Conversion of partially single-stranded replicating DNA to double-stranded DNA is delayed in megaloblastic anaemia

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Biochimica et Biophysica Acta, 607 (1980) 411--419 © Elsevier/North-Holland Biomedical Press

BBA 99647

CONVERSION OF PARTIALLY SINGLE-STRANDED REPLICATING DNA TO DOUBLE-STRANDED DNA IS DELAYED IN MEGALOBLASTIC ANAEMIA

R. G I T E N D R A

WICKREMASINGHE

and A. V I C T O R H O F F B R A N D

Department of Haematology, Royal Free Hospital,Pond Street,London, N W 3 2 Q G (U.K.) (Received September 11th, 1979) Key words: DNA replication; Megaloblastic anemia; Okazaki fragment; Single-stranded DNA; Double-stranded DNA

Summary DNA from phytohaemagglutinin-stimulated lymphocytes which had been pulse-labelled for 1 rain with [3H]deoxycytidine eluted as partially singlestranded DNA from columns of benzoylated napthoylated DEAE-cellulose. The label was transferred progressively into the double-stranded DNA fraction upon incubation in the presence of unlabelled deoxycytidine. The rate of transfer was slower in untreated lymphocytes from patients with megaloblastic anaemia than in corresponding control cells. A similar delay was also observed in normal lymphocytes treated with methotrexate or hydroxyurea. A close temporal correlation between the joining of Okazaki pieces (measured by alkaline sucrose gradients) and the transfer of the pulse label to double-stranded DNA suggested that the latter process represented the filling of gaps between Okazaki pieces. We suggest that this gap-filling step is retarded in megaloblastic anaemia and in cells treated with methotrexate or hydroxyurea.

Introduction Synthesis of at least one of the two daughter DNA strands at a replication fork occurs via the formation of short nascent fragments (Okazaki fragments) of up to approx. 300 nucleotides in mammalian cells [1--4]. These fragments are formed by the extension of short (about ten nucleotides) RNA primers [ 5--7 ]. DNA of higher molecular weight is formed by the excision of the RNA Abbreviations: dCyt, deoxycytidine; dThd, deoxythymidine.

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primers, filling of the gaps between adjacent fragments and ligation of the Okazaki pieces [8,9]. Pulse-labelled DNA of eukaryotic cells behaves as though it were partially single stranded [9--11]. This property of newly synthesized DNA has been attributed to the presence of unfilled gaps between Okazaki pieces. On continued incubation in unlabelled medium the pulse-labelled DNA is converted to the double-stranded form [10]. The primary defect in megaloblastic anaemia is though to be a lesion in DNA synthesis. Hoffbrand et al. [12] suggested that incomplete filling of gaps between Okazaki pieces causes the morphological and cell kinetic abnormalities associated with megaloblastic anaemia [13,14]. Affinity chromatography on benzoylated naphthoylated DEAE-cellulose permits the fractionation of fully double-stranded DNA from partially single-stranded DNA [15]. We have used this technique to demonstrate that the rate of conversion of pulse-labelled partially single-stranded DNA to double-stranded DNA is slower in cultured l y m p h o c y t e s from patients with megalobiastic anaemia than in control cells. Materials and Methods

Reagents. Benzoylated naphthoylated DEAE-cellulose was obtained from Boehringer-Mannheim GmbH, Mannheim, F.R.G. Other reagents were purchased from sources listed elsewhere [16,18]. Lymphocytes. Phytohaemagglutinin-stimulated l y m p h o c y t e s from patients with megaloblastic anaemia and control cultures obtained b y the addition of folinic acid were prepared as previously described [16]. Patients I.C. and A.M. had reduced serum vitamin B-12 (less than 100 n g . 1-1) due to pernicious anaemia. Patient J.S. had a reduced serum folate level (0.3 ~g • 1-1) due to adult coeliac disease. Normal l y m p h o c y t e s for experiments on the effects of drugs were prepared as described previously [ 16,17]. Pulse-chase procedures. Cells were concentrated to 8 . 1 0 7 . m1-1 in their culture medium and 0.15 ml aliquots labelled for 1 min at 37°C with [3H]d e o x y c y t i d i n e (20 Ci/mmol) at 750 pCi • m1-1 (37.5 • 10 -6 M). The pulse was terminated b y the addition of 8 ml of the appropriate used medium containing 10 #M unlabelled deoxycytidine and a 2 ml sample was mixed immediately into ethanol at --20°C. Further samples were taken during the incubation at the indicated times. Pulse-chase experiments on normal l y m p h o c y t e s treated with methotrexate or h y d r o x y u r e a were carried o u t similarly, except that [3H]thymidine (dThd) was used to label hydroxyurea-treated cells. Drugs were added to cultures 1 h prior to labelling, and were also present in the chase media. Uniformly labelled DNA was prepared using [ 3H]thymidine [ 18]. DNA was extracted from cells [18] and dissolved in 1 mM EDTA, 10 mM Tris-HC1 (pH 7.8) (buffer A) containing 0.3 M NaC1. DNA samples were cooled in an ice/water mixture and sonicated for 2 min in 30-s bursts using a MSE sonicator at a peak-to-peak amplitude of 20 ~A. This procedure yielded DNA fragments with an average size of 0.5 ~m as measured b y neutral sucrose gradient centrifugation (see below). Affinity chromatography on benzoylated naphthoylated DEAE-cellulose. DNA-samples (less than 10 ~g DNA) in 0.3 M NaC1/buffer A were applied to 8 × 30 mm columns of benzoylated naphthoylated DEAE-cellulose equilibrated

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in the same buffer. After rinsing with this buffer, double-stranded DNA was eluted with 1 M NaC1 in buffer A. Partially single-stranded DNA was eluted completely b y 1 M NaCl, 1.75% caffeine, 6 M guanidinium hydrochloride in buffer A. 1 ml aliquots of each fraction were counted in a toluene-based scintillation fluid containing 33% Triton X-100. Recovery of label was in excess of 85%. Sucrose gradient sedimentation. Neutral 5--20% sucrose gradients contained 1 M NaC1 in buffer A. Centrffugation was at 180 000 × g and 16°C for 2 h in the 6 × 5.5 ml rotor of the MSE Superspeed 75 centrifuge. 0.2 ml fractions were collected from the b o t t o m of the tube and processed for scintillation counting as described [16]. 5--20% alkaline sucrose gradients were run as described previously [16]. Sedimentation coefficients were obtained b y the calculation m e t h o d of Funding and Steensgaard [19]. Results

Fig. 1 summarizes data from pulse-chase experiments carried out on lymphocytes from three patients with megaloblastic anaemia. D N A was extracted from untreated or folinic acid-treated lymphocytes (controls)which had been pulselabelled for 1 rain with [SH]dCyt. W h e n sonicated to fragments of approx. 0.5 /~m, 65--85% of the pulse-labelled D N A eluted in the partially singlestranded D N A fraction during affinity chromatography. In contrast, only

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Time (min) F i g . . 2 . R a t e o f c o n v e r s i o n o f p u l s e - l a b e l l e d p a r t i a l l y s i n g l e - s t r a n d e d D N A t o d o u b l e - s t r a n d e d D N A in n o r m a l l y m p h o c y t e s p r e i n c u b a t e d f o r 1 h in t h e p r e s e n c e o f (a) 1 0 -6 M m e t h o t r e x a t e o r (b) 1 0 - 3 M h y d x o x y u r e a , e , d r u g - t r e a t e d cells; o, c o n t r o l cells. T h e p u l s e - l a b e l l i n g w a s w i t h [ 3 H ] d C y t in (a) a n d [ 3 H ] d T h d in (b). T h e a p p r o x i m a t e c p m l o a d e d o n e a c h c o l u m n w e r e (a) c o n t r o l , 1 3 0 0 0 c p m ; d r u g t r e a t e d , 2 0 0 0 c p m . (b) C o n t r o l , 8 0 0 0 c p m ; d r u g t r e a t e d , 1 0 0 0 c p m .

20% o f a sample of uniformly labelled sonicated DNA eluted as partially singlestranded DNA. Upon chasing in the presence of unlabelled dCyt, the proportion of label behaving as partially single-stranded DNA decreased in a timed e p e n d e n t manner, with a concomitant increase of label in the fully doublestranded DNA fraction (Fig. la--c). In the three experiments, the transfer of label from partially single-stranded DNA to double-stranded DNA was retarded in untreated megaloblastic lymphocytes compared to the rate in the control (folinic acid-treated) cells (Fig. 1). Experiments carried out using untreated and control lymphocytes from four further patients with megaloblastic anaemia (three pernicious anaemia, one folate deficiency) gave similar results. Analogous experiments were carried out using normal lymphocytes treated with methotrexate (Fig. 2a) or h y d r o x y u r e a (Fig. 2b). The transfer of pulse label from the partially single-stranded DNA to the double-stranded DNA fraction was retarded in cells treated with either drug when compared to control cells. H a y t o n et al. [10] have suggested that pulse-labelled DNA behaves as partially single-stranded DNA because of the existence of gaps between nascent Okazaki fragments. This view is supported by the fact that treatment of pulselabelled DNA with nucleases specific for single-stranded nucleic acids results in its conversion to double-stranded DNA with little solubilization of the pulse label [10,18]. We therefore attempted to demonstrate a temporal correlation

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between Okazaki piece joining and the transfer of pulse label from partially single-stranded DNA to double-stranded DNA. Normal l y m p h o c y t e s were concentrated to 8 • 10 ~ • m1-1, and a 120 #1 aliquot pulse-labelled with 750 p ~ i .

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m1-1 [3H]dThd (20 • 10 -6 M) for 20 s. A sample was taken into cold ethanol at the end of the 20 s pulse and the rest diluted with 5 ml used medium containing 100 ~M unlabelled dThd. Further aliquots were taken during the incubation. The cells were harvested by centrifugation and divided into two portions. One portion was analyzed by alkaline sucrose gradient centrifugation (Fig. 3). D N A was extracted from the other portion, sonicated and analyzed by affinity chromatography. 48% of the 20 s pulse-labelled D N A sedimented at less than 9 S (i.e. less than 1 0 0 0 nucleotides long) (Fig. 3a). A longer sedimentation of this same sample showed that the low molecular weight peak displayed a heterogenous size distribution centred at 4 S (150 nucleotides) and with an upper limit at about 6 S (350 nucleotides) (Fig. 4). These sizes are in g o o d agreement with estimates of Okazaki piece size in other eukaryotic systems [1--4,8]. On chasing, the label was chased into longer D N A strands (Fig. 3b--f). In Fig. 5 the loss of label from Okazaki pieces is shown together with data demonstrating loss of label from partially single-stranded D N A determined by affinity chromatography of corresponding D N A samples. Fig. 5 also demonstrates that if the D N A samples were only sonicated for 1 min, the timedependent transfer of label to the double-stranded D N A fraction was largely abolished.

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Discussion

The results here have demonstrated the time-dependent transfer of pulse label from partially single-stranded DNA to double-stranded DNA during DNA replication in phytohaemagglutinin-stimulated human lymphocytes. In untreated lymphocytes from patients with megaloblastic anaemia this transfer was retarded relative to the rate in corresponding control cells derived by the addition of folinic acid. A similar delay was seen in lyrnphocytes treated with either methotrexate or hydroxyurea. As a corollary of the discontinuous mode of replication of DNA [1--8], the DNA immediately behind the replication fork would be expected to exist in a partially single-stranded state. Pulse-labelled DNA has been shown by chromatography on benzoylated naphthoylated DEAE-cellulose [9--11,18] and by other methods [9,20] to behave as though it contained single-stranded regions. Furthermore, the conversion of pulse-labelled DNA to the fully doublestranded form by nucleases specific for single-stranded nucleic acids with little accompanying loss of label [10,18] suggests that labelled Okazaki pieces are annealed to the unlabelled template strand so that stretches of unlabelled template DNA are exposed. A close temporal correlation was demonstrated between the rate of Okazaki piece joining as measured by alkaline sucrose gradient sedimentation and the transfer of pulse label from partially single-stranded DNA to double-stranded DNA. This suggests (but does not prove) that the time-dependent transfer of pulse label to the double-stranded DNA fraction is related to the filling of gaps between Okazaki pieces. Single-stranded DNA regions can also arise at the replication fork due to unwinding of template DNA ahead of polymerization. However, these single-stranded regions would need to be stabilized by singlestranded DNA binding proteins [21] and are unlikely to persist in deproteinized DNA since they would reanneal on isolation. Gaps between Okazaki pieces are unable to reanneal on DNA isolation and are therefore the most likely candidates for conferring the partially single-stranded character on pulselabelled DNA. Therefore, we propose as a likely hypothesis that the rate of filling of gaps between Okazaki pieces is reduced in megaloblastic cells. For technical reasons, a direct demonstration of a decreased rate of Okazaki piece joining in megaloblastic lymphocytes was not possible. Pulse-chase experiments using [aH]dThd labelling could not be utilised for this purpose because of the likelihood that dThd would correct the defective DNA synthesis in megaloblastic anaemia [12]. Pulse labelling with [aH]dCyt did not result in a sufficient incorporation of radioactivity to permit analysis by alkaline sucrose gradient sedimentation. Treatment of eukaryotic cells with the deoxyribonucleotide pool-depleting drugs hydroxyurea [22--24] or fluorodeoxyuridine [25,26] has been shown to inhibit the joining of Okazaki pieces. The results here demonstrate that methotrexate or hydroxyurea result in a decreased rate of transfer of pulse label to the ~louble-stranded DNA fraction as was the case in megaloblastic lymphocytes. A decrease in the concentration of one or more deoxyribonucleotide triphosphates occurs in cells treated with methotrexate [27--29] or hydroxyurea

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[29,30] and presumably results in this decreased rate of Okazaki piece joining [22--26]. However, no significant decrease in any DNA precursor pool has been demonstrated in phytohaemagglutinin-stimulated lymphocytes or marrow cells in megaloblastic anaemia [29,31]. Recent reports, however, have suggested a functional compartmentalization of DNA precursor pools. Reddy and Matthews [32] have proposed that a kinetically coupled complex of enzymes maintains a high concentration of deoxyribonucleotides in the vicinity of the replication fork of the coliphage T4. Given the defect in dTTP biosynthesis in megaloblastic anaemia [33,34], it is plausible that the supply of this nucleotide at the replication fork is decreased without a measurable change in the total cellular dTTP pool. There is evidence for functional compartmentalization of deoxyribonucleotide pools in various eukaryotic cell types [35--37]. Our preliminary results suggest that a similar situation may pertain to phytohaemagglutinin-stimulated lymphocytes as well (Taheri, M.R., Wickremasinghe, R.G., Ganeshaguru, K. and Hoffbrand, A.V., unpublished results). However, the possibility that the lesions in DNA synthesis documented here and elsewhere [8,16] result from the more generalized defects caused by vitamin deficiency cannot be ruled out. However, it should be pointed out that the synthesis of both protein and RNA is relatively intact in megaloblastic anaemia [12]. Acknowledgement The authors thank the Medical Research Council of Great Britain for financial support. References 1 2 3 4 5 6 7 8 9 I0 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

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