273

Mutation Research, 52 ( 1 9 7 8 ) 273--284 © Elsevier/North-Holland Biomedical Press

ENHANCEMENT OF REPAIR REPLICATION IN MAMMALIAN CELLS BY H Y D R O X Y U R E A *

JUDITH M. CLARKSON

Univeristy of Texas System Cancer Center, Science Park--Research Division, Smithville, Texas 78957 (U.S.A.) (Received 16 February 1978) (Revision received 11 May 1978) (Accepted 22 May 1978)

Summary The effect of h y d r o x y u r e a on DNA repair replication has been studied in Chinese hamster ovary cells. Mitotic cells were treated with UV irradiation, methyl methanesulfonate or nitrogen mustard and incuated in the presence of each of the 4 [3H]deoxyribonucleosides plus BrdUrd and FdUrd for 2 h. The a m o u n t of repair replication was quantitated on CsC1 gradients and similar values were obtained for each nucleoside. In all cases addition of HU during the incubation period increased these values approximately 2-fold. Following MMS treatment, pool sizes for each of the nucleosides were estimated by varying the a m o u n t of exogenously supplied nucleoside. They were found to be insensitive to the addition of HU and it is concluded that the increased incorporation of [3H]deoxyribonucleosides in the presence of HU reflects an increased amount of repair replication.

Introduction

Treatment of mammalian cells with a great variety of agents results in DNA damage and subsequent DNA repair. This repair synthesis is usually detected as uptake of [3H]dThd, either as non-semi-conservative synthesis on CsC1 gradients [15], or, in the absence of normal DNA replication, by autoradiography or TCA extraction of the cellular material [14,15]. H y d r o x y u r e a (HU) is frequently used in these experiments to eliminate [3H] dThd uptake by normal semi-conservative replication. It is a p o t e n t inhibi* This work was supported by National Cancer Institute Grant CA-19281. A b b r e v i a t i o n s : BrdUrd, bromodeoxyttridine; FdUrd, fluorodeoxyuridine; HU, hydroxyuxea; dAdo, d e o x y a d e n o s i n e ; dCyd, deoxycytidine; dGuo, d e o x y g u a n o s i n e ; dThd, thymidine; MMS, methyl m e t h a n e s u l f o n a t e ; H N 2 , nitrogen mustard; CHO, Chinese hamster ovary.

274 tor of DNA synthesis [24] and is frequently used in cell cycle experiments to prevent G1 cells from entering S phase [18]. The primary cause of this inhibition is thought to be due to interference with ribonucleotide reduction, an effect that can be reversed by removal of the HU or by addition of deoxyribonucleosides [ 1,3,24]. Cleaver [5] demonstrated, using a variety of inhibitors of DNA synthesis, that repair replication of UV-damaged DNA was not necessarily affected by such drugs and, in particular, that HU was n o t an inhibitor of this synthesis. However, Djordjevic and Tolmach [9] had already shown t h a t HU decreased the survival of UV-irradiated HeLa cells. In addition, there have been conflicting reports on the effect of HU on other aspects of DNA repair. Regan et al. [16] f o u n d no reduction in the rate of loss of dimers from UV-irradiated human cells in the presence of HU and Wolff [23] could demonstrate no delay in the repair of X-ray-damaged chromosomes, despite previous reports by Prempree and Merz [13] showing an inhibition. Similar results were obtained by Ben-Hur and Ben-Ishal [2] who demonstrated, following UV irradiation, an inhibition of strand rejoining, the final stage of the DNA repair process. This inhibition was n o t confirmed for bovine cells [7] and could n o t be demonstrated in X-irradiated mammalian cells [10,17]. The effect of HU on strand rejoining described by Ben-Hur and Ben-Ishai [2] was found by them to be reversible by the addition of all 4 deoxyribonucleosides. They concluded that the primary effect of HU is on endogenous synthesis of the nucleotides resulting in a depletion of the pools. This conclusion was also reached in a recent paper by Collins et al. [8]. Similarly, inhibition of DNA synthesis by HU has been attributed to a decrease in pool size [19]. We have been aware for some time that HU affects the a m o u n t of 3H-dThd incorporated by repair replication. In view of the proposed use of unscheduled DNA synthesis as an assay system for the detection of potential carcinogens, it was felt that the indiscriminate use of HU might lead to inaccurate quantitation. There have been several reports [8,12,20] showing that the level of unscheduled DNA synthesis after UV irradiation was altered in the presence of HU and in one case [8], that this effect was dependent on the concentration of exogenous dThd used. In this study the uptake of all 4 deoxyriboncleosides by cells damaged by a variety of agents has been considered. Uptake of each nucleoside was increased in the presence of HU. Pool sizes were estimated by varying the a m o u n t of exogenously supplied nucleoside and in each case was found to be insensitive to the addition of HU. Materials and methods

Cell line and culture techniques CHO cells were maintained as monolayer cultures in McCoy's 5A Medium (GIBCO, Grand Island, N.Y.) with 20% fetal calf serum as described by Humphrey et al. [11]. For the experiments described here, mitotic cells were used in order to minimize [3H]dThd uptake for semi-conservative replication. 1.5 × 107 cells were seeded into 32 oz. prescription bottles and incubated overnight. For the double-label experiments [14C]dThd was added to a final concentration of 0.001 gCi/ml. Unattached and dead cells were shaken from the

275 monolayers and the medium replaced by that containing 0.06 pg/ml colcemid. The cells were incubated for an additional 2 h, thus allowing accumulation of mitotic cells [21]. Gently shaking the bottles caused these cells to be released into the medium [22]. The mitotic cells were pelleted at 1000 × g , washed once in fresh medium and resuspended in medium at a concentration of 106 cells/ml.

Drug treatment MMS was obtained from Eastman Kodak, Rochester, N.Y. and HN2 from Merck, Sharp and Dohme, West Point, Pa. Each was diluted with saline immediately prior to use and a concentrated solution added to the cell suspension. They were incubated for 30 min at 37°C and washed once in medium before plating in radioactive medium. UV irradiation Cells for UV irradiation were plated into fresh medium in the requisite number of 6 cm petri dishes. They were incubated for 1 h at 37°C to allow attachment. Irradiation was by means of two 15-watt General Electric germicidal lamps emitting predominantly 254 nm light at a fluence of 0.55 j/m2/sec. The cells at the edges of the plates, which would be shielded from the irradiation by the sides of the dishes, were removed with a sterile cotton swab and the remaining cells were washed twice with saline to remove traces of medium. Labeling o f cells Immediately following treatment the cells were incubated for 2 h in medium containing tritiated deoxyribonucleoside. For the gradient experiments, 10 pCi/ml [3H]nucleoside was present, together with 10 -s M BrdUrd, 10 -6 M FdUrd + 2 mM HU. For the remaining experiments two protocols were used. Either 5 #Ci/ml of [3H]nucleoside was added with varying concentrations of unlabeled nucleoside (10-6--10 -s M), or, the a m o u n t of labeled nucleoside was varied and the total amount of nucleoside was adjusted to 10 -6 M. FdUrd and/or HU were added as indicated in the figure legends. Radioactive deoxyribonucleosides were obtained from Schwarz-Mann (Orangeburg, N.Y.) and had the following specific activities: dAdo 6.5 Ci/mM; dCyd 22 Ci/mM; dGuo 6.5 Ci/mM and dThd 13 or 60 Ci/mM as indicated in the legends. Isolation o f DNA and CsCl gradients The cells were trypsinized and the nuclei isolated as described by Clarkson and Hewitt [4]. This procedure removed most of the RNA, protein and soluble nucleosides. The nuclei were resuspended in 10 mM Tris, 1 mM EDTA and lysed with sodium laurylsulfate.The suspension was made 0.1 N with NaOH and 4.5 ml added to 1 g Cs2SO4 and 4.8 g CsC1. Gradients were generated in a Beckman 50Ti angle rotor at 40,000 rpm for 45 h. Fractions were collected by piercing the b o t t o m of the tubes. Each was assayed for optical density at 260 nm and 100 lambda pipetted onto Whatman G F / A filters for radioactive determinations. Incorporation o f [3H]nucleosides into DNA measured in cell lysates The cells were trypsinized and resuspended in saline. 1% sodium dodecyl sat-

276 cosinate (Geigy) was used to lyse the cells. In experiments involving nucleosides other than thymidine, 50 pg/ml RNase (Sigma) or 0.1 N NaOH was added for 15 min at 60°C to degrade RNA. Identical results were obtained for both procedures. An equal volume of cold 10% TCA was then added. Samples were precipitated onto Whatman GF/C filters and washed with 5% TCA and ethanol. Dried filters were solubilized with NCS Amersham (Arlington Heights, Ill.) and a toluene fluor containing PPO and POPOP added for counting. Results

In Table 1 data are presented for the incorporation into DNA of each of the [3H]nucleosides by repair replication following treatment of mitotic cells with UV light, MMS or HN2. The data are expressed as dpm 3H/pg of DNA, assuming a 20% counting efficiency for 3H and an extinction coefficient of 0.028 cm2/pg for absorption of single-stranded DNA at 260 nm. In addition, an identical sample was incubated in the presence of 2 mM HU. For each t y p e of treatment the incorporation of the nucleosides is increased in the presence of HU resulting in approximately a doubling of the specific activity of the DNA. One explanation for this result is that HU reduces the pool size and thus increases the availability of exogenously supplied [3H]nucleoside for DNA synthesis. The pool size of each deoxyribonucleoside has been estimated by varying the concentration of nucleoside supplied to the cell (Figs. 1 and 2). Pool sizes are calculated from the following equation which utilizes data for [3H]nucleoside incorporation into ~4C-prelabeled DNA. Let X = dpm of 3H contributed by medium; A = total molarity of nucleoside (N) contributed by medium; and P = molarity of N contributed by pool. Thus, the specific activity of newly synthesized DNA = ( X / A + P) 3H dpm/mole of N incorporated. However, we are measuring the 3H to 14C ratio as a function of exogenous nucleoside, where 14C counts represent the a m o u n t of old DNA, and 3H counts represent the a m o u n t of new DNA synthesized. The following TABLE 1 T H E SPECIFIC A C T I V I T Y OF DNA L A B E L E D WITH [ 3 H ] D E O X Y R I B O N U C L E O S I D E S PORATED DURING REPAIR REPLICATION

INCOR-

M i t o t i c cells w e r e t r e a t e d w i t h MMS, U V light or H N 2 as d e s c r i b e d in Materials and m e t h o d s . T h e y w e r e t h e n i n c u b a t e d for 2 h in m e d i u m c o n t a i n i n g 10 -5 M B r d U r d , 10 -6 M F d U r d , 1 0 ~ C i ] m l [ 3 H ] n u c l e o s i d e +2 m M H U . T h e D N A w a s i s o l a t e d o n alkaline CsC1/Cs2SO 4 gradients and the s p e c i f i c a c t i v i t y o f the D N A b a n d i n g in t h e r e g i o n o f t h e gradient c o r r e s p o n d i n g t o u n s u b s t i t u t e d D N A , e s t i m a t e d . T h e d a t a is e x p r e s s e d as d p m / ~ g D N A and r e p r e s e n t s t h e m e a n of 3 d e t e r m i n a t i o n s for e a c h value. Nucleoside

Treatment 3 × 10 -3 M MMS

10 J m -2 U V

10 -4 M H N 2

dAdo dAdo + HU dCyd dCyd + HU

863 1950 2300 9090

955 3480 4623 9270

355 607 852 2260

dGuo dGuo + HU dThd dThd + HU

1208 1950 606 1462

1226 1986 1110 2390

416 843 253 444

277 o d Ado • dCyd

0.2

~

./'~9

/ /

dThd~

./" ~O.P

o

i

i

5x10 -s

lO-S

Total Nucleoside Concentrofion (M) Fig. 1. T h e u p t a k e o f [ 3 H ] d e o x y r i b o n u c l e o s i d e s d u r i n g r e p a i r s y n t h e s i s in t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s o f t o t a l n u e l e o s i d e . M i t o t i c cells ( p r e l a b e l e d w i t h [ 1 4 C ] d T h d ) w e r e t r e a t e d w i t h 3 X 1 0 - 3 M M M S f o r 3 0 r a i n a~nd i n c u b a t e d f o r 2 h in m e d i a c o n t a i n i n g 5 / ~ C i / m l [ 3 H ] d e o x y r i b o n u e l e o s i d e s a n d v a r y i n g c o n c e n t r a t i o n s ( 1 0 - 6 - - 1 0 -5 M) o f u n l a b e l e d n u c l e o s i d e . T h e cells w e r e t h e n h a r v e s t e d , s u s p e n d e d in saline, l y s e d a n d i n c u b a t e d f o r 1 5 m i n in 0 . I N N a O H a t 6 0 ° C , f o l l o w e d b y T C A p r e c i p i t a t i o n .

are needed to convert the above unit

(3H/14C).

(X/A +P) into the measured unit

Let r = ratio of new to old DNA; c = specific activity of old DNA; and E = 3H counting efficiency. Then, c rE

14C cpm mole N (old)

-

Since

×

mole N (old) mole N ( n e w )

3H dpm - 3Hcpm

X

(c/rE) is constant for all experimental conditions, let this = k. Rearrang13

D

0.2 d Ado 13 No Hu o + Hu

dThd~o

dCyd

--I.~¢: to

b

0.1

0

i 5x10-6

t 10-5

I

5xlO"

6

J

I0 -5

Y i

5x10-6

i

10-5

Totol Nucleoside Concenfrolion (M) Fig. 2. T h e e f f e c t o f H U o n t h e u p t a k e o f [ 3 H ] d e o x y r i b o n u c l e o s i d e s d u r i n g r e p a i r s y n t h e s i s . E x p e r i m e n t a l d e t a i l s axe i d e n t i c a l t o t h o s e in Fig. 1, H U b e i n g a d d e d t o s o m e o f t h e s a m p l e s d u r i n g t h e 2 - h i n c u b a tion.

278 ing the above: 3H cpm = 3H dpm X 14C cpm × k mole N incorporated = A +/7 Let 3H cpm -R 14C cpm Then, kR

X X A +pOr---kR

A +P

In Fig. 1, for each nucleoside 1/R is pl ot ted against A. X / k is a constant for each e x p e r i m e n t and thus, (A1 +P) • l / R 2 = (A2 + P ) • l / R 1. The values of 1/R for A = 10 -6 or 10 -s were substituted in this equation and P calculated. In Fig. 2, data are shown for a series of experiments in which 3 of the nucleosides were tested in the presence or absence of HU. The results from these two sets o f data are shown in Table 2. It is evident t hat dCyd and d T h d have pool sizes of 3--7 × 10 -7 M and that the pools of dA do and dG uo are approximately 10 times larger. In addition, it is clear that HU does n o t effect the pool size to any appreciable e x t e n t -- the greatest effect being on dCyd, an average value o f 4.7 X 10 -7 M being lowered to 3.6 X 10 -7 M. This would n o t a c c o u n t for a doubling of the specific activity of the DNA as shown in Table 1, and suggests th at HU does in fact affect the total incorporation of nucleosides into damaged DNA. In Figs. 3--5 [3H]dThd incorporation in the presence of cold BrdUrd or cold

TABLE 2 THE POOL SIZE FOR EACH OF THE 4 DEOXYRIBONUCLEOSIDES D A T A IN F I G S . 1, 2 A N D 5

CALCULATED

FROM

THE

I n Figs. 1 a n d 2 X a n d k are c o n s t a n t a n d P c a n b e c a l c u l a t e d b y u s i n g v a l u e s o f R c o r r e s p o n d i n g t o 2 diff e r e n t v a l u e s o f A, I n Fig. 5, k is a g a i n c o n s t a n t a n d f o r a n y g i v e n v a l u e o f X, R c a n be d e t e r m i n e d f o r A = 10 -5 or 10 -6 M. Nucleomde

dAdo dCyd

P o o l size (M)

Sottrce o f d a t a

--HU

+HU

3.6 X 1 0 -6 5.0 X 10 -6

3.5 X 10 -5

4.3 4.7 7.4 2.4

X × X X

10 10 10 10

-7 -7 -7 -7

dGuo

3.1 × 10 -6

dThd

3.1 5.2 7.0 4.2

X X X X

10 10 10 10

-7 -7 -7 -7

2.4 X 1 0 -7 4 . 8 X 10 -7

Fig. 1 Fig. 2 Fig. Fig. Not Not

1 2 shown shown

Fig. 1 5.0 X 10 - 7

Fig. Fig. Not Fig.

1 2 shown 5

279 o

dThd

o

dThd+Hu

o

* BrdUrd BrdUrd +Hu

~00

/

o200

/

/ [

~oo

/; 0

"

30

0

60

Concentration of 3H dThd (~Ci/ml) Fig, 3. T h e u p t a k e o f d i f f e r e n t c o n c e n t r a t i o n s o f [ 3 H ] d T h d d u r i n g r e p a i r s y n t h e s i s in the p r e s e n c e of u n l a b e l e d T d R o r B r d U r d + HU, Mitotic cells ( p r e l a b e l e d w i t h [ 1 4 C ] d T h d ) , w e r e t r e a t e d w i t h 3 X 10 -3 M MMS f o r 30 m i n a n d i n c u b a t e d f o r 2 h in m e d i u m c o n t a i n i n g a t o t a l c o n c e n t r a t i o n of 1 0 -6 M d T h d or d T h d + B r d U r d . A t t h e h i g h e s t c o n c e n t r a t i o n o f [ 3 H ] d T h d (60 ~ C i / m l ) n o u n l a b e l e d n u e l e o s i d e w a s a d d e d . F o r 6, 12 or 3 0 / ~ C i / m l t h e c o n c e n t r a t i o n w a s m a d e 1 0 -6 M b y a d d i n g u n l a b e l e d d T h d or B r d U r d . 2 m M H U w a s a d d e d as i n d i c a t e d .

200

200

o dThd o dThd+FdUrd • BrdUrd

6

o

olO- M

BrdUrd + FdUrd

/o

"B c)

L loo

,~ ioo

%

/

0

I

I

30

60

Concentrnfion of 3HdThd ( ~ C i / m l )

00

3O I

dThd],CVml

6O I

,

Fig. 4. T h e u p t a k e o f d i f f e r e n t c o n c e n t x a t i o n s o f [ 3 H ] d T h d during repair s y n t h e s i s in the p r e s e n c e o f u n l a b e l e d d T h d or BrdUrd + F d U r d . E x p e r i m e n t a l details are i d e n t i c a l to t h o s e in Fig, 3, 1 0 -6 M F d U r d replacing t h e H U in s o m e s a m p l e s . Fig. 5. T h e u p t a k e o f d i f f e r e n t c o n c e n t r a t i o n s of [ 3 H ] d T h d d u r i n g r e p a i r s y n t h e s i s in t h e p r e s e n c e of u n l a b e l e d B r d U r d . E x p e r i m e n t a l details r e s e m b l e t h o s e in Fig. 3, b u t t h e t o t a l c o n c e n t r a t i o n o f n u c l e o side was m a d e 10 -6 o r 1 0 -5 M w i t h u n l a b e l e d B r d U r d .

280 dThd is shown for a constant a m o u n t of total nucleoside (10 .6 or 10 -s M). In this case R is plotted against X. As demonstrated by Cleaver [6], it is evident that there is no preference for dThd over BrdUrd, all the points falling on a straight line, i.e., R is proporational to X. In addition, the data in Figs. 3 and 4 directly compare [3H]dThd uptake when the total nucleoside concentration is made 10 -6 M with dThd or BrdUrd. In Fig. 3, data are also shown for these same experimental points in the presence of 2 mM HU. The results presented previously are confirmed for both sets of data, HU increasing the incorporation 2-fold. In Fig. 4 the effect of FdUrd on the incorporation of exogenously supplied nucleosides is shown. FdUrd enhances the uptake of BrdUrd during normal, semi-conservative replication (data not shown) but these data suggest that it has no effect on the incorporation of either dThd or BrdUrd during repair synthesis. Fig. 5 shows that even in the presence of 10 -s M BrdUrd, [3H]dThd incorporation is proportional to its concentration in the medium. From this data, using the formula X / A + P = k R , P can again be calculated. For any given value of X, R 1 ( A j + P ) = R 2 ( A 2 + P ) . Using A = 10 -6 or 10 -s and the corresponding value for R, a value of 4.2 × 10 -7 M for P is obtained, consistent with the other estimates for the dThd pool shown in Table 2. In addition, it is possible to calculate k. For X = 6 0 gCi/ml and A = 1 0 -6M, R = 1 7 6 . Since there are 2 . 2 × 1 0 6 dpm/pCi, X = 60 × 2.2 × 106 dpm/ml. Then, X _ 60 × 2.2 × 106 dpm/ml = 1017 dpm/M = k R A +P 1.4 × 10 .6 M/1 Using Avagadro's number, k R = (1017/6 × 1023) dpm/nucleotide. Therefore, k = (1017/6 × 1023 × 176) = 10 -9 dpm/nucleotide. There are 10 l° nucleotides/ cell, so k = 10 dpm/cell. For each experiment approximately 2.5 × 10 s cells were used per sample, and each sample averaged 125 14C cpm. Therefore, c = 5 X 10 -4 cpm/cell E=0.2

It is now possible to calculate r; k - c rE '

therefore,

5 × 10 .4 cpm/cell r = 0.2 c p m / d p m X 10 dpm/cell = 2.5 × 10 .4 (0.025%)

This value can now be compared with the data obtained in Table 1. Since we have values for each of the pool sizes together with the specific activities of the [aH]nucleoside, it is possible to calculate the total a m o u n t of nucleoside incorporated. In Table 3 the specific activity of each nucleoside is adjusted to take into account the nucleoside contributed by the pool. Since there is no preference for dThd or BrdUrd (Figs. 3--5), the value for the effective specific activity of [3H]dThd relies heavily on the concentration of the BrdUrd (10 .5 M). The 3H dpm/pg DNA shown in Table 1 can now be converted to total nucleoside incorporated; e.g., following 3 × 10 .3 M MMS, the specific activity of DNA labeled with dAdo = 850 dpm/pg. Since there are 2.2 × 106 dpm/pCi this converts to 3.9 X 10 .4 pCi/#g.

281 TABLE 3 THE SPECIFIC ACTIVITY OF DEOXYRIBONUCLEOSIDES AVAILABLE FOR REPAIR REPLICAT I O N , C O R R E C T I N G F O R U N L A B E L E D N U C L E O S I D E C O N T R I B U T E D BY E N D O G E N O U S S Y N THESIS G i v e n t h e s p e c i f i c a c t i v i t y o f t h e [ 3 H ] n u c l e o s i d e , t h e t o t a l c o n c e n t r a t i o n o f n u c l e o s i d e a d d e d c a n b e estim a t e d . V a l u e s f o r t h e p o o l size a r e t a k e n f r o m T a b l e 2 a n d t h u s t h e t o t a l n u c l e o s i d e c o n c e n t r a t i o n a n d f i n a l specific a c t i v i t y are k n o w n . Nucleoside

Sp. A c t . (Ci/mM)

Nucleoside a d d e d (M) (10 pCi/ml)

P o o l (M)

Total nucleoslde concentration (M)

Final specific activity (Ci/mM)

dAdo dCyd dGuo dThd

6.5 22 6.5 60

1.5 X 10 -6 0.5 X 10 -6 1.5 X 10 -6 1 0 -5 (as B r d U r d )

4 5 3 5

5.5 X 1 0 -6 10 -6 4.5 X 10 -6 1 0 -5

1.8 10.0 2.2 1.0

X 10 X 10 X 10 X 10

-6 -7 -6 -7

The final specific activity o f [3H] d A d o = 1.8 C i / m M (Table 3) 3.9 X 10 -4 - 1.8 X 1 0 6 m M / p g = 0 . 2 2 X 10 -6

pM/pg

(MW o f n u c l e o s i d e is a p p r o x i m a t e l y 2 5 0 ) = 0 . 2 2 × 10 -6 X 2 5 0

pg/pg

= 5.5 X 10 -s pg d A d o / p g D N A ( A T c o n t e n t o f m a m m a l i a n cells is 60%) T h e r e f o r e , t h e total n u c l e o t i d e r e p l a c e m e n t = 1.8 X 10 -4

pg/pg D N A

= 0.018%. TABLE 4 THE DATA FROM TABLE 1 FOR ESTIMATES OF REPAIR SYNTHESIS CONVERTED FROM 3H d p m / ~ g D N A T O # g N U C L E O S I D E / ~ g D N A (X 1 0 5 ) A N D , IN P A R E N T H E S E S , % O F D N A R E P L A C E D U s i n g t h e final s p e c i f i c a c t i v i t i e s f r o m T a b l e 3, d p m c a n b e c o n v e r t e d t o /~M a n d t h u s ~ g o f n u c l e o s i d e . T h e % o f e a c h n u c l e o s i d e i n t h e D N A is t h e n u s e d t o give t h e t o t a l a m o u n t o f D N A r e s y n t h e s i z e d . Nucleoside

Treatment 3 X 1 0 - 3 M MMS

10 Jm -2

10 -4 M HN2

dAdo dAdo + HU dCyd dCyd + HU

5.5 12.5 2.8 10.0

(0.018%) (0.042%) (0.014%) (0.050%)

6.0 22.0 5.3 10.0

(0.020%) (0.073%) (0.027%) (0.050%)

2.3 3.8 1.0 2.5

(0.008%) (0.013%) (0.005%) (0.013%)

dGuo dGuo + HU dThd dThd + HU

6.2 10.0 6.8 17.0

(0.031%) (0.050%) (0.023%) (0.057%)

6.3 10.0 12.5 27.0

(0.032%) (0.050%) (0.041%) (0.090%)

2.0 4.3 2.8 5.0

(0.010%) (0.022%) (0.009%) (0.017%)

282 Table 4 shows the data for Table 1 converted from ~H dpm/pg DNA to pg nucleoside/pg DNA and, in parentheses, percentage of total nucleosides replaced for each treatment condition (+HU). The values derived from each nucleoside agree very closely, for 3 X 1 0 - 3 M MMS ranging from 0.014 to 0.031% without HU and 0.042 to 0.057% with HU. The value of r calculated above (0.025%) was for cells treated with 3 X 10 -a M MMS and incubated in the absence of HU. It is evident that both techniques yield very similar data. Discussion

The experiments presented here were designed to test the effect of HU on DNA repair synthesis. By using several different DNA damaging agents, and monitoring the incorporation of all 4 deoxyribonucleosides, it has been possible to conclude that enhancement of repair synthesis by HU is a general phenomenon. Using two different techniques, quantitating the incorporation of [3H]nucleoside into DNA, and measuring the uptake per cell, identical results were obtained, i.e. by substituting each set of data in the equation, a similar value for r (the percentage of newly synthesized DNA) was evident. The effect of HU on repair synthesis has been studied in UV-irradiated cells of several different origins. Smith and Hanawalt [20] found a 25--40% enhancement of repair synthesis in Wl-38 cells and Lampidis and Little [12] showed a greater than 2-fold increase in another human diploid line. However, Cleaver [5] showed no effect of HU on repair synthesis in HeLa cells and Collins et al. [3], using this same cell line, f o u n d that increased incorporation of exogenously supplied dThd was related to the concentration of dThd a d d e d - less than 5 pM dThd was necessary to demonstrate an effect. We have no evidence for a concentration-dependent e f f e c t - HU increasing the incorporation of [3H]dThd in the presence of both 10 -6 M and 10 -s M dThd or BrdUrd. In both of these reports for HeLa cells UV doses of 100 Jm -2 or higher were used. Thus, it is possible that at these high doses, or with this particular cell line, less conclusive results are possible. Several reports have intimated that HU effects both repair and S-phase synthesis by reducing endogenous synthesis of nucleotides [2,8,19], and indeed, the increased incorporation of exogenously supplied [3H]nucleosides is consistent with this. However, the data in Figs. 2 and 3 and Table 2 clearly show that HU does not significantly reduce the pool size. This is particularly striking in the case of [3H]dThd incorporation which is enhanced in the presence of 10 -s M BrdUrd -- a 20-fold excess over the pool size. By using GI cells in this study, we have eliminated the possibility that HU is acting by inhibiting normal semi-conservative DNA synthesis, thus increasing the availability of enzymes and DNA precursors that would otherwise be used for this synthesis. It seems, therefore, that HU acts directly on the DNA repair mechanism. This conclusion is consistent with the reduced survival of UVirradiated HeLa cells incubated in the presence of HU [9]. As mentioned in the Introduction, there has been little agreement in the reports for effects of HU on the different stages of DNA repair. The data most consistent with the observations given here concern the final, strand rejoining step. Ben-Hut and BenIshai [2] and Collins et al. [8] both reported an inhibition of this step by HU.

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