A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 6, 1988
D or ot a Wil ma ńs ka, K ry s ty n a Śl ask a- Ki ss , M ar e k G ni a zd ow sk i
4 ’, 6- D IA M ID IN E- 2- PHE NY LI ND OL E (DAPI) P RE F ER E N T I AL L Y INH IBI TS PO LY A SY N TH E SI S CA T AL Y S E D
BY B A C TE R I A L RN A PO LY MER AS E*
4’,6-Diamidine-2-phenylindole (DAPI), a dye interacting preferen-tially with dA : dT sequences of DNA and showing high affinity to the hybrid poly rA : poly dT exhibits a stronger inhibitory effect on poly A than on RNA synthesis in vitro catalysed by Escherichia coli DNA-de- pendent RNA polymerase.
4 ’, 6- D ia m id in e- 2- phe ny li nd ol e (DAPI) forms str on g c om p le xe s w it h D NA and in hib it s D NA - d e p e n de n t D NA and R NA s y nt he si s in v i -tro [2, 10-12, 14]. E xp e r i m e nt a l d ata have led to d if f er e nt m o -del s c o n c er n i n g the s tr u ct u re of the complex. In te r ca l at i ve [2, 3, 10-12] or g ro ove b i nd i n g of the d ru g to D NA [9] has bee n p o -st ulated. N eve r th e le s s, h ig h p r e f e r en c e to dA : dT se qu en ce s of D NA is g e n e r a l l y a ck no wl edg ed . S om e d at a i nd ic at e th at the d rug e xh i b it s h ig h a f f i n it y to the h y b ri d p o ly rA : p o ly d T [2]. The h ig h i n h i b i to r y e f fe c t of DAPI o n e n z y ma t ic r ea c ti o ns in vo lv in g this type of h y b r id has b e en p r e d i c t e d b y M i l dn e r et al. [13]. E s c h e r i c h ia c oli D N A d e p e n d e nt R N A p o ly m e r a se (r i bo n uc l e o s i de t r i -p h o s -p ha t e : R NA n uc l eo t id y l t ra n s f e r a se , E.C. 2.7.7.6) in cu ba te d w it h ATP s y nt h es i ze s p ol y A on shor t o l i g o t h y mi d y l i c se que nc es of DN A [4, 5] Hence, the r e a ct i on p r o vi d es an i nt e r e s ti n g m od e l to a s sa y the d ru g s pe cif icit y.
This work was supported by the Polish Academy of Sciences within the project 09.7.
MATERIALS AND METHODS
DA PI w as s y n t h e s i z e d b y Dr J. K ap uś ci ńs ki . T he o th er m a t e r ia l s an d E. co li R N A p o l y m e r a s e w e r e t he sa me as p r e v i o u s l y d e s c r ib e d [6, 8], R e a c t i o n m ix t u r e s as s pe c i f i e d [6] c o n t a i n e d e it h e r [14 C] A TP as a so le s u b s t r at e (poly A s ynt hesi s) or [14C ] AT P w i t h the ot he r t h re e u n l a b e l l e d n u c l e o s i d e t r i p h o s p h at e s (RNA s ynth esis ),
2 +
KC l at the c o n c e n t r a t i o n ind ic ate d, 2 m M Mn , 8 yg of na t iv e calf t hy m us D N A a nd a bo u t 1 Bu r ge s s' u ni t of the e n zy m e per a s sa y v o -lume (0.25 ml) w e r e used. T he o th e r a ss a y c o n d i ti o n s we r e as d e s c r i b e d be f o r e [6]. P o l y n u c l e o t i d e s y nt h e si s in the p r es e n c e of i n h i b i t or s w as a s s a ye d at t wo or th re e d i f f e r e n t d ru g c o n c e n t r a -ti on s an d e x p r e s s e d in p e rc e n t of the c o r r e s p o n d i n g c o nt r o l s (i.e as s a y s w i t h o u t an inh ibi tor ). D r u g c o n c e n t r a t i o n s d e c r e a s i n g the s y n t he s i s to 50% w e r e r ead f r om i n hi b i t i o n curves.
RESULTS AND DISCUSSION
R e l a t i v e ra te s of R N A a nd p o l y A s yn t h e se s an d i n h i b i to r y e f -fe ct s of D N A - i n t e r a c t i n g l i ga n ds d e p e n d o n the ion ic s t r e n g t h and th e a c ti v a t o r u s e d [15]. In th e p r e s e n t e d e x pe r i m e n t s the e ff e ct of D API is a s s a y e d in th e p r e s e n c e of n at i ve DN A and M n ^ + . Th e y i el d s of the t wo t yp es of t he f o rm e d p r o d u c t ar e c o m p a r a b l e u n -d er t h e s e c o n -d i t i o n s [15]. C o n c e n t r a t i o n of DA PI i n h i b i t i n g het e- jropolymer s y n t he s i s to 50% d e c r e a s e s f rom 35.4 yM ih th e ab se n ce of K Cl to 13.6 y M at 0.15 M K Cl it d oe s n ot p r a c t i c a l l y v a r y for the p o l y A s y n t he s is (Tab. 1). C o n c e n t r a t i o n s of th e d r u g d ec re -T a b l e 1 Concentration of DAPI ( yM) decreasing RNA and poly A synthesis to 50%
Stężenie DAPI ( y M) hamujące syntezę RNA i poly A do 50%
KCl (M) RNA Poly A
0.00 34.5 ± 8.8 (4) 5.5 ± 1.3 (3)
0.05 30.0 ± 8.5 (4) 6.6 t 1.8 (5)
0.15 13.6 ± 1.4 (4) 5.3 ± 1.5 (4)
N o t e : The averages of three-five independent experiments (as indicated in the parantheses) + standard deviations are shown.
as i ng R NA and the h om o p o l ym e r f or m at i on d if f er b y fact or of 6.5, 4.5 a nd 2.5 at 0.0, 0.05 an d 0.15 M KC1, re sp ect ivel y. Ot h er li -gan ds (e th id iu m br omide, a crif lavi n, d i s t a m y c i n A a nd netropsin) s ho w e it he r no d i f f e r e n ce s or lower d if f ere nc es . At m os t twice lower c o n c en t r a t i o n of d i s t a m y c i n has b een n ee d e d for p ol y A s y n -th esi s t ha n R NA s y nt h es i s to d e c r ea s e the ir rate to 50% [15]. S i m i l a r l y the h ig h er i nh i b it o r y e ff e ct of DAPI on p ol y A s y n t h e -sis w as fo un d u si n g d e n a t u r e d D NA as a t em pl a te (not shown).
As sh o wn by C h a m b e r l i n an d B e r g [4, 5] p o ly A c ha in s are s y n t h e si z e d w i t h AT P by r e pe t it i ve t r a n s c r i p -ti on of o l i g o t h y m i d y l i c s eq u en c es in D N A in the a b se n ce of oth er n uc l e os i d e tr ip ho sp h at e s. rA : d T st ru c tu r es s ho u ld be t r a ns i en t ly f or me d t he n a nd the c ou r se of the r e a ct i on is d e p e n de n t o n their f or m a ti o n a nd s e p a r a ti o n a l l ow i ng a reu se of the t em pl at e f r a g -m en t by the e nz ym e a nd on the e l o n g a t i o n of the s y n th e si z ed cha ins. Th e hi g he r i n h i bi t o r y ef f ec t of DAPI on p o l y A tha n on RN A f o r m at i on m a y be du e to a p r e f e r en t i a l b i n d i n g of the d ru g to th ese rA : dT s t ru c tu r es w h i c h i nc r eas es the s ta b il i t y of the pr o d u c t - te mp l at e comp lex, he nc e d ec r e a s i n g a rate of the s y n -thesis. A l t er n at i ve l y, DAPI m a y in hi b it p o l y A s yn t he sis b y i n -t e r a c-t i o n w i -t h o li g o d A : d T s eq u en c es of D N A p r e v e nt i n g thei r use in p o l y A synth esis . Th e latter m od e of i n te r ac t io n seems to be less im p or t a n t for the ef f ec t re c or d ed here as p ol y A s y nt h e -sis o cc urs p r i m a r i l y on s in gl e s t r an d ed re gi o ns of D NA [5]. C o n -s i d e ra b le d i f f er e n c e -s in i n h i b it i o n of R N A a nd p o l y A sy nt he sis are c o n s i st e n t w i t h the p o s t u l a t e d h i gh a f fi n i t y of the d ru g to dT : rA h y b r i d s t ru c tu r es [2].
ACKNOWLEDGEMENTS
The authors are grateful to Dr B. Skoczylas for her generous gift of DAPI and the discussion of the results, to Miss M. Affel- towicz and Mrs K. Myszkowska for their skilful technical assi-stance, and to Mrs J. Dyniak for reading the manuscript. Dr K. Ślaska-Kiss present adress is Biological Research Center, Szeged, Hungary.
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Department of General Chemistry Institute of Physiology and Biochemistry Medical Academy of Łódź
Dorota Wilmańska, Krystyna Ślaska-Kiss, Marek Gniazdowski
PREFERENCYJNE HAMOWANIE SYNTEZY POLI A KATALIZOWANE BAKTERYJNĄ. POLIMERAZĄ RNA PRZEZ V.6-DIAMIDYNO-2-FENYLOINDOL (DAPI)
4’,6-Diamidyno-2-fenyloindol (DAPI), związek wykazujący preferencją w
sto-sunku do sekwencji typu dA : dT i wysokie powinowactwo do hybrydu poli rA : : poli dT hamuje w znacznym stopniu syntezą poli A niż syntezą RNA katalizowa-ną in vitro przez zależkatalizowa-ną od DiJA polimerazą RNA E. coli.
