molecule (anaerobically and in the presence of ethanol as OH scavenger) gave smaller yields and only inactive binding sites (o.I per IOO eV) were produced.

INTRODUCTION

Tile role of DNA in tile enzymatic synthesis of RNA by tile RNA polymerase enzyme is now firmly established and various aspects of this synthesis have been recently reviewed 1. Some work has been carried out on the effect of chemical agents, ultraviolet and ?,-radiation on the I)NA acting as a template 2-n. It has been shown previously that radiation affects both the overall rate of RNA synthesis and to soine extent also brings about a change in the coding, i.e. in the base colnposition of the newly synthesized RNA 10.~. There is clearly considerable interest in these processes from a biological and radiobiological point of view and it was therefore decided to carry out a more quantitative study of this process. It has been shown firstly in this l",iochim. Bi()ph),s..4cla, x99 (197o) le6 138

RNA SYNTHESIS ON DAMAGEDD N A TEMPLATES

127

laboratory, and confirmed by m a n y other workers, that the effect of ionizing radiation, e.g. y-rays, on DNA in aqueous systems basically consists of (a) chemical changes of the pyrimidine and purine bases, and (b) some scission of the phosphodiester backbone of the DNA TM. It was clearly desirable to study these two different types of DNA damage separately in the enzymatic RNA synthesis. This could not be done by means of radiation as it is impossible to protect selectively one or other of these sites. However, it is possible to study the effect of the scission of the phosphodiester linkages independently by using the enzymes deoxFribonuclease I and deoxyribonuclease II which bring about such scissions leading to 5'- or 3'-phosphomonoester end groups, respectively. The plan of the work to be described in this paper was to study (i) theeffect of the deoxyribonucleases on the template activity, and (ii) the effect of radiation, which also includes the damage to the constituent bases. Although a considerable amount of work has been done on deoxyribonuclease action no satisfactory information was available concerning the number of end groups produced by the action of deoxyribonucleases under certain well-defined conditions. The first task was, therefore, to devise a relatively simple and reliable method to allow an unambiguous estimation of the number of end groups present in a given DNA preparation. As mentioned above, both deoxyribonuclease I and deoxyribonuclease II were used and it was found that two different experimental methods had to be used to measure the number of end groups produced.

MATERIALS AND METHODS Calf thymus DNA, beef pancreatic deoxyribonuclease I, pancreatic trypsin and soybean trypsin inhibitor were all of the highest purity supplied by Sigma Chemical Co., London. Deoxyribonuclease II, isolated from bovine spleen, and Escherichia coli alkaline phosphatase were purchased from Worthington Chemical Co., N. J., U.S.A. RNA polymerase from Micrococcus lysodeikticus was prepared by Procedure A of NAKOMOTOet al. is. Some of this was purchased from Miles Chemical Co., Elkhart, Ind., U.S.A. Nucleoside 5'-triphosphates, (ATP, CTP, GTP and UTP) were purchased from Schwartz Bioresearch, Orangeburg, N. Y., U.S.A. ~8-'aC]Adenosine 5'-triphosphate from the Radioehemical Centre, Amersham. All the above materials were used without further purification. Irradiations were performed on a 2oo-Ci 6°Co y-ray source. The DNA was dissolved in I mM NaC1. For experiments in the presence of low concentrations of ethanol (48 raM) which were carried out to scavenge the radiation-produced OH radicals, the DNA solution and ethanol were flushed with pure N 2 before mixing and then irradiated in sealed tubes. In y-irradiation under these conditions essentially only hydrated electrons are left to attack the DNA; such DNA will be referred to as e2q-reacted DNA. For the measurements of the deoxyribonuclease I-induced hyperchromicity and phosphodiester-bond scission the enzymatically produced end groups were measured Biochim. Biophys. ,4cta, 199 (x97o) I26.-I38

I28

J.P. GODDARDel al.

by means of a recording pH stat (Radiometer type ABU 16) 14. 2oo/~g of deoxyribonutlease I, dissolved in 0.2 M NaC1, were added to 15o ml of o.~ ?o I)NA in 0.2 M NaCl, 7 mM MgSO 4, which had previously been neutralised to pH 7.0. As degradation proceeded, carbonate free o.o2 M NaOH was added by o.5-ml auto burette to hold the pH constan~ at 7.0, ~he volume added being recorded ~() an accuracy of o.ooi ml. The solution was kept free of C() a l)y a c(mstant passage of N 2. The at)sorbance at 259 nm was measured by taking aliquots (2.o nil) of the solution at intervals, and adding inlmediately to a known volume of ED'FA to terminate tile deoxyribonuclease I action, l:rom the absorbance measurements the percentage hyperchronlicity was calculated on the basis of a 38°..0 increase in absorbance (at 259 nm) to be equivalent to IOO0o hyperchromicity. For the subsequent DNA priming experiments the EI)'I'A and deoxyribonuclease I had to be removed. This was accomplished by incubation with trypsin after quenching with EDTA at known times, followed by addition of trypsin inhibitor. Zn 2+ instead of Mg2¢ was used as the activating metal ion for deoxyribonuclease I and excess EDTA was neutralized with additional Zn °''-. Since the Zn 2 ¢- E D T A complex is more stable than that with Mn '~¢-, no interference was caused in the subsequent priming experiments in which Mn"- was used. Control experiments (without deoxyribonuclease I) showed that such a treatment did not affect the priming activity. An aliquot of the original deoxyribonuclease I incubation mixture was placed in a spectrophotometer and its absorbance followed with time (l;ig. I). In this way a functional relationship could be established between the absorbance (hytx'rchromicity) and the progressing deoxvribonuclease action. For the deoxyribonuclease I l action, DNA was dissolved to a concentration of I mg/ml and an equal w)lume of 0.2 M acetate buffer (pH 4.6) was added. I6O/2 g of deoxyribonuclease II, dissolved in the same acetate buffer, were added to 25 ml of the DNA solution and its action terminated by raising the pH to 7.8 by addition of Tris base. As a control a DNA solution containing deoxyribonuclease II at pH 7.8 was used. Aliquots (2.0 ml) were removed from tile experimental and control samples for absorbance measurements at 26o nm using 2-rain cells. The absorbance of the DNA-deoxyribonuclease II system remained constant at pH 7.8 for at least i day. Absorbarite measurements were made about ~o times for each sample and percentage hyi)erchronficity and its standard deviation calculated. The remaining (25 lnl) solution was used to estimate the degree of phosphodiester-bond scission in the deoxyribonuclease I I-treated DNA by measuring the total I)1 released by (enzymatic) hydrolysis of the newly-formed phospholnonoester groups. l;or this purpose, the deoxyribonuclease II-treated DNA sample was denatured by alkali to increase accessibility of the phosphomonoester groups for hydrolysis and glycine was added to plt 8.9. 200/~g of E. coli alkaline phosphatase in 0.2 M glycineNaOH (pH 8.9) were added and the mixed solution incubated in polythene bottles at 37 °. After 24, 36 and 48 h incubation, io-ml aliquots were removed and adjusted to pH i.o by addition of 25 °,o HC104. The supernate was analysed for I)t by the method of MARTINAND DOTY16, slightly modified with respect to sample and solvent volumes to afford ep(67o nm) = 18 97 ozl: 1565 for the. final blue solution. Each analysis was performed using untreated DNA, taken through an identical procedure, as blank. The mean result of three phosphate analyses and their standard deviation was calculated. Biochim. Biophys. Acta, x99 (x97o) i26--138

RNA SYNTHESIS ON DAMAGED D N A TEMPLATES

12 9

For priming experiments using deoxyribonuclease II-treated DNA as template the samples quenched at known times by raising the pH to 7.8, could be used directly in the incubation mixture. Again the absorbance of an aliquot was measured versus time, (Fig. I). Unless otherwise stated in the legends, RNA synthesis was carried out in incubation mixtures of total volume of o.25 ml containing o.625/tmole Mn('la; 37-5/,moles Tris, p H 7.5; o.25 Fmole of UTP, CTP and GTP; o.o 7 #mole ATP of known specific radioactivity, RNA polymerase and the primer being tested. Incubation, washing and assay were performed as previously describedX°.aL For a study of some aspects of inhibition kinetics, a fixed small amount of more highly degraded DNA was added for each run to variable amounts of native DNA. Actual degrees of degradation for the deoxyribonucleases are shown in Table III. Strict precautions were taken to ensure consistency within each set of experiments and the enzyme activity of each reaction mixture was assayed as follows. A standard reaction mixture (o.25 nal) containing ioo t~g of DNA and the same amount of enzyme as that used in the concomitant experiment, was incubated at 3o ° for 2o rain. One unit of enzyme was arbitrarily defined as that which catalysed incorporation of labelled AMP into acid-insoluble RNA, to a concentration of I/tmole AMP per 1 of the above reaction mixture, after 2o min incubation at 3 o~'. One unit thus defined corresponds to approx, o.5 units defined by NAKOMOTO et al. ~3.

RESULTS AND DISCUSSION

(z) Chain scission by the action o/deoxvribonuclease I and I I The effect of the action of the deoxyribonuclease I and II on the absorbance of DNA solutions is shown in Fig. I. The relation between the concomitant increase in absorbance (hyperchromicity) and the number of phosphomonoester end groups z,O 1700

168O

1660

j

J

/

Z 2o >~

E = c~ "~ 164(, 10

"~620

{

2'o =b 6~ 8~ . 4 o - ~ ]rrne ( m ~ ]

~o

~o

a5

"~o

1.~,

2~o

2t5

3%

B o n d S c i s s l o n (°1o)

Fig. i. ]{elation b e t w e e n t i m e of d e o x y r i b o n u c l e a s e action a n d increase of a b s o r b a n c e (hyperc h r o m i c i t y ) of D N A . O , d e o x y r i b o n u c l e a s e 1; O , d e o x y r i b o n u c l e a s e l I. (Limits of error s h o w n indicate a p p r o x i m a t e a c c u r a c y o | t h e i n s t r u m e n t . ) Fig. 2. ]{elation b e t w e e n p h o s p h o d i e s t e r - b o n d scission a n d h y p e r c h r o m i c i t y after action of deoxyribonuclease 1 ( O ) a n d d e o x y r i b o n u c l e a s e 11 ( 1 ) on D N A .

Riochim. lliophys..4eta, I99 (I97O) 126-138

13o

j . P . GODDARD et al.

is shown in Fig. 2. This gives the relationship that scission of i °/o of phosphodiester bonds corresponds to a lO.5 °o increase in hyperchromicity which holds for both deoxyribonuclease I and II. This thus allows one to correlate directly, on a purely experimental basis, the hyperchroinicity with the number of end groups present. (2) Kinetics o / R N A synthesis The D N A - R N A polymerase-triphosphate system is rather complex and it is not at all clear a priori to what extent classical Michaelis-Menten kinetics can be applied in this casd6-18.The polymerase is bound quite strongly on sites of the DNA molecule. It has been shown by several workers that these sites are pyrimidinc-rich clusters on the DNA ag-'~, the base in the first nucleotide incorporated being predominantly a purine 22m. Let us start by considering the binding of the enzyme E (polymerase) to the DNA template D according to; E-..I) ~

t.2D

(i)

If one attempts to apply Michaelis-Menten kinetics to this, it must be borne in mind that the relevant units here are the binding sites provided by the template and not DNA itself or its constituent nucleotides (which differ from the former only by a factor, i.e. the average number of nucleotides per DNA molecules) ~6. The relation between the concentration [D] of DNA in nucleotide base pairs and the concentration of polymerase binding sites B ] is given by: !/~] = iD]In

(2)

where n is the number of nucleotide base pairs per binding site, so that Eqn. I should be written as: E-t-

J3 ~ / - : ~

(3)

P.'B

(triphosphates) Mna÷ >- p r o d u c t s

and, (3a)

Deriving the expression for tile velocity v one cannot neglect here the concentration of the complex compared with the concentration of the binding sites and omitting the quadratic term one obtains: V t~ i

v - :¢,, ~ [t;i--: [B!

(4)

where V denotes the maxinmm velocity. For strong binding of the enzyme, K m will be very small so that in this case: K= << LE

(5)

and K m can be neglected compared with iE], which is the reverse of the conditions prevailing under usual circumstances. For the reciprocal relation (LINEWEAVERBORK~ plot), rearranging Eqn. 4 and with Eqn. 2 one obtains the expression: 1

t

I F.]n

~, ~ p :,- ..~.

l

x :B f

(6)

where the intercept on the x-axis is now: ~E)z = ~

Biochim. Biophys..4cta, 199 (197 o) I 2 6 - I 3 8

(7)

RNA SYNTHESIS ON DAMAGED DNA TEMPLATES

131

which replaces K m in the conventional Michaelis-Menten equation. This, as will be shown below, is in agreement with the experimental results. V is given in general by: v

=

ki1-!v

(8)

k being a second order rate constant, and r is the fraction of the total number of binding sites which are active in the RNA synthesis. Separate experiments showed that the ratio of the rates of total RNA synthesis primed by treated DNA's to native DNA remained unchanged during the incubation period, (20 rain) so that the synthesis after 20 min can be regarded as the actual rate of RNA synthesis. Native D N A The slope of the LI,XEWEAVER-BURK24 plots should be independent of iEi, since: (Vn = m a x i m u m velocity for native DNA) I I !L'!~. 1 v = V~ ~- Vn !D-I

(9)

Substituting for Vn from Eqn. 8 one obtains: n slope -- -k- - v -

(io)

Unlike the case of some phage DNA, e.g. T 4 phage 4°, for DNA of calf thymus, the proportion of active to inactive binding sites is not known. The following discussion is based on the simple assumption that the number of inactive sites is negligible, i.e. v = I in Eqn. 8. Thus the slope (Eqn. IO) would become (n/k). If on the other hand v ~.C I, tile values for n will be increased by a factor (I/V) and the yields in the radiolysis of DNA would be correspondingly decreased by the factor v. The independence of this slope on enzyme concentration has been shown by MARUSHIGE AND BONNER 25, and confirmed in this laboratory (J. P. GODDARD, J . J . W E I S S AND C. M. W H E E L E R , unpublished results). It should be emphasized that the experiments reported here refer only to DNA's such as calf thymus DNA. In m a n y cases transcription of bacteriophage DNA's is asymmetric 2e-al or can be changed to symmetric by various treatments 27.s°. For tile DNA's of higher organisms the experimental results show no evidence of asymmetric transcription. KARKAS AND CHARGAFF 32 showed that denaturation by heat, acid and alkali, and also sonic degradation caused no change in the composition of the synthesized RNA compared to that from untreated DNA. Similarly, it has been observed that rat-liver chromatin shows changes in base composition of synthesized RNA after removal of the protein 25, and this has been ascribed to an alteration from asymmetric to symmetric synthesis. Thus it would appear that bacteriophage DNA is partly under positive control for RNA synthesis ~ (e.g. the recently discovered a factor) a whereas mammalian DNA is under negative control by histones 35. Consequently, the type of kinetics derived above cannot be applied to bacteriophage DNA systems. Moreover, as such experiments have been carried out with excess of enzyme, inactive binding sites would escape notice since these would simply bind some of tile excess enzyme. Biochim. Biophys. Acta, 199 (I97 o) 126-t38

I32

j.P. GODDARDet al.

Deoxvribonuclease-treated D N A The results shown in Fig. 3 indicate that V and ~v decrease in the same proportion with increasing degrees of chain scission of the template. LIXEWEAVER-Bt, RK24 plots were found to be parallel.

C

.-

0

BoeO 0.4

02

1

£ •o (;'

2 0 2'

'

4

3

o.4 '..._._L-~

scission (°;o3 0

7-

Co

02

1

E "",~ 4OC 0

2

0.4

3

02

a 04

20O 0 4 O 1 r4yDerchrom c:t y ~°,.)

1

2

3

4

l:ig. 3. Chanl~e in D N A t e m p l a t e p r o p e r t i e s b y d e o x y r i b o n u c l e a s e t r e a t m e n t , a. l l e c i p r o c a l of m a x i m u l n v e l o c i t y (Ve - l ) a s a f u n c t i o n of i n c r e a s i n g d e o x y r i b o n u c l e a s e 1 t r e a t m e n t , b. ~,, ~ as a f u n c t i o n of i n c r e a s i n g d e o x y r i b o n u c h ~ a s e I t r e a t m e n t , c. Ve -~ as a f u n c t i o n of i n c r e a s i n g d c o x y r i b o n u c l e a s c 11 t r e a t m e n t , d. g,e -~ as a f u n c t i o n of i n c r e a s i n g d e o x y r i b o n u c l e a s e I I t r e a t m e n t . C o n d i t i o n s as d e s c r i b e d in t h e t e x t , 6 u n i t s of l e N A p o l y m e r a s e p e r 0.2 5 m l r e a c t i o n m i x t u r e for a a n d b a n d 6. 7 u n i t s fnr c ~.tnd (t. l l y p e r c h r o m i c i t y a n d b o n d s c i s s i o n a r e r e l a t e d b y t h e d a t a g i v e n ill l"ig. 2.

Thus if a corresponds to the number of binding sites originally present and x to the number of new binding sites produced by deoxyribonuclease action and assuming that all these new sites are inactive, the fraction of active sites present is: a

v'

=

a-t- x

fill

The maxinmm velocity for the deoxyribonuclease-treated templates (Ve) then is: Vo = kF':;v"

(~')

r" e Vn =

(13)

and V'

At the same time the number of nucleotide base pairs per binding site will be decreased from n to n ( a / ( a - ' x ) ) so that from Eqn. 7 one obtains: (~oe denotes the ~o value for deoxyribonuelease-treated templates)

Therefore, I,% -

~0,.

V, =

t/'n

05/

i.e. the sh)pes of the LIYEWEAVER-BURK24 plots should be parallel. This is in agreement with tile experimental findings and thus confirms that the new sites produced by deoxyribonuclease action are in fact inactive sites. l¢iochim. Biophys. ,4eta, I 9 9 (197 ° ) I 2 6 - t 3 8

RNA

S Y N T H E S I S ON D A M A G E D D N A

TEMPLATES

133

If the reciprocals of Ve and ~?e for the deoxyribonuclease-treated templates are plotted vs. % hyperchromicity, i.e. the number of phosphomonoester end groups produced, and x which stands for the inactive binding sites is identified with the number of such end groups, by plotting the maximum velocities and ~ values vs. x, (Figs. 3a-3d) according to the equations: I

I

Ve

~

~n

I

I

I --

X

-~- V n ' ~ I L

(16)

)." • -

(17)

one can derive values for a, which is simply the reciprocal of n, viz." a

=

I n

(IS)

From the experiments on native DNA one obtains values of n = 41o and 500 nucleotide base pairs per active binding site. This would correspond to 24 and 20 active sites respectively, for a DNA molecule of mean molecule weight 6. IOe daltons. l'or deoxyribonuclease [ the plots are linear (Figs. 3a and 3 b) while for deoxyribonuclease II this is generally not the case (Figs. 3c and 3d). The most likely source for this deviation is that some RNA-release mechanism is operative which leads to an increase in the rates. This could be due to very small amounts of ribonuclease contamination of the deoxyribonuclease II (ref. 36) since the former is known to cause an increase in the rate of RNA synthesis 37. ,/-irradiated D N A

LINEWEAVER-BURK21 plots from DNA templates after 7-irradiation under aerobic conditions are also linear but not parallel to that for native DNA. This, as is shown below, leads to the conclusion that `/-irradiation also produces some active sites for the RNA polymerase in addition to inactive ones. TABI.E

I

~e VALUES AND MAXIMUM VELOCITIES (Ve) FOR DEOXYRIBONUCLEASE-TREATED ]).~N'A TE.XlPI.ATES FOR CONSTANT POLYMERASE CONCENTRATION

Phosphodtesterbond scission ( % )

~e

Ve

(ktM)

(ttM/2o rain)

32.i 27. 3 27. 3 10. 7 I4.6 I1. 3 8. 7

IO.O 8.8 8.8 5.,5 4.8 3.8 2.8

31.8 18.2 r 1.5 6. 4 5.o 4.4

IO.O 6.5 4'5 2.9 2.3 1. 7

l)eoxyribonuclease 1 o o.o15 o.o39 0.068 o.139 o.231 0.302

Deoxyribonuclease II o 0.027 0.053 o. 145

0.273 0"420

l~iochim. Biophys..4cta, 199 ( I 9 7 ° ) t 2 6 - 1 3 8

134

j.P.

GOI)DARI) et al.

T A B I . E ii l~r V A L U E S A N D M A X I M U M MERASE CONCENTRATION

VELOCITIES

Dose (krads)

(Vr)

FOR IRRADIATED

DNA

TEMPLATES

FOR CONSTANT

~r

V,.

(I.*.T1)

(laM,i2o mi~z)

3r.8 I4.6 8.7

POLY-

Aerobic

(}.22

7"7

6.65 8.to 1 r..~o

3.4 2.5 2.7

io 7.3 5.7 5 .8 4"5 4"3 4 "o 3 .0 3.6 -.I

i1.88

i .2

1.4

o

o.{}7 r .95 2.70 4.38 5.22

l l "7

9 .0 6-3

Anaerobic (e~q-reacted} 31.i 33.9 30.9 I9.3 20.8 IO. 4 9.8 5.7

o

4.I 9.0 ,2.0 4.5.2 67.7 9o.u

I3Z.o

If by y-irradiation

I{}.O 9.4 I0.0 5.o 6. 3 2. 7 24 t.5

the total

number

of binding

s i t e s is i n c r e a s e d

from a to

( a + x ) t h e a v e r a g e n u m b e r of n u c l e o t i d e b a s e p a i r s p e r b i n d i n g s i t e will b e d e c r e a s e d f r o m n t o n ( a / ( a + x ) ) . If a l l t h e n e w b i n d i n g s i t e s w e r e i n a c t i v e , t h e s l o p e o f t h e r e c i procal plots would remain

constant

From the values of the maxinmm

5.0

,[' ~2.

lO0

7~

5

.

~

~00

, o

o

•

~"

~

for irradiated

action. templates

±

500

:5

a s in t i l e c a s e o f t h e d e o x y r i b o n u c l e a s e

v e l o c i t i e s a n d ~v's d e n o t e d

°

0

5

,

A

'0

~5

20.

iO

Dose(kroJ}

Fig. 4. C h a n g e in D N A t e m p l a t e properties by" 7-irradiation. a. I'rlp'n/Vn~r as a f u n c t i o n of r a d i a t i o n close (R) {under aerobic conditions), b. Vr * as a f u n c t i o n of r a d i a t i o n close (R) (under aerobic conditions). T h e results, o b t a i n e d from t h r e e i n d e p e n d e n t e s t i m a t i o n s , were corrected for difference in reaction m i x t u r e e n z y m e c o n c e n t r a t i o n by s t a n d a r d i z a t i o u to io u n i t s R N A p o l y m e r a s e . 3 , I4.1 uIaits R.N'A polymera-se; A, I6.9 u n i t s R N A polymera-se; 0.25 ~ m o l e A'I'I', (Yl'l } a n d U T P ; 62 nnloles rail 1CTP (5"3 " I o s c o u n t s / r a i n per/~mole); arid G , 10.8 u n i t s R N A p o l y m e r a s e , c. Vr -t as a f u n c t i o n of r a d i a t i o n dose (R) u n d e r a n a e r o b i c c o n d i t i o n s (e--reacted). d. Vr -1 as a f u n c t i o n of r a d i a t i o n close (h') u n d e r a n a e r o b i c c o n d i t i o n s (e---reacted). R e a c t i o n m i x t u r e s c o n t a i n e d 8 units RNA polymerase.

Biochim. Biophys. Acta, 199 (t97 o) IzO-- I38

RNA

S Y N T H E S I S ON I)AMAGEI) D N A

TEMPLATES

135

by Vr and ~r given in Table I I and from Figs. 4 a and 4 b it is evident that the slopes decrease as Vr decreases. This would be the case if the factor by which Vr decreases is smaller than that for the corresponding V/r values (at constant enzyme concentrations). If the number of radiation-produced active sites is denoted by a~, then Vr = klE~ a.+a, a+Xr

(I9)

where Xr corresponds to the total number of new binding sites produced by the radiation, I ~Pr = [ E ; - - - - a-T Xr

(20)

and the slope is given by the ratio: ~r Wr

I h(aTal)

(2x)

which decreases with increasing radiation-produced active sites, a x. All radiation-produced sites m a y be assumed to be proportional to the radiation dose R: Xr = g R

(22)

a t -- gl R

(23)

where the proportionality factors g and g~ are related to the radiation yields, e.g. the number of (chemical) events produced by the absorption of IOO eV of radiation (the so called G values). Thus: 2

=

i

~r

~n

+ g

n

(24)

~na

In combination with the corresponding ratios of the maximum velocities one obtains for the radiation-produced active sites: Vr .~ tpn

--

Vn

,

gtl~

...... ~r a

~- i

(25)

Thus if n (the number of nucleotide base pairs per active site for native DNA) has previously been determined the yields (G values) for the radiation-induced production of total and active binding sites can be calculated from plots such as given in Fig. 4. These experiments give a value of 2.1 total sites per ioo eV out of which 0.6 site is active so that, in fact, the fraction of (1.5/2.1) = o . 7 is inactive in RNA synthesis.

Action o/ inhibitors Tile above considerations are also supported by the action of suitable inhibitors. The simplest type of inhibitor would clearly be a substrate which binds tile polymerase enzyme to form an inactive site but with a very similar binding constant. Such a substrate would be provided by DNA highly degraded by deoxyribonuclease action or by irradiation because although this would bind the enzyme in a similar way, the enzyme bound on such a degraded DNA would not lead to RNA s3mthesis. Biochim. Biophys. Acta, I 9 9 ( I 9 7 o) 1 2 6 - 1 3 8

J . P . GODDARD el al.

I36

If one were to a d d to the s y s t e m an a m o u n t of i n h i b i t o r of d e g r a d e d D N A corresp o n d i n g to a c o n c e n t r a t i o n of 2I! nucleotide base pairs one o b t a i n s for the ~vt value in t h e presence of the i n h i b i t o r an expression of the s a m e form as in Michaelis-Menten kinetics: /ill

=

~ n ( I !-

rt;iw,)

(26)

where lk,i denotes the W value for tim i n h i b i t o r itself. F o r d e n a t u r e d D N A a c t i n g as inhibitor: W, -

(27)

J'i~

where n is the n u m b e r of nucleotide base pairs per binding sites of the inhibitor. I n t r o d u c i n g this into Eqn. 26 one obtains:

{sin (~,~-~,) . . . .

(28)

where J J / m is the c o n c e n t r a t i o n of the binding site of the inhibitor. Since i l i and m are known the n for n a t i v e D N A can again be calculated. A small correction is necessary owing to the presence of active n a t u r a l sites on the a d d e d inhibitor. This was achieved either by successive a p p r o x i m a t i o n , or b y using a value of n d e t e r m i n e d from the e x p e r i m e n t s involving Va and V values and observing if consistent results were o b t a i n e d . l"or i r r a d i a t e d DNA, Eqn. 26 becomes: g'i

~n "- 1£ g R n

(29)

again enabling G values of t o t a l site f o r m a t i o n to be calculated. In these e x p e r i m e n t s small fixed a m o u n t s of D N A , after a s o m e w h a t e x t e n d e d t r e a t m e n t with deoxyribonuclease, or after r e l a t i v e l y high doses of radiation, were a d d e d to n a t i v e D N A and the ~o a n d V values were d e t e r m i n e d (at c o n s t a n t e n z y m e c o n c e n t r a t i o n ) . The V values in the presence of i n h i b i t o r are, of course, the same as in its absence. The V' values of d e o x y r i b o n u c l e a s e - p r o d u c e d inhibitor are given in T a b l e l I I and in Table IX" for r a d i a t i o n - t r e a t e d D N A a c t i n g as inhibitor. I n c l u d e d in ] ' a b l e I I I are the values of n for n a t i v e D N A , c a l c u l a t e d from the inhibitor experiment, and in Table IX' the c a l c u l a t e d G values are given. The a g r e e m e n t between TABLE

Ill

t~Ol VALUES FOR ( N A T I V E ) D N A "FE.X,I P L A T E S INIIII:HTE;I) BY" ])EOXYRII~ONIJCLEASE-TRFATI,;D D N A l.-oR CONSTANT POLYMERASE CONCENTRATION

Phosphodiestcvbond scission

Treated D.Y'.'I (t~g per tube)

~ot

(,uM)

Number o/ nuch'otide base pairs per actiw' site (n) (calc.)

o.~ 0.2 0. 5

60.5 6o.5 57. t

455 50o ~,25

0.96

o.I

o.5o

0.2

o.26

0. 5

60.4 54.5 5°.0

455 417 435

(%)

l)eoxyvibonuclease I 0.87 o.69 0.29

Dcoxyribonuclease I I

ltiochim. Itiophys..4eta,

199 (x97 o) I 2 6 t 3 8

RNA SYgTHESIS ON DAMAGED DNA TEMPLATES TABLE

137

IV

lpl VAI-UI~]S A N D R A D I A T I O N Y I E L D S ((~ V A L U E S ) FOR D N A D N A FOR C O N S T A N T P O L Y M E R A S E C O N C E N T R A T I O N

Dose (krads )

TEMPLATES

INHIBITED

Irradiated D N A (#g/tube)

tpt (t~.~l )

G (talc.)

2.0 5.o 10.O

,'~o. 3 I53.o 244.0

2. 3 2. 3 2.0

BY ~-IRRADIATED

.4 erobic 2.0 2.0 2.0

.q naerobic (eaq-reacted) I8S

0. 4

05.8

0.09

97.3

I .O

80.0

O. I O

97.3

2.o

128.8

o.o8

the sets of n and of the G values are very satisfactory. Values of n could also be obtained from deoxyribonuclease II-treated material since only very small amounts of this were used, these values agreed with those obtained from the deoxyribonuclease I treated inhibitor. Conclusions These results indicate that binding sites are spaced on average about 500 pairs apart. This agrees well with estimates made for other DNA's by several workers using different techniques, e.g. 55o-11oo (ref. 38) and 700 for T 7 DNA, 550-850 for polyoma DNA, 650-750 for papilloma DNA, and 35o-7o0 for ). DNA sg. Since denatured DNA can initiate active synthesis of RNA, although at a reduced ratO °, the reason for the lack of synthesis when RNA polymerase is attached to a site formed by deoxyribonuclease I action, must reside in the 3'-OH or 5'- phosphate ends formed by main chain scission. Deoxyribonuclease-induced binding sites being inactive, RNA synthesis takes place almost completely on double-strand DNA, since the degree of denaturation caused by deoxyribonuclease treatment is very small. Hybridisation experiments with replicative form q)XI74 DNA as a template have shown that no new RNA is formed from DNA containing single-strand breaks 41, which would imply that if any polymerase is attached to these sites, it is inactive. Shearing of q)XI74 DNA changes its mode from asymmetric to symmetric synthesis, indicating that more of the DNA is available for RNA synthesis 2v. This, however, does not necessarily imply that sites of shear are themselves active; it may be that here changes in conformation of the DNA allow more sites to bind the enzyme. Printing by T 4 DNA, after sonication under conditions of one enzyme molecule to 5oo-iooo nucleotide pairs, leads to a lower rate of synthesis of RNA but to no new initiation sites 4°. This again could be interpreted as being due to binding of RNA polymerase molecules which are inactive for initiation and synthesis. For DNA y-irradiated aerobically, the radiation yields (G values) of total site formation are approximately equal to the known total attack on DNA under these conditions. The active site formation corresponding to a G value of about 0.6 site/ IOO eV presumably arises from some type of base damage since chain scission does not apparently lead to active sites, although it is not possible to say exactly what type of damage is responsible. For DNA attacked by hydrated electrons the low G value of site formation corresponds to a low G value of base damage 42, although again the specific site of attack is not known. Biochim. Biophys. Acta, I 9 9

(197 ° ) I26-138

138

j.P. GODDARD et al.

A('KNOWLEDGEMENTS

We should like to express our gratitude to the Nuffield Foundation for financial support for this work. One of us (J.P.G.) held a Science Research Council Studentship during the course of this work.

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Biochiln. Biophys. Acta, 199 ( i 9 7 ° ) 1 2 6 - 1 3 8