BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 496-503
Vol. 180, No. 2, 1991 October 31, 1991
INHIBITORY BINDING THE DNA PRIMER
Kalman
OF ADENOSINE DIPHOSPHORIBOSYLTRANSFERASE SITE OF REVERSE TRANSCRIPTASE TEMPI~ATES
G.
Buki,
Pal
I.
Bauer
and
Ernest
TO
Kun*
L a b o r a t o r y for E n v i r o n m e n t a l T o x i c o l o g y and Chemistry, and the Octamer Research Foundation, Romberg Tiburon Centers, S a n F r a n c i s c o State University, Tiburon, CA 94920
Received September 9, 1991
Purified adenosine d i p h o s p h o r i b o s e t r a n s f e r a s e protein binds to RNA-DNA hybrid templates of reverse t r a n s c r i p t a s e at the DNA p r i m e r s i t e and i n h i b i t s RT a c t i v i t y of H I V and M M u RTs. T h i s action is p r e v e n t e d by a u t o - p o l y - A D P - r i b o s y l a t i o n of the transferase but is reinduced by inhibitory ligands of the enzyme. © ~991 Academic Press, Inc.
Adenosine diphosphoribosyl
transferase
(ADPRT, E.C.2.4.2.30)
highly abundant non-histone nuclear protein of higher and
there
is
synthesizing
convincing
function of
evidence this
that
protein
the
oligo(ADP-ribose)
synthesis
represents
only
a
few
(1,2).
This is
of the D N A - i n d e p e n d e n t
rates of
(3), which
even in the 56 kDa p o l y p e p t i d e
eukaryotes
poly(ADP-ribose)
p e r c e n t of its m o l e c u l a r activity in intact cells in a g r e e m e n t with the m a g n i t u d e
is a
can be readily
fragment of ADPRT
determined
(4) that has no
DNA r e c o g n i t i o n sites
(5). C o n s e q u e n t l y the r e l a t i v e l y slow auto-
poly-ADP-ribosylation
of A D P R T
in
intact
cells
function
by
covalently
to
the
ADPRT
the b i n d i n g
DNA r e g i o n s
topological
serves
providing bound
demonstrating internal
(6)
changes
and slow t u r n o v e r
most
probably
(ADPR)n
Considerable (I0,ii),
from the binding
that
evidence
of A D P R T to DNA t e r m i n i
and c r u c i f o r m DNA result
a self-regulatory
DNA-repelling (7).
of the p o l y m e r
as well
is
exists as to
and s i g n i f i c a n t
of ADPRT
to circular
To whom correspondence should be addressed. ADPRT, A d e n o s i n e d i p h o s p h o r i b o s y l transferase; RT, r e v e r s e t r a n s c r i p t a s e ; TE, T r i s - E D T A buffer; NA, n u c l e i c acid; GSH, reduced glutathione; DTT, dithiothreitol; (ADPR)n , polyadenosine diphosphoribose; MMu RT, Moloney murine leucosis virus RT. Abbreviations:
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
496
Vol. 180, No. 2, 1991
DNA,
a process
inhibitory
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
ligands
has a regulatory has
also
Klenow
been
(12).
also
regulated
Recently, that
is i n h i b i t e d
by a u t o - A D P - r i b o s y l a t i o n
The p r e s e n t with
the
primer
report,
binding
shown
(8),
DNA
and
(28), and it
synthesis
and
this
enzyme
that A D P R T by the
inhibition
of ADPRT or by removal
to R N A - D N A
sites of reverse transcriptase M~terials
NAD
of Zn 2+
(9).
as an e x t e n s i o n
of A D P R T
both
of SV40 DNA
in v i t r o
by A D P R T
from one of its two zinc fingers
by
it has been
effect on the synthesis
demonstrated
fragment
modulated
is
of p r e v i o u s hybrids,
studies,
deals
in p a r t i c u l a r
to
templates.
and M e t h o d s
MMu RT, p ( r A ) . ( d T ) 1 2 _ 1 8 and p( rC) . (dG)l 2-1 8 w e r e o b t a i n e d from Pharmacia (Piscataway, NJ), and polyA (average length of 300 b), h i s t o n e s (Cat. #7755), oligo(dT) 12,18, d G T ~ 5 and TTP frp~ Sigma (St. Louis, MO). Trac~ nucleot-ides [ ~ - ~ P ] T T P , [a-JzP]dGTP (each 650 Ci/mmol) and [ P]NAD (250 Ci/mmol) were purchased from ICN (Irvine, CA). ADPRT was prepared from calf thymus by a published method (14). HIV reverse transcriptase (recombinant, clone BH-10) was a gift from the AIDS R e s e a r c h and R e f e r e n c e R e a g e n t P r o g r a m of AIDS, NIAID, NIH (Rockville, MD). O c t a m e r i c d s D N A (GCATGCAT) was p r e p a r e d by a n n e a l i n g s y n t h e t i c c o m p l e m e n t i n g s t r a n d s (15). C o e n z y m i c DNA (CoDNA) c o n s i s t e d of 2 0 0 - 1 0 0 0 bp dsDNA, a s i d e p r o d u c t of the i s o l a t i o n of A D P R T (13), w i t h an average of 500 bp (325 kDa) size. Nitrocellulose membranes (type HA, 2.4 cm d i a m e t e r , 0 . 4 5 ~m) w e r e from M i l l i p o r e (Bedford, MA) and filter b i n d i n g assays were p e r f o r m e d as p u b l i s h e d (12). The labeled hybrid polynucleotide p(rA).p([32p]dT) was p r e p a r e d by a n n e a l i n g 66 ~ g of poly(rA) w i t h 0.8 ~ g oligo(dT) 1 2 _ 1 8 in 40 ~ i of TE buffer at room temperature for 30 min, then 200 ~ i of 5xHIV buffer (25~^mM Tris-HCl, 300 mM KCl, 35 mM MgCl2, pH 8.2) and 70 ~ C i of [e-~zP]TTP and 650 ~ i of sterile water were added. Finally 0.5 ~ g of H I V - R T in i00 ~ i of RT d i l u t e r (50 mM p h o s p h a t e , pH 7.2, 50 mM KCl, 50% glycerol and 0.2% Triton X-100) was admixed and the system incubated at 25°C for 40 min, followed by extraction w i t h phenol/chloroform, 1 : l(v/v) and w i t h chloroform. The nucleic acid was precipitated with 2 volumes of 100% ethanol o v e r n i g h t at -20°C, c e n t r i f u g e d and the p r e c i p i t a t e dissolved in 40 ~ i of TE buffer. Spectrophotometric assay for nucleic acid c o n c e n t r a t i o n was based on 1 A260 = 40 ~ g n u c l e i c acid, and the specific activity was 28,500 cpm/~g. Gel retardation was assayed in b o t h 1.2% a g a r o s e and 4% a c r y l a m i d e gels as d e s c r i b e d (16). A s s a y s for A D P R T a c t i v i t y were done in a l i q u o t s (50 ~I) containing 16.5 pmol of ADPRT in IxHIV buffer supplemented with 8 mM DTT, 2 mM GSH, and 0.i mM [32p]NAD (0.i ~ C i ) . After incubation at 2 5 ° C for 4 min, t h e r e a c t i o n w a s s t o p p e d by 2 ml of 20% t r i c h l o r o a c e t i c acid and the precipitate collected by filtration and r a d i o a c t i v i t y c o u n t e d by liquid s c i n t i l l a t i o n s p e c t r o m e t r y (13). The A D P - r i b o s y l a t e d ADPRT was made in 6 ml of reaction mix c o n t a i n i n g 25 mM Tris. HCl (pH7.7), 1 mM EDTA, 0,25 mM NAD, 0.2% Tween 20, 240 ~ g of A D P R T and 0.i ~ g of o c t a m e r i c dsDNA. A f t e r incubation for 2 h o u r s at 25°C, the m i x w a s c o n c e n t r a t e d in C e n t r i c o n 30 (Amicon, Beverly, MA) to 3 m g / m l and the m e d i u m changed to 25 mM Tris.HCl (pH 7.7), 1 mM EDTA, I0 mM 2-mercaptoe t h a n o l , 0 . 2 % T w e e n 20 with the concomitant r e m o v a l of t h e o c t a m e r i c DNA and NAD. HIV RT a c t i v i t y was a s s a y e d in 50 ~ i of 497
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
i x H I V b u f f e r s u p p l e m e n t e d w i t h 8 mM DTT, 2 m M GSH, 50 )/M [uzP]TTP or [a-JZ]dGTP and 2 //g of p ( r A ) . (dT) 1 2 - 1 8 or p(rC). ( d G ) 1 2 _ 1 8 ; 0.03 lag of H I V RT s t a r t e d p o l y m e r i z a t i o n at 25°C, w h i c h was s t o p p e d a f t e r i0 min w i t h cold TCA, p r e c i p i t a t e c o l l e c t e d on f i l t e r s , and r a d i o a c t i v i t y c o u n t e d as for A D P R T assays above. In experiments (Fig. 3B, 4B), where the concentrat i o n of (dT) 12_18 p r i m e r was varied, the poly(rA) was kept cons t a n t ( 3 . 3 ) a g / t u b e ) a n d a n n e a l i n g w a s a l l o w e d for 30 m i n at ambient temperature in TE b u f f e r p r i o r to a d d i t i o n of o t h e r components of the RT assay. With MMu RT, the reaction was done at 37°C. RNase H activity was tested as p u b l i s h e d (17) and partially z i n c - d e p l e t e d ADPRT was prepared as reported (9).
Results
Binding constants hybrid
were
of A D P R T
determined
system containing tions
by a filter b i n d i n g
1.5 nM A D P R T
of the hybrid between
analysis for
and HIV RT to the p o l y ( r A ) . (dT) 12_18 technique
or HIV RT and v a r y i n g
0.075 and 3 ~g/ml.
r e v e a l e d h i g h a f f i n i t y K d values,
ADPRT
and
0.31
±
0.i
nM
for
HIV
in a
concentra-
Scatchard binding
namely
RT,
(12)
as
0.26 ± 0.I nM
determined
in
5
parallel tests.
Binding of A D P R T to the hybrid nucleic acid was also d e m o n s t r a t e d with the gel r e t a r d a t i o n the
retardation
tions its
of the
of ADPRT,
substrate
When
the
(18)
(Fig.
labeled
whereas
i.)
hybrid
preincubation
- completely
enzymatic
benzamide
assay
abolished
activity
The
by
first
increasing
of A D P R T
with
retardation
of A D P R T
5 lines show
was
by
-
6-10). either
(19) A D P R T was
bound to the hybrid as shown by u n i m p a i r e d r e t a r d a t i o n 20).
1 mM NAD
(lanes
inhibited
or 5 - i o d o - 6 - a m i n o - l , 2 - b e n z o p y r o n e
concentra-
(lanes Ii-
It was d e t e r m i n e d
in separate tests that b e n z a m i d e at 0.4 mM
or 5 - I - l , 2 - b e n z o p y r o n e
at 0.5 mM had no direct effect on isolated
RT enzymes hybrid
(not shown).
(not shown).
HIV RT also retarded the m i g r a t i o n of the
Since binding of ADPRT to DNA termini
gene-
rally increases the p o l y m e r a s e activity of ADPRT,
we compared the
coenzymatic
activators
ADPRT, (15).
function
coenzymic Results
are
DNA
of
the
(14)
hybrid
with
and a s y n t h e t i c
illustrated
in Fig.
2
known
octameric wherein
it is e v i d e n t
that despite the strong b i n d i n g of the hybrid to ADPRT, small c o e n z y m a t i c
activation
of
DNA d u p l e x
of the p o l y ( A D P - r i b o s e )
only very polymerase
function of ADPRT is conferred by the hybrid. The s u p p r e s s i v e effect of ADPRT on HIV RT activity is illustrated in Fig.3 show
a
(A and B). Two different templates were used and results stronger
suppression
with
498
p(rA). (dT)12_18
than
with
Vol. 1 80, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60 ~
O CoDNA oe%amer
50 --
/
40 o o .~
30
0
20
I
10
Origin --
300 bp --
,-, I
0
10-+ 10-~ 10-2 10-1 100 101 102 10~
(9
1 2 3 4 5 6 7 8 910111213141516171819~
~.~
nucleic a c i d , nM
F i g u r e I % Gel r e t a r d a t i o n a s s a y for t h e b i n d i n g of A D P R T to p(rA}.p([~ZP]dT) hybrid polynucleotide. Gel: 1.2% agarose. NA: 35 ng (1000 cpm)/lane, Lanes 1-5 show the retardation of labeled hybrid NA by increasing amounts (0, 5, 10, 20, 40 ng) of ADPRT. (Note: due to a handling error, lane 4 contained 40 ng and lane 5 20 ng of ADPRT) Lanes 6-10 are with the same p r o g r e s s i o n of ADPRT amounts but preincubated with 1 mM NAD for i0 min at ambient temp; lanes 11-15 are the same but with benzamide added (0.4 mM); while lanes 16-20 have 5-iodo-6-amino-l,2-benzopyrone (0.5mM) added. Fiqure 2. Coenzymic activity of CoDNA, octameric deoxyrlbonucleotide duplex (octamer DNA) and p(rA). (dT)12_18 on the rates of auto-poly-ADP-ribosylation of ADPRT. Assay for ADPRT activity described in Methods.
p(rC). (dG)12_18; slightly binding
above
HIV
RT
by
hybrid.
ADPRT
action
of A D P R T
(compare
inhibitory Zn 2+
(9),
of
ADPRT
parable
to
the
its
RT
inhibitory enzyme
0.3
latter
template.
two
by
diminishes
RT
but
HIV
RT
(Fig.
of
partial
effect The
on
the
effect
DNA of
499
of
presumably site the
second
of
a the
inhibitory curve
from
inhibitory
action
Poly-ADP-ribosylated
ADPRT
(Fig.
does
of
obtained
of inhibition
3B),
with
and
preference
primer
no d i r e c t
former
results
reinforce
4A),
pretreatment
influence
(9).
the
compare
have
The
with
(Fig.
for
curves).
on H I V
ADPRT,
to
3B;
the
kinetics
that histones of
for
agrees
ADPRT
themselves
action
~M
competition
and
lower
from
binding
Klenow
is
The apparent
(top c u r v e
has
benzopyrone
(i0).
histones
RT
of
the
is of i n t e r e s t
whereas
no
ADPRT
simulates
on H I V
tion
for
between
It
of
to p ( r A ) . ( d T ) 1 2 _ 1 8
dsDNAs
competition
top)
1 ~M
of A D P R T
with various
I50
not
and
with abolish
4B) . T h e s e zinc
the
on
MMu
deple-
inhibitory
results
depletion
polymerase ADPRT
partial
6-nitroso-l,2-
of
activity RT
are
com-
ADPRT of
activity
on the is
Vol.
180,
No.
2, 1 9 9 1
BIOCHEMICAL
AND
BIOPHYSICAL
A
RESEARCH COMMUNICATIONS
B
100 oiodG emplte05
/
° r1406°0°4 p(rA).oUgo(dT)ternpla'~teI ~ .
20
0.2
--
0.1
0 0.01
........
I 0. I
........
I I
i
i -200
0
ADPRT concentration (/~M)
200
l 600
400
i/[prirner],
800
I000
/~M-*
F i q u r e 3. A: T h e inhibitory e f f e c t of A D P R T on HIV RT at v a r y i n g c o n c e n t r a t i o n s o f A D P R T w i t h t w o t y p e s o f R N A - D N A h y b r i d s as t e m p l a t e s . H u n d r e d % a c t i v i t i e s w e r e 53 p m o l a n d 21 p m o l of n u c l e o t i d e s incorporated for the A.T and C.dG templates respectiv e l y , a n d 3 jsg h i s t o n e s were present in a l l a s s a y s . B: L i n e w e a v e r - B u r k p l o t of H I V R T a c t i v i t y at v a r y i n g c o n c e n t r a t i o n s of p r i m e r . Ordinate ( l / V ) is in u n i t s of pmol-" of TMP i n c o r p o r a t e d in 10 min. 0, no a d d i t i v e ; V , p l u s h i s t o n e s (3 Jug) ; • , plus ADPRT (0.67 JuM) ; ¥ , plus histones and ADPRT.
summarized
in T a b l e
inhibitory
action
tory,
and
i. of
preference
Just
as w i t h
HIV
ADPRT
without
being
of
p(rA).(dT)12_18
A
B
100 - o
| 0.2 ~-
RT,
histones
by to
promote
themselves
p(rC).(dG)12_18
~ NOBP-treatedADPRT • ADPRT
/
"--o ~
8o
O
~
60
•
4-0
~
2o
0.1 O
L
0
0.01
O ADP-rlbosylated
--
•
• native
,
,
i ,,,,,[
i
,
, ,lJl,[
0.1
1
-200
ADPRT, /~M
0
200
400
1/[primer],
600
800 -1
1000
/~M
Figure 4. A : T h e e f f e c t of native A D P R T a n d of a u t o - A D P - r i b o s y l a t e d A D P R T o n H I V R T activity. C o n d i t i o n s w e r e the same as in Fig. 3A, a u t o - A D P - r i b o s y l a t e d ADPRT was made as d e s c r i b e d in Methods, 100% activity: 65 pmoles TMP incorporated. B: The effects on H I V R T a c t i v i t y of n a t i v e A D P R T and A D P R T w h i c h is 50% z i n c - d e p l e t e d b y t r e a t m e n t w i t h 6 - n i t r o s o - l , 2 - b e n z o p y r o n e (NOBP). C o n d i t i o n s were the same as in Fig. 3B.
500
the
inhibiwas
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
1
The effect of ADPRT on the activity of MMu RT RT activity with Exper. conditions
p(rC)-(dG)I2-18
No ADPRT,
120 (100%)
+ ADPRT,
No Histone No Histone
No ADPRT, + ADPRT,
p(rA)-(dT)I2_I8
81 (38% inhib.)
+ Histone
109
+ Histone
56.8
(100%)
12.9
(87% inhib.)
58.2
30 (75% inhib.)
6.7
(88% inhib.)
RT concentration was 0.02 ~g/test consisting of ~ m M Tris-HCl ~ H 8.5 at 25~C), 8 mM SgCl2, i0 mM DDT, 0.5 mM [ ~ - ~ P ] T T P or [aP]dGTP, 1 ~ g of p(rA). (dT)12_18 or p(rC). (dG)12_18 in 50 ~i volume. ADPRT: 0.67 uM; histones: 5 ~g/test, if present. Incubation was carried out at 37°C for 5 min and acid p r e c i p i t a b l e radioactive product assayed as described in Methods. Values in the table are given as pmoles of deoxyribonucleotides incorporated in 5 min at 37°C. Data are the averages of 3 parallel assays, not differing more than ±10% from the mean value.
also
shown.
its R N a s e
Contrary
H activity
to
was
the
DNA
synthetic
activity
by ADPRT
(results
unaffected
of
HIV
RT,
n o t shown).
Discussion The
inhibitory
site
of R T
template
DNA
hybrids.
has
only
adds per
effect
to a l r e a d y
which
we
probably
observe ADPRT
1 ~M
Therefore
ADPRT
to
RT
relevance. nuclear the
cytosol
species lase
templates
(27)
cellular
(21)
(22).
of A D P R T (23-26) in
range
conclude
within
However
the
were
isolated
antiretroviral
cell
evidence
identified
may
RT
RT
as w e l l
is a selfof A D P R T
are
well
at
within
which
binding
is of
pharmacological
is a p r e d o m i n a n t l y
is m a i n l y that
as
properties
in nuclei,
have
inasmuch
of A D P R T
501
observation
a drug-induced
exists
ribonucleoprotein action
of
acid
in p h y s i o l o g i c a l l y
that ADPRT
retroviral
is n o t e x c l u s i v e ,
This
formation
existing
to R N A -
nucleic
concentrations
that
thought
whereas
(ADPR)n
The
of A D P R T
colligative role
inhibition
concentration
It is g e n e r a l l y
enzyme
lization
ADPRT.
we
that
at t h e p r i m e r
hybrid
ADPRT.
a prominent
enzymatic
significant
the actual (20).
of
the
on
evidence
play
binding
binding,
efficacy
and that
activity
on D N A s y n t h e s i s
a novel
strong
coenzymic
cells
regulatory
the
accumulating
s_ee of A D P R T
functioning
illustrates
Despite
minimal
of A D P R T
the
operative
in
nuclear
loca-
as c y t o p l a s m i c
ADPRT
(ADPR) n g l y c o h y d r o -
particles. ligands,
which
Therefore may
a
induce
Vol. 180, No. 2, 1991
the
binding
example
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of A D P R T
of which
to
RT
is reported
templates,
cannot
in an accompanying
be
excluded,
paper
an
(19).
Acknowledgments This r e s e a r c h was s u p p o r t e d by A F O S R 8 9 - 0 2 3 1 and by funds of O c t a m e r R e s e a r c h Foundation. The f o l l o w i n g r e a g e n t was o b t a i n e d t h r o u g h the AIDS R e s e a r c h and R e f e r e n c e R e a g e n t Program, NIAID, NIH: H I V r e v e r s e t r a n s c r i p t a s e f r o m the Univ. of A l a b a m a at Birmingham, Center for AIDS Res., Gene Expression Core Facility.
References
i. 2. 3. 4.
5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16.
17. 18. 19. 20.
Yamanaka, H., Penning, C.A., Willis, E.H., Wasson, D.B. and Carson, D.A. (1988) J. Biol. Chem. 263, 3879-3883. Ludwig, A., Behnke, B., Holtlund, J. and Hilz, H. (1988) J. Biol. Chem. 263, 6993-6999. Bauer, P.I. and Kun, E. (1985) in "ADP-ribosylation of Proteins (eds. Althaus, F.R., Hilz, H. and Shall, S.) springer-Verlag, Berlin, Heidelberg, pp. 63-74. Simonin, F., de Murcia, J.-M., Poch, O., Muller, S., Gradwohl,G., Molinete, M., Penning, C., Keith, G. and de Murcia, G. (1990) J. Biol. Chem. 256, 19249-19256. Buki, K.G. and Kun, E. (1989) Biochemistry 27, 5990-5995. Romaschin, A.D. and Kun, E. (1981) Biochem. Biophys. Res. Commun. 102, 952-957. Zahradka, P. and Ebisuzaki, K. (1982) Eur. J. Biochem. 127, 579-585. Nobori, T., Yamanaka, H. and Carson, D.A. (1989) Biochem. Biophys. Res. Commun. 163, 1113-1118. Buki, K.G., Bauer, P.I., Mendeleyev, J., Hakam, A. and Kun, E. (1991) FEBS Lett. (in press). Sastry, S.S., Buki, K.G. and Kun, E. (1989) Biochemistry 28, 5670-5680. Sastry, S.S. and Kun, E. (1990) Biochem. Biophys. Res. Commun. 167, 842-847. Sastry, S.S. and Kun, E. (1988) J. Biol. Chem. 263, 15051512. Buki, K.G., Kirsten, E. and Kun, E. (1987) Anal. Biochem. 167, 160-166. Bauer, P.I., Buki, K.G. and Kun, E. (1990) FEBS Lett. 273, 6-10. Hakam, A., McLick, J., Buki, K.G. and Kun, E. (1987) FEBS Lett. 212, 73-78. Current Protocols in Molecular Biology (1987) (eds. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., seidman, J.G., Smith, J.A. and Struhl, K.) Green Publishing Associates and wiley Interscience, New York, p2.5.1. Starnes, M.C. and Cheng, Y-chi (1989) J. Biol. Chem. 264, 7073-7077. Purnell, M.R. and Whish, W.J.D.(1980) Biochem. J. 185, 775777. Cole, G.A., Bauer, G., Kirsten, E., Mendeleyev, J., Bauer, P. I., Buki, K.G., Hakam, A. and Kun, E. (1991) Biochem. Biophys. Res. Commun. 180, 504-514. Bauer, P.I., Buki, K.G., Hakam, A. and Kun, E. (1990) Biochem. J. 270, 17-26. 502
VOI. 180, No. 2, 1991
21.
22. 23. 24. 25. 26. 27. 28.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Althaus, F.R. and Richter, C. (1987) in "ADP-ribosylation of Proteins" Mol. Biol. Biochem. Biophys. Ser. 37, SpringerVerlag, New York. Haseltine, W.A. (1991) The FASEB J. 5, 2349-2360. Furneaux, H.M. and Pearson C.K. (1980) Biochem. J. 187, 91-103. Roberts, J.H., Stark, P., Giri, C.P. and Smulson, M. (1975) Arch. Biochem. Biophys. 171, 305-315. Elkaim, R., Thomassin, H., Niedergang, C., Egly, J.M., Kempf, J. and Mandel, P. (1983) Biochimie 65, 653-659. Thomassin, H., Niedergang, C. and Mandel, P. (1985) Biochem. Biophys. Res. Commun. 133, 654-661. Thomassin, H. and Mandel, P. (1989) in "ADP-ribose Transfer Reactions" (Jacobson, M.K. and Jacobson, E.L., eds.) Springer-Verlag, New York, pp 125-129. Eki, T. and Hurwitz, J. (1991) J.Biol. Chem. 266 3087-3100.
503
Vol. 180, No. 2, 1991 October 31, 1991
INHIBITORY BINDING THE DNA PRIMER
Kalman
OF ADENOSINE DIPHOSPHORIBOSYLTRANSFERASE SITE OF REVERSE TRANSCRIPTASE TEMPI~ATES
G.
Buki,
Pal
I.
Bauer
and
Ernest
TO
Kun*
L a b o r a t o r y for E n v i r o n m e n t a l T o x i c o l o g y and Chemistry, and the Octamer Research Foundation, Romberg Tiburon Centers, S a n F r a n c i s c o State University, Tiburon, CA 94920
Received September 9, 1991
Purified adenosine d i p h o s p h o r i b o s e t r a n s f e r a s e protein binds to RNA-DNA hybrid templates of reverse t r a n s c r i p t a s e at the DNA p r i m e r s i t e and i n h i b i t s RT a c t i v i t y of H I V and M M u RTs. T h i s action is p r e v e n t e d by a u t o - p o l y - A D P - r i b o s y l a t i o n of the transferase but is reinduced by inhibitory ligands of the enzyme. © ~991 Academic Press, Inc.
Adenosine diphosphoribosyl
transferase
(ADPRT, E.C.2.4.2.30)
highly abundant non-histone nuclear protein of higher and
there
is
synthesizing
convincing
function of
evidence this
that
protein
the
oligo(ADP-ribose)
synthesis
represents
only
a
few
(1,2).
This is
of the D N A - i n d e p e n d e n t
rates of
(3), which
even in the 56 kDa p o l y p e p t i d e
eukaryotes
poly(ADP-ribose)
p e r c e n t of its m o l e c u l a r activity in intact cells in a g r e e m e n t with the m a g n i t u d e
is a
can be readily
fragment of ADPRT
determined
(4) that has no
DNA r e c o g n i t i o n sites
(5). C o n s e q u e n t l y the r e l a t i v e l y slow auto-
poly-ADP-ribosylation
of A D P R T
in
intact
cells
function
by
covalently
to
the
ADPRT
the b i n d i n g
DNA r e g i o n s
topological
serves
providing bound
demonstrating internal
(6)
changes
and slow t u r n o v e r
most
probably
(ADPR)n
Considerable (I0,ii),
from the binding
that
evidence
of A D P R T to DNA t e r m i n i
and c r u c i f o r m DNA result
a self-regulatory
DNA-repelling (7).
of the p o l y m e r
as well
is
exists as to
and s i g n i f i c a n t
of ADPRT
to circular
To whom correspondence should be addressed. ADPRT, A d e n o s i n e d i p h o s p h o r i b o s y l transferase; RT, r e v e r s e t r a n s c r i p t a s e ; TE, T r i s - E D T A buffer; NA, n u c l e i c acid; GSH, reduced glutathione; DTT, dithiothreitol; (ADPR)n , polyadenosine diphosphoribose; MMu RT, Moloney murine leucosis virus RT. Abbreviations:
0006-291X/91 $1.50 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
496
Vol. 180, No. 2, 1991
DNA,
a process
inhibitory
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
that
ligands
has a regulatory has
also
Klenow
been
(12).
also
regulated
Recently, that
is i n h i b i t e d
by a u t o - A D P - r i b o s y l a t i o n
The p r e s e n t with
the
primer
report,
binding
shown
(8),
DNA
and
(28), and it
synthesis
and
this
enzyme
that A D P R T by the
inhibition
of ADPRT or by removal
to R N A - D N A
sites of reverse transcriptase M~terials
NAD
of Zn 2+
(9).
as an e x t e n s i o n
of A D P R T
both
of SV40 DNA
in v i t r o
by A D P R T
from one of its two zinc fingers
by
it has been
effect on the synthesis
demonstrated
fragment
modulated
is
of p r e v i o u s hybrids,
studies,
deals
in p a r t i c u l a r
to
templates.
and M e t h o d s
MMu RT, p ( r A ) . ( d T ) 1 2 _ 1 8 and p( rC) . (dG)l 2-1 8 w e r e o b t a i n e d from Pharmacia (Piscataway, NJ), and polyA (average length of 300 b), h i s t o n e s (Cat. #7755), oligo(dT) 12,18, d G T ~ 5 and TTP frp~ Sigma (St. Louis, MO). Trac~ nucleot-ides [ ~ - ~ P ] T T P , [a-JzP]dGTP (each 650 Ci/mmol) and [ P]NAD (250 Ci/mmol) were purchased from ICN (Irvine, CA). ADPRT was prepared from calf thymus by a published method (14). HIV reverse transcriptase (recombinant, clone BH-10) was a gift from the AIDS R e s e a r c h and R e f e r e n c e R e a g e n t P r o g r a m of AIDS, NIAID, NIH (Rockville, MD). O c t a m e r i c d s D N A (GCATGCAT) was p r e p a r e d by a n n e a l i n g s y n t h e t i c c o m p l e m e n t i n g s t r a n d s (15). C o e n z y m i c DNA (CoDNA) c o n s i s t e d of 2 0 0 - 1 0 0 0 bp dsDNA, a s i d e p r o d u c t of the i s o l a t i o n of A D P R T (13), w i t h an average of 500 bp (325 kDa) size. Nitrocellulose membranes (type HA, 2.4 cm d i a m e t e r , 0 . 4 5 ~m) w e r e from M i l l i p o r e (Bedford, MA) and filter b i n d i n g assays were p e r f o r m e d as p u b l i s h e d (12). The labeled hybrid polynucleotide p(rA).p([32p]dT) was p r e p a r e d by a n n e a l i n g 66 ~ g of poly(rA) w i t h 0.8 ~ g oligo(dT) 1 2 _ 1 8 in 40 ~ i of TE buffer at room temperature for 30 min, then 200 ~ i of 5xHIV buffer (25~^mM Tris-HCl, 300 mM KCl, 35 mM MgCl2, pH 8.2) and 70 ~ C i of [e-~zP]TTP and 650 ~ i of sterile water were added. Finally 0.5 ~ g of H I V - R T in i00 ~ i of RT d i l u t e r (50 mM p h o s p h a t e , pH 7.2, 50 mM KCl, 50% glycerol and 0.2% Triton X-100) was admixed and the system incubated at 25°C for 40 min, followed by extraction w i t h phenol/chloroform, 1 : l(v/v) and w i t h chloroform. The nucleic acid was precipitated with 2 volumes of 100% ethanol o v e r n i g h t at -20°C, c e n t r i f u g e d and the p r e c i p i t a t e dissolved in 40 ~ i of TE buffer. Spectrophotometric assay for nucleic acid c o n c e n t r a t i o n was based on 1 A260 = 40 ~ g n u c l e i c acid, and the specific activity was 28,500 cpm/~g. Gel retardation was assayed in b o t h 1.2% a g a r o s e and 4% a c r y l a m i d e gels as d e s c r i b e d (16). A s s a y s for A D P R T a c t i v i t y were done in a l i q u o t s (50 ~I) containing 16.5 pmol of ADPRT in IxHIV buffer supplemented with 8 mM DTT, 2 mM GSH, and 0.i mM [32p]NAD (0.i ~ C i ) . After incubation at 2 5 ° C for 4 min, t h e r e a c t i o n w a s s t o p p e d by 2 ml of 20% t r i c h l o r o a c e t i c acid and the precipitate collected by filtration and r a d i o a c t i v i t y c o u n t e d by liquid s c i n t i l l a t i o n s p e c t r o m e t r y (13). The A D P - r i b o s y l a t e d ADPRT was made in 6 ml of reaction mix c o n t a i n i n g 25 mM Tris. HCl (pH7.7), 1 mM EDTA, 0,25 mM NAD, 0.2% Tween 20, 240 ~ g of A D P R T and 0.i ~ g of o c t a m e r i c dsDNA. A f t e r incubation for 2 h o u r s at 25°C, the m i x w a s c o n c e n t r a t e d in C e n t r i c o n 30 (Amicon, Beverly, MA) to 3 m g / m l and the m e d i u m changed to 25 mM Tris.HCl (pH 7.7), 1 mM EDTA, I0 mM 2-mercaptoe t h a n o l , 0 . 2 % T w e e n 20 with the concomitant r e m o v a l of t h e o c t a m e r i c DNA and NAD. HIV RT a c t i v i t y was a s s a y e d in 50 ~ i of 497
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
i x H I V b u f f e r s u p p l e m e n t e d w i t h 8 mM DTT, 2 m M GSH, 50 )/M [uzP]TTP or [a-JZ]dGTP and 2 //g of p ( r A ) . (dT) 1 2 - 1 8 or p(rC). ( d G ) 1 2 _ 1 8 ; 0.03 lag of H I V RT s t a r t e d p o l y m e r i z a t i o n at 25°C, w h i c h was s t o p p e d a f t e r i0 min w i t h cold TCA, p r e c i p i t a t e c o l l e c t e d on f i l t e r s , and r a d i o a c t i v i t y c o u n t e d as for A D P R T assays above. In experiments (Fig. 3B, 4B), where the concentrat i o n of (dT) 12_18 p r i m e r was varied, the poly(rA) was kept cons t a n t ( 3 . 3 ) a g / t u b e ) a n d a n n e a l i n g w a s a l l o w e d for 30 m i n at ambient temperature in TE b u f f e r p r i o r to a d d i t i o n of o t h e r components of the RT assay. With MMu RT, the reaction was done at 37°C. RNase H activity was tested as p u b l i s h e d (17) and partially z i n c - d e p l e t e d ADPRT was prepared as reported (9).
Results
Binding constants hybrid
were
of A D P R T
determined
system containing tions
by a filter b i n d i n g
1.5 nM A D P R T
of the hybrid between
analysis for
and HIV RT to the p o l y ( r A ) . (dT) 12_18 technique
or HIV RT and v a r y i n g
0.075 and 3 ~g/ml.
r e v e a l e d h i g h a f f i n i t y K d values,
ADPRT
and
0.31
±
0.i
nM
for
HIV
in a
concentra-
Scatchard binding
namely
RT,
(12)
as
0.26 ± 0.I nM
determined
in
5
parallel tests.
Binding of A D P R T to the hybrid nucleic acid was also d e m o n s t r a t e d with the gel r e t a r d a t i o n the
retardation
tions its
of the
of ADPRT,
substrate
When
the
(18)
(Fig.
labeled
whereas
i.)
hybrid
preincubation
- completely
enzymatic
benzamide
assay
abolished
activity
The
by
first
increasing
of A D P R T
with
retardation
of A D P R T
5 lines show
was
by
-
6-10). either
(19) A D P R T was
bound to the hybrid as shown by u n i m p a i r e d r e t a r d a t i o n 20).
1 mM NAD
(lanes
inhibited
or 5 - i o d o - 6 - a m i n o - l , 2 - b e n z o p y r o n e
concentra-
(lanes Ii-
It was d e t e r m i n e d
in separate tests that b e n z a m i d e at 0.4 mM
or 5 - I - l , 2 - b e n z o p y r o n e
at 0.5 mM had no direct effect on isolated
RT enzymes hybrid
(not shown).
(not shown).
HIV RT also retarded the m i g r a t i o n of the
Since binding of ADPRT to DNA termini
gene-
rally increases the p o l y m e r a s e activity of ADPRT,
we compared the
coenzymatic
activators
ADPRT, (15).
function
coenzymic Results
are
DNA
of
the
(14)
hybrid
with
and a s y n t h e t i c
illustrated
in Fig.
2
known
octameric wherein
it is e v i d e n t
that despite the strong b i n d i n g of the hybrid to ADPRT, small c o e n z y m a t i c
activation
of
DNA d u p l e x
of the p o l y ( A D P - r i b o s e )
only very polymerase
function of ADPRT is conferred by the hybrid. The s u p p r e s s i v e effect of ADPRT on HIV RT activity is illustrated in Fig.3 show
a
(A and B). Two different templates were used and results stronger
suppression
with
498
p(rA). (dT)12_18
than
with
Vol. 1 80, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
60 ~
O CoDNA oe%amer
50 --
/
40 o o .~
30
0
20
I
10
Origin --
300 bp --
,-, I
0
10-+ 10-~ 10-2 10-1 100 101 102 10~
(9
1 2 3 4 5 6 7 8 910111213141516171819~
~.~
nucleic a c i d , nM
F i g u r e I % Gel r e t a r d a t i o n a s s a y for t h e b i n d i n g of A D P R T to p(rA}.p([~ZP]dT) hybrid polynucleotide. Gel: 1.2% agarose. NA: 35 ng (1000 cpm)/lane, Lanes 1-5 show the retardation of labeled hybrid NA by increasing amounts (0, 5, 10, 20, 40 ng) of ADPRT. (Note: due to a handling error, lane 4 contained 40 ng and lane 5 20 ng of ADPRT) Lanes 6-10 are with the same p r o g r e s s i o n of ADPRT amounts but preincubated with 1 mM NAD for i0 min at ambient temp; lanes 11-15 are the same but with benzamide added (0.4 mM); while lanes 16-20 have 5-iodo-6-amino-l,2-benzopyrone (0.5mM) added. Fiqure 2. Coenzymic activity of CoDNA, octameric deoxyrlbonucleotide duplex (octamer DNA) and p(rA). (dT)12_18 on the rates of auto-poly-ADP-ribosylation of ADPRT. Assay for ADPRT activity described in Methods.
p(rC). (dG)12_18; slightly binding
above
HIV
RT
by
hybrid.
ADPRT
action
of A D P R T
(compare
inhibitory Zn 2+
(9),
of
ADPRT
parable
to
the
its
RT
inhibitory enzyme
0.3
latter
template.
two
by
diminishes
RT
but
HIV
RT
(Fig.
of
partial
effect The
on
the
effect
DNA of
499
of
presumably site the
second
of
a the
inhibitory curve
from
inhibitory
action
Poly-ADP-ribosylated
ADPRT
(Fig.
does
of
obtained
of inhibition
3B),
with
and
preference
primer
no d i r e c t
former
results
reinforce
4A),
pretreatment
influence
(9).
the
compare
have
The
with
(Fig.
for
curves).
on H I V
ADPRT,
to
3B;
the
kinetics
that histones of
for
agrees
ADPRT
themselves
action
~M
competition
and
lower
from
binding
Klenow
is
The apparent
(top c u r v e
has
benzopyrone
(i0).
histones
RT
of
the
is of i n t e r e s t
whereas
no
ADPRT
simulates
on H I V
tion
for
between
It
of
to p ( r A ) . ( d T ) 1 2 _ 1 8
dsDNAs
competition
top)
1 ~M
of A D P R T
with various
I50
not
and
with abolish
4B) . T h e s e zinc
the
on
MMu
deple-
inhibitory
results
depletion
polymerase ADPRT
partial
6-nitroso-l,2-
of
activity RT
are
com-
ADPRT of
activity
on the is
Vol.
180,
No.
2, 1 9 9 1
BIOCHEMICAL
AND
BIOPHYSICAL
A
RESEARCH COMMUNICATIONS
B
100 oiodG emplte05
/
° r1406°0°4 p(rA).oUgo(dT)ternpla'~teI ~ .
20
0.2
--
0.1
0 0.01
........
I 0. I
........
I I
i
i -200
0
ADPRT concentration (/~M)
200
l 600
400
i/[prirner],
800
I000
/~M-*
F i q u r e 3. A: T h e inhibitory e f f e c t of A D P R T on HIV RT at v a r y i n g c o n c e n t r a t i o n s o f A D P R T w i t h t w o t y p e s o f R N A - D N A h y b r i d s as t e m p l a t e s . H u n d r e d % a c t i v i t i e s w e r e 53 p m o l a n d 21 p m o l of n u c l e o t i d e s incorporated for the A.T and C.dG templates respectiv e l y , a n d 3 jsg h i s t o n e s were present in a l l a s s a y s . B: L i n e w e a v e r - B u r k p l o t of H I V R T a c t i v i t y at v a r y i n g c o n c e n t r a t i o n s of p r i m e r . Ordinate ( l / V ) is in u n i t s of pmol-" of TMP i n c o r p o r a t e d in 10 min. 0, no a d d i t i v e ; V , p l u s h i s t o n e s (3 Jug) ; • , plus ADPRT (0.67 JuM) ; ¥ , plus histones and ADPRT.
summarized
in T a b l e
inhibitory
action
tory,
and
i. of
preference
Just
as w i t h
HIV
ADPRT
without
being
of
p(rA).(dT)12_18
A
B
100 - o
| 0.2 ~-
RT,
histones
by to
promote
themselves
p(rC).(dG)12_18
~ NOBP-treatedADPRT • ADPRT
/
"--o ~
8o
O
~
60
•
4-0
~
2o
0.1 O
L
0
0.01
O ADP-rlbosylated
--
•
• native
,
,
i ,,,,,[
i
,
, ,lJl,[
0.1
1
-200
ADPRT, /~M
0
200
400
1/[primer],
600
800 -1
1000
/~M
Figure 4. A : T h e e f f e c t of native A D P R T a n d of a u t o - A D P - r i b o s y l a t e d A D P R T o n H I V R T activity. C o n d i t i o n s w e r e the same as in Fig. 3A, a u t o - A D P - r i b o s y l a t e d ADPRT was made as d e s c r i b e d in Methods, 100% activity: 65 pmoles TMP incorporated. B: The effects on H I V R T a c t i v i t y of n a t i v e A D P R T and A D P R T w h i c h is 50% z i n c - d e p l e t e d b y t r e a t m e n t w i t h 6 - n i t r o s o - l , 2 - b e n z o p y r o n e (NOBP). C o n d i t i o n s were the same as in Fig. 3B.
500
the
inhibiwas
Vol. 180, No. 2, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table
1
The effect of ADPRT on the activity of MMu RT RT activity with Exper. conditions
p(rC)-(dG)I2-18
No ADPRT,
120 (100%)
+ ADPRT,
No Histone No Histone
No ADPRT, + ADPRT,
p(rA)-(dT)I2_I8
81 (38% inhib.)
+ Histone
109
+ Histone
56.8
(100%)
12.9
(87% inhib.)
58.2
30 (75% inhib.)
6.7
(88% inhib.)
RT concentration was 0.02 ~g/test consisting of ~ m M Tris-HCl ~ H 8.5 at 25~C), 8 mM SgCl2, i0 mM DDT, 0.5 mM [ ~ - ~ P ] T T P or [aP]dGTP, 1 ~ g of p(rA). (dT)12_18 or p(rC). (dG)12_18 in 50 ~i volume. ADPRT: 0.67 uM; histones: 5 ~g/test, if present. Incubation was carried out at 37°C for 5 min and acid p r e c i p i t a b l e radioactive product assayed as described in Methods. Values in the table are given as pmoles of deoxyribonucleotides incorporated in 5 min at 37°C. Data are the averages of 3 parallel assays, not differing more than ±10% from the mean value.
also
shown.
its R N a s e
Contrary
H activity
to
was
the
DNA
synthetic
activity
by ADPRT
(results
unaffected
of
HIV
RT,
n o t shown).
Discussion The
inhibitory
site
of R T
template
DNA
hybrids.
has
only
adds per
effect
to a l r e a d y
which
we
probably
observe ADPRT
1 ~M
Therefore
ADPRT
to
RT
relevance. nuclear the
cytosol
species lase
templates
(27)
cellular
(21)
(22).
of A D P R T (23-26) in
range
conclude
within
However
the
were
isolated
antiretroviral
cell
evidence
identified
may
RT
RT
as w e l l
is a selfof A D P R T
are
well
at
within
which
binding
is of
pharmacological
is a p r e d o m i n a n t l y
is m a i n l y that
as
properties
in nuclei,
have
inasmuch
of A D P R T
501
observation
a drug-induced
exists
ribonucleoprotein action
of
acid
in p h y s i o l o g i c a l l y
that ADPRT
retroviral
is n o t e x c l u s i v e ,
This
formation
existing
to R N A -
nucleic
concentrations
that
thought
whereas
(ADPR)n
The
of A D P R T
colligative role
inhibition
concentration
It is g e n e r a l l y
enzyme
lization
ADPRT.
we
that
at t h e p r i m e r
hybrid
ADPRT.
a prominent
enzymatic
significant
the actual (20).
of
the
on
evidence
play
binding
binding,
efficacy
and that
activity
on D N A s y n t h e s i s
a novel
strong
coenzymic
cells
regulatory
the
accumulating
s_ee of A D P R T
functioning
illustrates
Despite
minimal
of A D P R T
the
operative
in
nuclear
loca-
as c y t o p l a s m i c
ADPRT
(ADPR) n g l y c o h y d r o -
particles. ligands,
which
Therefore may
a
induce
Vol. 180, No. 2, 1991
the
binding
example
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
of A D P R T
of which
to
RT
is reported
templates,
cannot
in an accompanying
be
excluded,
paper
an
(19).
Acknowledgments This r e s e a r c h was s u p p o r t e d by A F O S R 8 9 - 0 2 3 1 and by funds of O c t a m e r R e s e a r c h Foundation. The f o l l o w i n g r e a g e n t was o b t a i n e d t h r o u g h the AIDS R e s e a r c h and R e f e r e n c e R e a g e n t Program, NIAID, NIH: H I V r e v e r s e t r a n s c r i p t a s e f r o m the Univ. of A l a b a m a at Birmingham, Center for AIDS Res., Gene Expression Core Facility.
References
i. 2. 3. 4.
5. 6. 7. 8. 9. i0. ii. 12. 13. 14. 15. 16.
17. 18. 19. 20.
Yamanaka, H., Penning, C.A., Willis, E.H., Wasson, D.B. and Carson, D.A. (1988) J. Biol. Chem. 263, 3879-3883. Ludwig, A., Behnke, B., Holtlund, J. and Hilz, H. (1988) J. Biol. Chem. 263, 6993-6999. Bauer, P.I. and Kun, E. (1985) in "ADP-ribosylation of Proteins (eds. Althaus, F.R., Hilz, H. and Shall, S.) springer-Verlag, Berlin, Heidelberg, pp. 63-74. Simonin, F., de Murcia, J.-M., Poch, O., Muller, S., Gradwohl,G., Molinete, M., Penning, C., Keith, G. and de Murcia, G. (1990) J. Biol. Chem. 256, 19249-19256. Buki, K.G. and Kun, E. (1989) Biochemistry 27, 5990-5995. Romaschin, A.D. and Kun, E. (1981) Biochem. Biophys. Res. Commun. 102, 952-957. Zahradka, P. and Ebisuzaki, K. (1982) Eur. J. Biochem. 127, 579-585. Nobori, T., Yamanaka, H. and Carson, D.A. (1989) Biochem. Biophys. Res. Commun. 163, 1113-1118. Buki, K.G., Bauer, P.I., Mendeleyev, J., Hakam, A. and Kun, E. (1991) FEBS Lett. (in press). Sastry, S.S., Buki, K.G. and Kun, E. (1989) Biochemistry 28, 5670-5680. Sastry, S.S. and Kun, E. (1990) Biochem. Biophys. Res. Commun. 167, 842-847. Sastry, S.S. and Kun, E. (1988) J. Biol. Chem. 263, 15051512. Buki, K.G., Kirsten, E. and Kun, E. (1987) Anal. Biochem. 167, 160-166. Bauer, P.I., Buki, K.G. and Kun, E. (1990) FEBS Lett. 273, 6-10. Hakam, A., McLick, J., Buki, K.G. and Kun, E. (1987) FEBS Lett. 212, 73-78. Current Protocols in Molecular Biology (1987) (eds. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., seidman, J.G., Smith, J.A. and Struhl, K.) Green Publishing Associates and wiley Interscience, New York, p2.5.1. Starnes, M.C. and Cheng, Y-chi (1989) J. Biol. Chem. 264, 7073-7077. Purnell, M.R. and Whish, W.J.D.(1980) Biochem. J. 185, 775777. Cole, G.A., Bauer, G., Kirsten, E., Mendeleyev, J., Bauer, P. I., Buki, K.G., Hakam, A. and Kun, E. (1991) Biochem. Biophys. Res. Commun. 180, 504-514. Bauer, P.I., Buki, K.G., Hakam, A. and Kun, E. (1990) Biochem. J. 270, 17-26. 502
VOI. 180, No. 2, 1991
21.
22. 23. 24. 25. 26. 27. 28.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Althaus, F.R. and Richter, C. (1987) in "ADP-ribosylation of Proteins" Mol. Biol. Biochem. Biophys. Ser. 37, SpringerVerlag, New York. Haseltine, W.A. (1991) The FASEB J. 5, 2349-2360. Furneaux, H.M. and Pearson C.K. (1980) Biochem. J. 187, 91-103. Roberts, J.H., Stark, P., Giri, C.P. and Smulson, M. (1975) Arch. Biochem. Biophys. 171, 305-315. Elkaim, R., Thomassin, H., Niedergang, C., Egly, J.M., Kempf, J. and Mandel, P. (1983) Biochimie 65, 653-659. Thomassin, H., Niedergang, C. and Mandel, P. (1985) Biochem. Biophys. Res. Commun. 133, 654-661. Thomassin, H. and Mandel, P. (1989) in "ADP-ribose Transfer Reactions" (Jacobson, M.K. and Jacobson, E.L., eds.) Springer-Verlag, New York, pp 125-129. Eki, T. and Hurwitz, J. (1991) J.Biol. Chem. 266 3087-3100.
503
