Volume 12 Number 16 1984
Nucleic Acids Research
Enzymatic excision from 7-irradiated polydeoxyribonucleotides of adenine residues whose imidazole
rings have been ruptured
Lars H.Breimer
Imperial Cancer Research Fund, Mill Hill Laboratories, London NW7 IAD,
UK
Received 29 June 1984; Accepted 24 July 1984
ABSTRACT
The main forms of base d a m a g e i np o l y d e o x y a d e n y l i c acid
•y-i r r a d i a t e d u n d e r h y p o x i c c o n d i t i o n s a r e d u e t o s a t u r a t i o n
and f r a g m e n t a t i o n of t h e a d e n i n e i m i d a z o l e r i n g . A n
i r r a d i a t e d p o l y m e r w a s a n n e a l e d w i t h an e q u i m o l a r a m o u n t o f
poly (dT) to g e n e r a t e a d o u b l e - s t r a n d e d p o l y d e o x y r i b o nucleotide containing scattered damaged base residues. O n
i n c u b a t i o n of the l a t t e r w i t h p a r t i a l l y p u r i f i e d cell
e x t r a c t s of E. coli , i m i d a z o l e r i n g - o p e n e d a d e n i n e , i.e.
4,6-diamino-T-formamidopyrimidine, was released i nfree form
by a DNA g l y c o s y l a s e a c t i v i t y .
The enzyme has been purified
4,500-fold, has M r = 2 9 , 0 0 0 , and a p p e a r s to be identical with
the previously described DNA repair enzyme formamidopyrimidine-DNA glycosylase.
INTRODUCTION
X-irradiation was the first environmental agent
d e m o n s t r a t e d to i n c r e a s e t h e f r e q u e n c y of m u t a t i o n ( 1 ) . It
has since been shown to c a u s e several types of l e s i o n s i n
DNA, including single- and double-strand b r e a k s , base-free
s i t e s , and a l t e r a t i o n s of the n i t r o g e n o u s b a s e s ( 2 , 3 ) .
T h o u g h r a d i a t i o n - i n d u c e d b a s e d a m a g e in D N A is k n o w n t o m a k e
an i m p o r t a n t c o n t r i b u t i o n to t h e m u t a g e n i c and l e t h a l e f f e c t s
o b s e r v e d in v i v o ( 4 ) , f o r t e c h n i c a l r e a s o n s m o s t s t u d i e s o n
DNA lesions induced b yionizing radiation have until recently
been c o n c e r n e d w i t h the f o r m a t i o n and fate of strand
b r e a k s . I o n i z i n g r a d i a t i o n is t h o u g h t to d a m a g e b a s e s t h r o u g h
g e n e r a t i o n of hydroxyl r a d i c a l s , to which p y r i m i d i n e r e s i d u e s
are somewhat more sensitive than purine residues ( 5 ) . A DNA
g l y c o s y l a s e w h i c h c a t a l y z e s the e x c i s i o n of r i n g - s a t u r a t e d ,
fragmented and ring-contracted thymine bases from oxidized
and y - i r r a d i a t e d D N A h a s b e e n f o u n d b o t h in b a c t e r i a a n d i n
© IR L Press Limited, Oxford, England.
6359
Nucleic Acids Research
mammalian cells (6-9). However, this enzyme does not act on
a l t e r e d p u r i n e r e s i d u e s ( 1 0 ) . It h a s b e e n s h o w n f o r a d e n i n e
damaged by ionizing radiation that t h e t w o main products
formed in D N A a r e 8-oxoadenine (saturation of t h e imidazole
ring) and 4,6-diamino-5-formamidopyrimidine
(fragmentation of
the imidazole ring) (5,11).
Chetsanga and Lindahl (12) have described a DNA
glycosylase which releases 7-methyl guanine residues whose
imidazole rings have been opened
(2,6-diamino-4-hydroxy-5-Nmethylformamido-pyrimidine) from alkylated DNA. This enzyme
a l s o o c c u r s b o t h in b a c t e r i a a n d m a m m a l i a n c e l l s ( 1 3 ) , a n d
the E.coli e n z y m e h a s been h i g h l y p u r i f i e d ( 1 4 ) . W h i l e t h e
enzyme has been termed formamido- pyrimidine-DNA glycosylase,
it h a s n o t b e e n k n o w n i f t h i s ( o r a n y o t h e r ) D N A g l y c o s y l a s e
could act on t h e ring-opened forms o f u n m e t h y l a t e d adenine
a n d g u a n i n e g e n e r a t e d b y r a d i o l y s i s ( F i g . 1 ) . In t h e p r e s e n t
paper, the enzymatic excision of such y-irradiation products
of a d e n i n e r e s i d u e s is i n v e s t i g a t e d .
MATERIALS
AND METHODS
Synthesis of polydeoxyribonucleotides
Poly(dA) and poly(dT) were synthesized using calf
terminal deoxynucleotidy1 transferase ( 1 5 ) purchased from PL
B i o c h e m i c a l s . [ 8 - 3 H ? dATP ( 2 4 . 3 Ci • m m o l " 1 ) a n d [ U - 1 4 C ? dATP
(450 mCi • mmol
) were obtained from Amersham.
T h e poly
([ H ] d A ) h a d a s p e c i f i c a c t i v i t y of 7 0 0 , 0 0 0 c p m . nmol
and
1 4
1
the poly ( [ C ] d A ) 8,500 cpm • n m o l ' .
Irradiation of poly(dA)
Radioactively labelled poly(dA) w a s dialysed against 1
mM p o t a s s i u m p h o s p h a t e , pH 7 . 4 , w h i c h h a d b e e n d e g a s s e d a n d
then s a t u r a t e d w i t h n i t r o g e n . T h e p o l y m e r w a s d i l u t e d in this
buffer to a final concentration of 0.2 m M . The solution w a s
sealed in a m i c r o r e a c t i o n V g l a s s vial ( W h e a t o n S c i e n t i f i c ,
M i l l v i l l e , N . J . , U S A ) under an a t m o s p h e r e of N - , a n d
irradiated with a dose of 2 0 0 Gy at 8 ° C t o 1 2 ° C using a
Co
source of flux 37 Gy • m i n " (Gamma beam 6 5 0 - Atomic Energy
of C a n a d a L t d . , O t t a w a , C a n a d a ) . T h e p o l y m e r w a s p r e c i p i t a t e d
w i t h t w o v o l u m e s o f e t h a n o l , in t h e p r e s e n c e o f 0 . 5 M N a C l ,
6360
Nucleic Acids Research
V
NH
"
dR
(n)
dR
(iv)
F i g . l . R i n g - o p e n e d forms of a d e n i n e and the a l k y l a t i o n
p r o d u c t 7 - m e t h y 1 g u a n i n e in D N A . I: A d e n i n e ; I I : 4 , 6 - d i a m i n o 5 - f o r m a m i d o p y r i m i d i n e ; I I I : 7 - m e t h y l g u a n i n e ; IV: 2 , 6 - d i amino-4-hydroxy-5-N-methylformamidopyrimidine. Note that
t h e s e f o r m a m i d o p y r i m i d i n e s are not a t t a c h e d to d e o x y r i b o s e i n
t h e s a m e f a s h i o n as n a t u r a l l y o c c u r r i n g p y r i m i d i n e s in D N A .
r e d i s s o l v e d a n d t h e n d i a l y s e d at 4 ° C a g a i n s t 1 M N a C l / 1 0 mM
p o t a s s i u m p h o s p h a t e , p H7.4 for 20 h, f o l l o w e d by 0.1 mM
E D T A / 1 0 mM T r i s • HC1 , p H 7 . 4 , for 4 h w i t h s e v e r a l c h a n g e s .
The recovery o fradioactive polymeric material after
irradiation and dialysis was 60%. Aliquots for base analysis
of i r r a d i a t e d p o l y m e r s were h y d r o l y s e d with formic acid a t
6 0 ° C for 16 h ( 1 1 ) .
Reference compounds
8 - 0 x o a d e n i n e was prepared a c c o r d i n g to the method o f
I k e h a r a e_t^ £ j _ . ( 1 6 ) , a n d t h e i m i d a z o l e r i n g o p e n e d f o r m o f
7 - m e t h y l g u a n i n e w a s m a d e a s d e s c r i b e d b y H a i n e s e_t_ a_l_. ( 1 7 ) .
4,6-Diamino-5-formamidopyrimidine was obtained from 4,5,6triaminopyrimidine (Sigma) b yreaction with formic acid a t
56°C for 2 h, f o l l o w e d by removal of t h e f o r m i c acid t h r o u g h
evaporation under reduced pressure ( 1 8 ) . A sexpected, the
f o r m i c acid t r e a t m e n t of t r i a m i n o p y r i m i d i n e g e n e r a t e d a
c o m p o u n d w i t h t h e H P L C f r a c t i o n a t i o n p r o p e r t i e s of 4 , 6 d i a m i n o - 5 - f o r m a m i d o - p y r i m i d i n e ( 1 1 ) . DNA containing ring
o p e n e d 7 - m e t h y 1 g u a n i n e w a s p r e p a r e d as d e s c r i b e d by C h e t s a n g a
and Lindahl ( 1 2 ) .
6361
Nucleic Acids Research
Enzymatic release of radioiysis products of adenine
The irradiated poly(dA) was annealed with an equimolar
amount of poly(dT) to generate a double-stranded polymer. The
s t a n d a r d e n z y m e r e a c t i o n m i x t u r e ( 5 0 ul ) c o n t a i n e d 0 . 1 M
KC1/0.07 M Hepes • K O H , pH 7.8/1 mM EDTA/1 mM dithiothreitol/I
nmol o f d o u b l e - s t r a n d e d ,
H-labelled polydeoxyribonudeotide/
50ng ( 1 0 " u n i t s , r e f . 1 2 ) o f p u r i f i e d E .coli f o r m a m i d o pyrimidi ne-DNA glycosylase. After 20 m i n at 37°C, t h e
r e a c t i o n m i x t u r e s w e r e c h i l l e d t o 0 ° C a n d 5 ul 2 M N a C l , 1 0
ul h e a t - d e n a t u r e d c a l f t h y m u s D N A ( 2 0 m g - m l " ) a n d 1 5 0 u l
cold e t h a n o l w e r e a d d e d . After 2 0 m i n at - 7 0 ° C , t h e s a m p l e s
w e r e c e n t r i f u g e d f o r 1 5 m i n in a n E p p e n d o r f c e n t r i f u g e a n d
1 5 0 pi o f e a c h s u p e r n a t a n t w e r e r e c o v e r e d . T h e s e s o l u t i o n s
were reduced to dryness under vacuum and redissolved in H , 0
for chromatographic analysis.
The enzymatic liberation of ring-opened
7-methylguanine
residues from alkylated DNA w a s assayed as described (12).
Analytical
chromatography
R e v e r s e phase HPLC analysis w a s p e r f o r m e d on a V a r i a n
5000 Liquid C h r o m a t o g r a p h e q u i p p e d with a M C H - 1 0 column (4 mm
x 30 cm, Micro-Pac). The column was eluted with distilled H,0
c
_1
at 2 ml . m i n
for t h e first 10 m i n , and then with a 0-100%
l i n e a r m e t h a n o l g r a d i e n t at 1 ml • m i n " f o r 2 0 m i n . I o n e x c h a n g e HPLC a n a l y s i s w a s p e r f o r m e d i s o c r a t i c a l l y at 2 ml•
m i n " 1 o n a P a rti si 1 1 0 / 2 5 S C X c o l u m n ( W h a t m a n ) w i t h 2 5 m M
ammonium phosphate (pH 3.5) as eluant.
In e i t h e r c a s e 1 ml
fractions were collected. Descending paper chromatography on
Whatman 3MM paper (7) was with n-butanol: formic acid: water,
10:2:15.
T h e solvent w a s allowed to run off the paper to
increase separation. (R, values were: Adenine 0.21, 4,6diamino-5-formamidopyrimidine 0 . 0 7 ,and 8-oxoadenine 0.27).
Enzyme purification
Highly purified formamidopyrimidine-DNA glycosylase was
obtained as a side fraction during t h e purification of endonuclease III ( 1 0 ) . T h e enzyme activity w a s measured with an
alkylated DNA substrate containing ring opened 7-methylg u a n i n e residues ( 1 2 ) . Briefly, a crude cell extract of
E. col i B w a s t r e a t e d w i t h P o l y m i n - P a n d f r a c t i o n a t e d s t e p 6362
Nucleic Acids Research
wise
by p h o s p h o c e l 1 u l o s e
previously
chromatography
( 1 0 ) . The 15-fold
fractionated
by linear
gradient
DNA-cel1ulose
appeared
at 0.5 M N a C l , and they w e r e
further
fractionated
active
fraction
purified,
column.
The most
600-fold
by A c A - 5 4
after
this
described
activity
chromatography
stranded
by u l t r a f i 1 1 r a t i o n . T h i s
as
purified
on a
active
pooled
purified
was then
and
concentrated
material
gel f i l t r a t i o n .
was then
The most
step was approximately
and was obtained
single-
fractions
4,500-fold
in 1 0 % y i e l d .
RESULTS
4 ,6-Diamino-5-formamidopyrimidine
Y-irradiation
of
Radioactively
Y-irradiated,
hydrolysed
of t h e a d e n i n e
pyrimidine
instead
labelled
ethanol
with
was observed.
previously
Enzymatic
by Bonicel
release
irradiated
When
stranded
with
free
after
of
material
prepared
poly
form
of
in
polymers.
(dA) gradually
during
storage,
as
noted
4,6-diamino-5-formamidopyrimidine
which
had been
of an e q u i m o l a r
highly
purified
double-
of
poly(dT),
formamidopyrimidine-DNA
precipitation
phase
also
HPLC
with
reference
this
not shown).
Small
and analysis
(Fig.2).
co-eluted
on ion-exchange
with
made
amount
4,6-diamino-5-formamidopyrimidine
5-formamidopyrimidine
(data
no
4,6-diamino-5-
a s it w a s n o t d e t e c t e d
co-migrated
graphy
labelled,
The ring-opened
products
poly(dA),
ethanol
by r e v e r s e
released
C]
poly(dA)
irradiated
incubated
About 0.16%
e_t_ £ j _ . ( 1 1 ) .
of free
by t h e addition
glycosylase,
detected
labelled
radiation
by H P L C .
[U-
of unirradiated , freshly
base
had been
poly(dA) was irradiated,
in t h e y i e l d
[8- H ] adenine
accumulated
by
4,6-diamino-5-formamido-
is a Y - I r r a d i a t i o n p r o d u c t
However,
natant
into
of 200 Gy. When
difference
hydrolysates
are formed
and dialysed was
and analysed
of [8- H] labelled,
adenine
was
acid
was converted
formamidopyrimidine
from
poly(dA) which
precipitated
formic
by a dose
significant
residues
poly(dA)
super-
The enzymatically
authentic
HPLC
compound
amounts
was
of
4,6-diamino-
(Fig. 2) and
on paper
of material
chromatoco-
6363
Nucleic Acids Research
300
200
o 100
10
20
Time
30
( mm )
F i g . 2 . A n a l y t i c a l c h r o m a t o g r a p h y of e t h a n o l s o l u b l e m a t e r i a l
released from y-irradiated poly(dA) b ypurified E.coli
formamidopyrimidine-DNA glycosylase {50 ngprotein added t o
the standard reaction mixture). The ^H-labelled poly(dA) was
e x p o s e d t o 2 0 0 G y u n d e r N2 a n d t h e n a n n e a l e d w i t h n o n - r a d i o active poly(dT) prior t oincubation with the enzyme. (Top)
Reverse phase HPLC, Micro-pac MCH-10 ( C ^ ) . (bottom) ionexchange HPLC, Partisil 10/25 SCX. Brackets indicate the
elution o fauthentic markers, a = 4,6-diamino-5-formamidopy r i m i d i n e , b = 8 - o x o a d e n i n e , c = a d e n i n e a n d d = 2 , 4 , 6 triaminopyrimidine. Filled symbols, enzyme added; open
symbols, noenzyme. The 4,6-diamino-5-formamidopyrimidine
released e n z y m a t i c a l l y represents 0.03%, and the n o n e n z y m a t i c a l ly l i b e r a t e d a d e n i n e 0 . 1 0 % , of t h e t o t a l r a d i o a c t i v i t y in the r e a c t i o n m i x t u r e . The two a n a l y s e s were
performed o ndifferent reaction mixtures.
6364
Nucleic Acids Research
25.7K
13.7K
M
F i g . 3 . S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s o f f o r m a m i d o p y r i m i di n e - D N A g l y c o s y l a s e p u r i f i e d f r o m E . c o l i . T h e
p r o t e i n s a m p l e s were d e n a t u r e d by h e a t i n g at 100°C for 9 0 s
in t h e p r e s e n c e o f 2 % S D S a n d 5 % m e r c a p t o e t h a n o l , a n d t h e n
e l e c t r o p h o r e s e d in T r i s / g l y c i n e / S D S b u f f e r , p H 8 . 3 ( r e f . 2 2 )
on a 1 0 % p o l y a c r y l a m i d e s l a b gel t o g e t h e r w i t h r e f e r e n c e
p r o t e i n s . The protein bands were visualised by staining with
Coomassie blue. Lane a, chymotrypsinogen a n dribonuclease ; b ,
4,500-fold purified enzyme; c, 600-fold purified enzyme. The
enzyme fraction in lane b is estimated t o b e about 9 0 % pure.
migrating with unmodified adenine were released nonenzymatica11y (Fig.2). This apparent release of adenine may
be due t o d e p u r i n a t i o n , o r r e p r e s e n t r e v e r s i o n t o a d e n i n e o f
a labile radiation product.
The release of 4,6-diamino-5-formamidopyrimidine is effected
by t h e D N A g l y c o s y l a s e w h i c h l i b e r a t e s r i n g - o p e n e d 7 - m e t h y l guani ne.
The E . coli f o r m a m i d o p y r i d i m i n e - D N A g l y c o s y l a s e , an
activity of about M = 30,000 (12, 1 4 ) , has been purified
4,500-fold from E.coli . A single major protein band o f M r =
2 9 , 0 0 0 and 2 - 3 faint b a n d s w e r e o b s e r v e d o n S D S - p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s ( F i g . 3 ) , i n d i c a t i n g t h a t t h e
enzyme preparation was about 9 0 % pure. This D N A glycosylase
fraction catalyzed the release o f both ring-opened 7-methylg u a n i n e and 4 , 6 - d i a m i n o - 5 - f o r m a m i d o p y r i m i d i n e . The enzyme
apparently liberated the former compound about 1 0 times more
effectively than the latter one. However, these data m a y n o t
be directly comparable as o n esubstrate was derived from
alkylated high molecular weight DNA, while the other was a
6365
Nucleic Acids Research
heavily
ratio
irradiated
between
15-fold
activities).
fraction
by D N A - c e l 1 u 1 o s e
extracts
These
data
enzyme was responsible
ring-opened
remained
phosphocel1ulose
purified
chromatography
cell
polydeoxyribonudeotide.
the two activities
purified
the 4,500-fold
in c r u d e
synthetic
purine
were
fraction
obtained
after
due to
indicate
for t h e e x c i s i o n
when the
was compared
that
of both
with
additional
and gel f i l t r a t i o n .
unreliable
strongly
unchanged
The
(Assays
interfering
the same
kinds of
residues.
DISCUSSION
R i n g - o p e n e d p u r i n e s in D N A a r e o f i n t e r e s t a s p o t e n t i a l
cell-killing or mutagenic lesions after exposure to ionizing
radiation. However, no direct e v i d e n c e is a v a i l a b l e on their
potential m i s c o d i n g or non-coding p r o p e r t i e s . An apparently
analogous lesion is generated in a l k y l a t e d D N A as a secondary
product of 7-methy1guanine. Boiteux a n d Laval ( 1 9 ) , using an
alkylated polydeoxyribonudeotide containing thp latter type
of l e s i o n , have s h o w n that t h e r i n g - o p e n e d p u r i n e d e r i v a t i v e
b l o c k s c h a i n e l o n g a t i o n by E . c o l i D N A p o l y m e r a s e I j_n_ v i t r o .
Their data consequently indicate that t h e altered purine m a y
be regarded as a cell-killing l e s i o n .
The experiments described here have confirmed that one
of t h e c o n s e q u e n c e s o f y - i r r a d i a t i o n is s a t u r a t i o n a n d
rupture of t h e i m i d a z o l e ring of a d e n i n e s in D N A . T h e extent
of r i n g - o p e n i n g o b s e r v e d as a f u n c t i o n o f r a d i a t i o n dose w a s
s i m i l a r t o t h a t o b s e r v e d b y B o n i c e l «rt a J K ( 1 1 ) f o r D N A , a n d
by v a n H e m m e n a n d B l e i c h r o d t ( 2 0 ) f o r f r e e a d e n i n e . In
contrast, Chetsanga and Grigorian recently reported (21) a
much greater extent of conversion, and they were unable to
detect the enzymatic release of 4,6-diamino-5-formamidopyrimidine from y-irradiated DNA using a partially purified
p r e p a r a t i o n o f E . col i f o r m a m i d o p y r i m i d i n e - D N A g l y c o s y l a s e . In
the present work, the 4,6-diamino-5-formamidopyrimidine
g e n e r a t e d in a p o l y d e o x y r i b o n u d e o t i d e b y r-i rradi ati on h a s
been shown to be liberated by a D N A g l y c o s y l a s e a c t i v i t y .
This appears t o be a function of t h e previously described
formamidopyrimidine-DNA glycosylase (12), since the two
6366
Nucleic Acids Research
a c t i v i t i e s c o - p u r i f y at a c o n s t a n t r a t i o . T h e r e l a t i v e
i m p o r t a n c e o f t h i s e n z y m e in D N A r e p a i r a f t e r i o n i z i n g
radiation should n o w be evaluated by genetic m e t h o d s .
ACKNOWLEDGEMENTS
I thank C h r i s MacKay of t h e National I n s t i t u t e f o r
Medical R e s e a r c h , Mill Hill, L o n d o n , f o r a s s i s t a n c e with
Y - i r r a d i a t i o n a n d D r s . T. Lindahl a n d P. K a r r a n f o r helpful
di s c u s s i o n s .
REFERENCES
1. M u l l e r , H . J . ( 1 9 2 7 ) S c i e n c e 6 6 , 8 4 - 8 7 .
2. W a r d , J . F . a n d K u o , I. ( 1 9 7 6 ) R a d i a t . R e s . 6 6 , 4 8 5 - 4 9 8 .
3. T e o u l e , R . a n d C a d e t , J. ( 1 9 7 8 ) C h a p t e r 2 i n E f f e c t s o f
Ionizing R a d i a t i o n on D N A p p . 1 7 1 - 2 0 3 , H u t t e r m a n , J.,
Kohnlein, W. and Teoule, R., Eds., Springer Verlag, Berlin.
4. C e r u t t i , P . A . ( 1 9 7 6 ) in P h o t o c h e m i s t r y a n d P h o t o b i o l o g y of
Nucleic A c i d s , Wang, S.Y., E d . , V o l . 1 1 . , p p . 375-401,
Academi c Press , New York.
5. S c h o l e s , G. ( 1 9 7 6 ) in P h o t o c h e m i s t r y a n d P h o t o b i o l o g y o f
Nucleic A c i d s , Wang, S.Y., E d . , V o l . 1 . , p p . 5 2 1 - 5 7 7 ,
Academic Press, New York.
6. D e m p l e , B . a n d L i n n , S . ( 1 9 8 0 ) N a t u r e ( L o n d . ) 2 8 7 , 7 7 3 - 7 7 5 .
7. B r e i m e r , L . H . a n d L i n d a h l , T . ( 1 9 8 0 ) N u c l e i c A c i d s R e s . 8 ,
6199-6211.
8. K a t c h e r , H . L . a n d W a l l a c e , S . ( 1 9 8 3 ) B i o c h e m i s t r y 2 2 ,
4071-4082.
9. B r e i m e r , L . H . ( 1 9 8 3 ) B i o c h e m i s t r y 2 2 , 4 1 9 2 - 4 1 9 7 .
1 0 . B r e i m e r , L . H . a n d L i n d a h l , T . ( 1 9 8 4 ) J. B i o l . C h e m . 2 5 9 ,
5543-5548.
11. B o n i c e l , A . , M a r i a g g i , N . , H u g h e s , E . a n d T e o u l e , R. ( 1 9 8 0 )
Radiat. Res. 8 3 , 19-26.
1 2 . C h e t s a n g a , C . J . a n d L i n d a h l , T . ( 1 9 7 9 ) N u c l e i c A c i d s R e s . 6,
3673-3684.
13. M a r g i s o n , G . P . and Pegg, A . E . (1981) Proc. Natl. Acad. S c i .
USA 7 8 , 8 6 1 - 8 6 5 .
14. C h e t s a n g a , C.J., Lozon, M . , M a k a r o f f , C. a n d S a v a g e , L.
(1981) Biochemistry 2 0 , 5201-5207.
15. Kato, K . I . , G o n c a l v e s , J.M., H o u t s , G . E .and Bollum, F.J.
( 1 9 6 7 ) . J. B i o l . C h e m . 2 4 2 , 2 7 8 0 - 2 7 8 9 .
16. Ikehara, M . , Tada, H . , and M u n e y a m a , K. (1965) Chem. Pharm.
Bull. (Tokyo) 13, 1140-1142.
1 7 . H a i n e s , J . A . , R e e s e , C . B . a n d T o d d , L o r d ( 1 9 6 2 ) J. C h e m .
Soc. 5281-5288.
18. C a v a l i e r i , L . F . , T i n k e r , J . F . a n d B e n d i c h , A . ( 1 9 4 9 ) J. A m .
Chem. S o c .7 1 , 533-536.
1 9 . B o i t e u x , S . a n d L a v a l , J. ( 1 9 8 3 ) B i o c h e m . B i o p h y s .
Res. Commun. 110, 552-558.
20. van Hemmen, J.J. and Bleichrodt, J.F. (1971) Radiat. R e s .
46, 444-456.
2 1 . C h e t s a n g a , C . J . a n d G r e g o r i a n , C . ( 1 9 8 3 ) I n t . J. R a d i a t .
Biol. 4 4 , 321-331.
22. Laemmli, U. (1970) Nature (Lond.) 227, 680-685.
6367