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A probable mechanism for apurinic acid inhibition of DNA synthesis
: '!-'{,~olutn~ 4-I; number
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:
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~
...-
.
..
:
May
FEBS LETTERS
2
-
.
-..-~
1974
.
and
The University of Texax, Graduate School o f Biomedical 5¢tence~ at Houston. Houston, Texas 77025. USA Received 25 Februar~ 19"/4
1. Introduction
H e i k e ' s [1] o b s e r v a t i o n t h a t a p u r i n i c a c i d ( A P A ) inhibits D N A s y n t h e s i s b o t h in vivo a n d in v i t r o was investigated a n d c o n f i r m e d b y Mfiller et al. [2] w h o found that the inhibition of DNA polymerase from L 5 1 7 8 Y ( m o u s e l y m p h o m a ) cells w a s n o n - c o m p e t i tive w i t h r e s p e c t t o D N A t e m p l a t e c o n c e n t r a t i o n . This g r o u p also r e p o r t e d t h a t the m o s t e f f e c t i v e f r a c t i o n o f A P A o n a "molar basis h a d an average m o l e c u l a r weigh~ o f 19 5 0 0 w h i l e o n a w e i g h t - t o - v o l u m e basis a fractior of ~LW. 2 8 8 0 e x e r t e d t h e strongest i n h i b i t i o n . F u r t h e r , an APA with~ e d u c e d aldehyde g r o u p s w a s m u c h less effective a n d ~he nucleotide, n u c l e o s i d e a n d nucleobase d e c o m [ ~ ; i o n p r o d u c t s h a d n o i n h i b i t o r y effect. T h e ~ b s e r , ' a t i o n s suggested t o us t h a t A P A m i g h t
fully a n n e a l a s a p r i m e r . H o w e v e r , ahno~t all o f t h e t e r m i n a l 3':p6~iti0ns a r e o ~ ' u p l e d b y p h o s p h a t e o r p h o s p h a t e - - c a r b o h y d r a t e r e m n a n t ~ a n d a r e n o t available f o r a d d i t i o n o f s u b s t r a t e . I f A P A inhibition o f D N A p o l y m e r m e is the r e ~ t o f a ' b l o c k e d printer' m e c h a r d m ~ , t r e a t m e n t o f t h e APA with a pho~phatase or dlegterase or b o t h should r e d u c e o r reverse the ability t o i n h i b i t . T h e s e e x p e c t a t i o n s arc, in fact, ~ o n f i r m e d b y t h e e x p e r i m e n t ~ t o b e described
2. Methods
A p u r i n t c acid was p r e p a r e d , b y t[~e m e t h o d o f
~i~i~!!:~' ~!ii~i::!:i!~: '..:....~... ~:~. ~, . ..si : ~,.~~.~:~:~! . .,~:~:?::~ =~/~ '¸ ~:~!~i~i~¸,¸ ~ •~i~~.!@ ~ ~,~,~,~.,~, ! ~ , ~!•!i:~i~o ~~. ~,~,~ ~~~.,!~~.:~/~ ~~i~ ,~. ~!~y~!~]:~!~ ~.~,~~:~i~i .,~,~:, ,.~~~ i ~ ~i,i::~iii~ !i~:~¸::~i:~~:~i:~, i i'~:~!i!i~!~!~ii i i:i~ili!i;'~:~ !~i~ii~!i,!~~i~ ~?~ii~:!~ii~i!!~!~iii~ii!i~! ~'~iii~i~ii~~:~ii~iii i~i:~!ii~i~i:ii~iii~:~i~i~!~!ii:~!~i~i~i~i~~i~ii~:i~iii!ii~'~,~i:~!i~i~!~!i~ii: ~i~ii!~iii,~!ii~!~iiii~iiiii,~!iii~i~i~ii~!iiiii~!~:i]~
slow~/.C0oled t : o ~ o m temp.ez.atu~ e t o increase file like..'. N m o d o f a n n e a l i n g t ~ e . p . ~ e r : . a n d t e m p l a t e : T h e .
' .on fiae 3 ~:pos!~0r~ B a c t e ~ a l k ~ i n e _ p h o s p h a t a s e is , " " " a c t i v e o n a .v#,de n a r i e t y o f . p h o s p h a t e i~S~ers ,nnd ~ e a t - : . : • . naen~.of .APA With ~ )en;zb~ne.. q a i t g ob~.:ously a b 0 - / - . • g s h e s A P A i n ] a l b i f i o ~ , o f D N A p b l y m e r a s e . The-c.ombi-
. xn solutionswere reXxedin , atios Of
DNA
m g t o . i:0 n~g a n d 2 5
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• . " -. - ..iafion p r o d u c e d . b y 3<0]-] e.]igodeoxynuc]eofides ~Jm " " The ,ex~n~s ~'~rO W~Ch ~ e ~ 0 U S ~ A s-oluti0n~ o n r , e ~ l i e ~ : s ~ . d y w a s ,also ~Ound : t , o . b e , s p e c l ~ ¥ p l o - : . al~e~ed a s~anda~d D N A p o l y m e r a s e ~eacfi.on are ,.given ~en:t ~ ..~_e i n i t i ' a l ;ph,nse of thc-..,syn~etic :remc.tion: - :M~llez e t al.-{2], c o m m e n t o n an alkaline n u t ] e a s e " ~ab],e I. Emsh ~ A s o l u t i o n was ~ s o t e s t e d in.an " w N e h ±h,T feel ~ a y : : b e ~espons~b]e f o r .the. ~,ecovery.. assay withon~ t e m p l a t e a n d n o ~nCorporafi~n c o u l d b e ,of.cell p r o l i f e r a t i o n a f t e r A~PA a d m i r A s f r a f i o ~ O n r , o N ,detecled h0. a n y o f t h e s e case~. T h u s , A P A o r A P A deriservations raise ~e p~s~b~$y ~aI a non-specific ~,Id~.vatives C , o ~ l d n o ~ s e r v e a s b o t h i e m p l a ~ e and prinae~. -. " .The e n z y m e u s e d in '.these. assays was p:a~.:iatly purified n,e p h o s g h a l e m i g h t also pie}" a role :in..zeversi~g ,from fiae c y t o p l a s m i c .fraction o f %¥alkex 2 5 6 c a r e i n ~ i n h i b i t i o n ~n vivo. _ . .. -: s a r c o m a .ti~su,e (rat), 'This e n z y m e is m o s t active .with "3"I~ tin,fling b y t h e M:ffm z ~ o u p h h a ~ A ~ A w ~ . n o r ~ - a D N A te~aplate wh}ch has b e e n pro'dally de~.a.ded " -compefitNe w i t h r e s p e c t ~oi~e~ers~ b y D N A is -~.'So . enzyma~icaily, b u t sigrdficaut inco,rpora~ion ~ s o o c e n r s compaiible"wl,~h , 0 u ] r ~evidenc.e t h a t A P A c~m n o t snI~ v]]fli ~enaiuxed D N A [ • • " - " . s t L ~ l e as ~emplaie.-Thus0 A ~ A w o a ] d ~nly c o m p e t e " Oa~i fi~.ding .of app;ox.~mai,ely 3 " ~ i.n]-fibi'ti,ma ,~ztien " - a r i t l a e n d o g e n o u s p ~ m e r p r o b a b l y p r e s e n t as a m~,.or t ~ c . e as n m c h A P A as D N A w a s p~esen~ agrees w e ~ cont. ,mninant ,~f fl~e e n z y m e p ~ p a r a f i . o n a n d ~he zatio- w i t h t h e observations. Of Mt~ller el a!. [ 2 ] . O a r s , ~ s u l ~ s ox -pnmex t o Senapla~c w o u l d n o r be a]*.e~e,d B y adding
it~'m~t m n a l ~ . e v e n o n ~
indicate
:dies~exase
a]on~
~s . i n e f ~ e c -
0 ~ t h e basis .of .,these consiflerations, Lhen, ~ e c0n- e l u d e tha!.a likely m e c h a n i s m ~or fl~e A]:AinIerfe~ence. The
Table ] aolnfions on DNA
.~ffest of-APA
' " poly.nae~'as~
"
p,rime~ r n o ] e c n b s w i t h c : o ~ e q u e n I b ] o e k a g o ~f~hain 'e ] o n g a t i o m O f c . o .rose0 ~ in n o Way ~ s h e a ~ e --. t~ghly significan!t observali•on b y - t h e • M a i n z g m ~ p . i h a t s u c h m o l e c u l e s are ~aken u p ~ m o ]i~Ang e~Tflsto prc~- • duce.cy~ostasis,-fo~,ifourmeehmnism•h cone=% i!. : ~o~alfl b e ~e~afiv,ely e a s y t o des~gn.a~ ALPA o r p o ] y - , .. py~-'nidlne deriv.atbie w i t h a g r o i i p o n i h e ~ e n n l n a ] . 3 L p b s i f i o n t h a t is v e ~ res~sta.u~ t o ermymat~c >cleavagd :
Amoun~ Pe~cem IahiMtion JkPA s,o]ut~on A~A
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This'~esearch Was sUppor~e:d;in.pa~t~ b y !G~-aflf :---:-:: .,:
[3] Koznbeig,
A . J(lg~39) Science 1 6 3 , 1 4 1 0 2
.
. .
-
.
.
o (:~:~.._
:
,
~
...-
.
..
:
May
FEBS LETTERS
2
-
.
-..-~
1974
.
and
The University of Texax, Graduate School o f Biomedical 5¢tence~ at Houston. Houston, Texas 77025. USA Received 25 Februar~ 19"/4
1. Introduction
H e i k e ' s [1] o b s e r v a t i o n t h a t a p u r i n i c a c i d ( A P A ) inhibits D N A s y n t h e s i s b o t h in vivo a n d in v i t r o was investigated a n d c o n f i r m e d b y Mfiller et al. [2] w h o found that the inhibition of DNA polymerase from L 5 1 7 8 Y ( m o u s e l y m p h o m a ) cells w a s n o n - c o m p e t i tive w i t h r e s p e c t t o D N A t e m p l a t e c o n c e n t r a t i o n . This g r o u p also r e p o r t e d t h a t the m o s t e f f e c t i v e f r a c t i o n o f A P A o n a "molar basis h a d an average m o l e c u l a r weigh~ o f 19 5 0 0 w h i l e o n a w e i g h t - t o - v o l u m e basis a fractior of ~LW. 2 8 8 0 e x e r t e d t h e strongest i n h i b i t i o n . F u r t h e r , an APA with~ e d u c e d aldehyde g r o u p s w a s m u c h less effective a n d ~he nucleotide, n u c l e o s i d e a n d nucleobase d e c o m [ ~ ; i o n p r o d u c t s h a d n o i n h i b i t o r y effect. T h e ~ b s e r , ' a t i o n s suggested t o us t h a t A P A m i g h t
fully a n n e a l a s a p r i m e r . H o w e v e r , ahno~t all o f t h e t e r m i n a l 3':p6~iti0ns a r e o ~ ' u p l e d b y p h o s p h a t e o r p h o s p h a t e - - c a r b o h y d r a t e r e m n a n t ~ a n d a r e n o t available f o r a d d i t i o n o f s u b s t r a t e . I f A P A inhibition o f D N A p o l y m e r m e is the r e ~ t o f a ' b l o c k e d printer' m e c h a r d m ~ , t r e a t m e n t o f t h e APA with a pho~phatase or dlegterase or b o t h should r e d u c e o r reverse the ability t o i n h i b i t . T h e s e e x p e c t a t i o n s arc, in fact, ~ o n f i r m e d b y t h e e x p e r i m e n t ~ t o b e described
2. Methods
A p u r i n t c acid was p r e p a r e d , b y t[~e m e t h o d o f
~i~i~!!:~' ~!ii~i::!:i!~: '..:....~... ~:~. ~, . ..si : ~,.~~.~:~:~! . .,~:~:?::~ =~/~ '¸ ~:~!~i~i~¸,¸ ~ •~i~~.!@ ~ ~,~,~,~.,~, ! ~ , ~!•!i:~i~o ~~. ~,~,~ ~~~.,!~~.:~/~ ~~i~ ,~. ~!~y~!~]:~!~ ~.~,~~:~i~i .,~,~:, ,.~~~ i ~ ~i,i::~iii~ !i~:~¸::~i:~~:~i:~, i i'~:~!i!i~!~!~ii i i:i~ili!i;'~:~ !~i~ii~!i,!~~i~ ~?~ii~:!~ii~i!!~!~iii~ii!i~! ~'~iii~i~ii~~:~ii~iii i~i:~!ii~i~i:ii~iii~:~i~i~!~!ii:~!~i~i~i~i~~i~ii~:i~iii!ii~'~,~i:~!i~i~!~!i~ii: ~i~ii!~iii,~!ii~!~iiii~iiiii,~!iii~i~i~ii~!iiiii~!~:i]~
slow~/.C0oled t : o ~ o m temp.ez.atu~ e t o increase file like..'. N m o d o f a n n e a l i n g t ~ e . p . ~ e r : . a n d t e m p l a t e : T h e .
' .on fiae 3 ~:pos!~0r~ B a c t e ~ a l k ~ i n e _ p h o s p h a t a s e is , " " " a c t i v e o n a .v#,de n a r i e t y o f . p h o s p h a t e i~S~ers ,nnd ~ e a t - : . : • . naen~.of .APA With ~ )en;zb~ne.. q a i t g ob~.:ously a b 0 - / - . • g s h e s A P A i n ] a l b i f i o ~ , o f D N A p b l y m e r a s e . The-c.ombi-
. xn solutionswere reXxedin , atios Of
DNA
m g t o . i:0 n~g a n d 2 5
5 0 r a g , ~espectiwely, a n d
m gto
..
.
.. ,
•
.
• " highe,~ A P A : i o D N A ~a-t~os; 4~e .reac~cn is aetaaally-en-
-
• . " -. - ..iafion p r o d u c e d . b y 3<0]-] e.]igodeoxynuc]eofides ~Jm " " The ,ex~n~s ~'~rO W~Ch ~ e ~ 0 U S ~ A s-oluti0n~ o n r , e ~ l i e ~ : s ~ . d y w a s ,also ~Ound : t , o . b e , s p e c l ~ ¥ p l o - : . al~e~ed a s~anda~d D N A p o l y m e r a s e ~eacfi.on are ,.given ~en:t ~ ..~_e i n i t i ' a l ;ph,nse of thc-..,syn~etic :remc.tion: - :M~llez e t al.-{2], c o m m e n t o n an alkaline n u t ] e a s e " ~ab],e I. Emsh ~ A s o l u t i o n was ~ s o t e s t e d in.an " w N e h ±h,T feel ~ a y : : b e ~espons~b]e f o r .the. ~,ecovery.. assay withon~ t e m p l a t e a n d n o ~nCorporafi~n c o u l d b e ,of.cell p r o l i f e r a t i o n a f t e r A~PA a d m i r A s f r a f i o ~ O n r , o N ,detecled h0. a n y o f t h e s e case~. T h u s , A P A o r A P A deriservations raise ~e p~s~b~$y ~aI a non-specific ~,Id~.vatives C , o ~ l d n o ~ s e r v e a s b o t h i e m p l a ~ e and prinae~. -. " .The e n z y m e u s e d in '.these. assays was p:a~.:iatly purified n,e p h o s g h a l e m i g h t also pie}" a role :in..zeversi~g ,from fiae c y t o p l a s m i c .fraction o f %¥alkex 2 5 6 c a r e i n ~ i n h i b i t i o n ~n vivo. _ . .. -: s a r c o m a .ti~su,e (rat), 'This e n z y m e is m o s t active .with "3"I~ tin,fling b y t h e M:ffm z ~ o u p h h a ~ A ~ A w ~ . n o r ~ - a D N A te~aplate wh}ch has b e e n pro'dally de~.a.ded " -compefitNe w i t h r e s p e c t ~oi~e~ers~ b y D N A is -~.'So . enzyma~icaily, b u t sigrdficaut inco,rpora~ion ~ s o o c e n r s compaiible"wl,~h , 0 u ] r ~evidenc.e t h a t A P A c~m n o t snI~ v]]fli ~enaiuxed D N A [ • • " - " . s t L ~ l e as ~emplaie.-Thus0 A ~ A w o a ] d ~nly c o m p e t e " Oa~i fi~.ding .of app;ox.~mai,ely 3 " ~ i.n]-fibi'ti,ma ,~ztien " - a r i t l a e n d o g e n o u s p ~ m e r p r o b a b l y p r e s e n t as a m~,.or t ~ c . e as n m c h A P A as D N A w a s p~esen~ agrees w e ~ cont. ,mninant ,~f fl~e e n z y m e p ~ p a r a f i . o n a n d ~he zatio- w i t h t h e observations. Of Mt~ller el a!. [ 2 ] . O a r s , ~ s u l ~ s ox -pnmex t o Senapla~c w o u l d n o r be a]*.e~e,d B y adding
it~'m~t m n a l ~ . e v e n o n ~
indicate
:dies~exase
a]on~
~s . i n e f ~ e c -
0 ~ t h e basis .of .,these consiflerations, Lhen, ~ e c0n- e l u d e tha!.a likely m e c h a n i s m ~or fl~e A]:AinIerfe~ence. The
Table ] aolnfions on DNA
.~ffest of-APA
' " poly.nae~'as~
"
p,rime~ r n o ] e c n b s w i t h c : o ~ e q u e n I b ] o e k a g o ~f~hain 'e ] o n g a t i o m O f c . o .rose0 ~ in n o Way ~ s h e a ~ e --. t~ghly significan!t observali•on b y - t h e • M a i n z g m ~ p . i h a t s u c h m o l e c u l e s are ~aken u p ~ m o ]i~Ang e~Tflsto prc~- • duce.cy~ostasis,-fo~,ifourmeehmnism•h cone=% i!. : ~o~alfl b e ~e~afiv,ely e a s y t o des~gn.a~ ALPA o r p o ] y - , .. py~-'nidlne deriv.atbie w i t h a g r o i i p o n i h e ~ e n n l n a ] . 3 L p b s i f i o n t h a t is v e ~ res~sta.u~ t o ermymat~c >cleavagd :
Amoun~ Pe~cem IahiMtion JkPA s,o]ut~on A~A
umz.ea~t,e_~ "
SVD
¢.sng).
l~,ealed APA
' •
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.
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6,1
9.4
L'}4.6"
~ f x:lup~Scale ~ a y ~
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• * Negative 5)~h~iti~n.stm;Sfies
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:; T h c a ~ c r a g e . c n z y m a ~ : a , c ~ v Y I y • addifi0h
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This'~esearch Was sUppor~e:d;in.pa~t~ b y !G~-aflf :---:-:: .,:
[3] Koznbeig,
A . J(lg~39) Science 1 6 3 , 1 4 1 0 2
.
. .
-
.
.