Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L

Experimental Cell Research 158 (1985) 500-508

Effects of Psoralen on Replicon Size and Mean Rate of DNA Synthesis in Partially Synchronized Cells of Pisum sativum L. D. FRANCIS,’ NANETTE D. DAVIES,’ J. A. BRYANT,’ S. G. HUGHES,2 D. R. SIBSON1*2 and P. N. FITCHETT’ ‘Department of Plant Science, Uniwrsity College, Card@ CFI IXL and 2Unilever Research, Colworth House, Sharnbrook, Be#ord, MK44 ILQ, UK

We have examined by libre autoradiography the spacing of replicons in pea root me&ems during synchronized entry into S phase from arrest at the Gl/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication. @ 1985 Academic PRSS. IIK.

Both the initiation and the duration of S phase play a central role in controlling the cell cycle and are thus implicated in the regulation of plant development. DNA replication in eukaryotes occurs at multiple replicons tandemly arranged along the DNA molecule [l-3]. In all higher plants so far investigated a replicon consists of an origin from which two replication forks diverge in order to bring about replication of the DNA in the replicon [4, 51. Eukaryote nuclei are further characterized by the many replicons which they contain. For example, average numbers have been calculated for Xenopus laeuis, 30000 [6]; Pisum satiuum, 180000; and Secale cereale (cv Petkus Spring), 240 000 [7]. The duration of S phase is determined by the number of replicons, replicon spacing, the rate of fork progression and the pattern of replicon activation. Replicons are organized into families, members of which are to some extent clustered. Each replicon in a family is initiated synchronously at a specific point in S phase with each family having its own particular period of activity within S phase [4]. This may reflect a level of order within the genome in which specific, regularly spaced sequences define initiation points for DNA synthesis (replication origins), and also assign replicons to families. What is less clear, however, is the nature of the replicon origin itself. This is because of the lack of an assay or isolation system which unequivocally identifies origins. DNA sequences from a variety of eukaryotic organisms including yeast, Xenopus, mammals and plants have been shown to function as replication origins * To whom offprint requests should be sent. Address: Department of Plant Science, College, P.O. Box 78, Cardiff, CFl IXL, UK.

University

Copyrigbl @ 1985 by Academic Press, Inc. Au rights of reproduction in any form rcsemd 001448827/85$03.00

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in plasmids introduced into yeast cells [8-121. Such sequences are known as ARS (Autonomously Replicating Sequences). The number of ARS isolated from the nuclear genome of the yeast Saccharomyces cereuisiae is similar to the number of replication origins estimated by other means [9]. This suggests that, at least for yeast, ARS detected by their ability to allow replication of yeast plasmids are indeed replicon origins. The ARS isolated from a small number of organisms have been further characterized by sequence analysis, and all these have a similar, short A-T-rich sequence flanked by other A-T-rich sequences and by G-C-rich sequences [lo, 12, 131. This consensus of structure may thus represent the general features of eukaryotic replicon origins. On the other hand, it may simply represent structural features selected out by the yeast plasmid assay, having no relation at all to the replicon origins in the organism in question. What is needed, therefore, is a system for isolating the DNA which actually functions in very early S phase, and which should thus be highly enriched for DNA sequences which function as origins. Recently, it has been shown that the furocoumarin, psoralen, can penetrate living cells and if the cells are also exposed to long-wavelength (320-380 nm) UV light, psoralen can cross-link DNA [14, 151.Moreover, psoralen can prevent fork extension after the initiation of replication at the origin in Ehrlich ascites tumour cells [ 161. Because the double helix is unable to unwind further, origin DNA and short flanking sequences are trapped. Thus, cross-linking of DNA with psoralen offers a means of blocking the translocation of replicating forks so that short, early-replicated sequences can be isolated [ 161. By coupling this approach to a culture procedure resulting in synchronous initiation of DNA synthesis [17], we expect to be able to characterize DNA sequences which represent the initiation points of the replicon families which function early in S phase. As a preliminary study we have examined, by fibre-autoradiography, the intluence of psoralen on the pattern of deployment of initiation points during a synchronous entry into S phase. Our results show that psoralen cross-linking is effective in arresting the progress of replication forks in this system and does influence the spacing of initiation events. MATERIALS

AND METHODS

Tissue Culture A modification of procedures used to synchronize cells of pea root meristems at the Gl/S boundary [17) was used at 2O”C, unless otherwise indicated. Five-mm-long primary root tips of 4-day-old, aseptically grown seedlings of Pisum satiuum (cv Feltham First) were cultured in White’s medium for 48 h. The root tips were subsequently subcultured in White’s +10e6 M 5-fluorodeoxyuridine (FUdR) for 2 h at YC, followed by White’s + FUdR +2 % (w/v) sucrose for 24 h. Finally, the root tips were rinsed in fresh White’s for a further 24 h before transfer to White’s +2% sucrose.

Microdensitometry and Labelling Index At the end of each stage of the synchrony procedure, roots were fixed in 3 : 1 v/v absolute ethanol : glacial acetic acid from which Feulgen-stained squashes of the apical 1 mm of the roots were Erp Cell Res IS8 (1985)

502 Francis et al. prepared. Each stained preparation was analyzed at a wavelength of 560 nm with a Vickers M85A scanning microdensitometer, interfaced with a 32K Commodore microcomputer. Slides were normalized by taking readings of ‘/z telophase and prophase figures to fix the 2C and 4C levels respectively. Fifty interphase nuclei were measured per slide, and three slides (150 nuclei) per sampling time. Another batch of roots was exposed to [methyl-3H]thymidine @.a. 5 Ci mmol-‘; concentration 1 uCi ml-‘) for 15 min at the end of the final incubation in White’s medium. On transfer to White’s +2% sucrose, roots were also labelled for 15,30,45 or 60 min. The root tips were fixed and prepared as squashes as before. Permanent autoradiographs were prepared [18] and the labelling index (% frequency of nuclei labelled with [3H]TdR) was measured in a series of transects across the width of the coverslip; 600 nuclei per slide and five slides per treatment were scored at each fixation time.

Psoralen Cross-Linking, Labelling Protocol and Fibre Autoradiography Following the final subculture in White’s medium, roots were incubated in White’s supplemented with 2% sucrose for 15 min in the absence (controls), or presence of long-wavelength ultraviolet light (365 nm) (UV controls, where UV exposure is 15 min). UV light was provided by 2x160 mm, 8W bulbs with a high intensity 365~nmfilter at a distance of 150 mm from the roots giving an intensity of 5 mW cmm2. High sp. act. (70-90 Ci mmol-‘) [‘H]TdR was subsequently added (1 mCi ml-‘) for 30 or 60 min. A third batch of roots was treated in the same way but with a single addition of psoralen throughout the UV and [3H]TdR exposures. Thus, psoralen was added for either 45 or 75 min. Psoralen (trimethyloxalen) was dissolved in absolute ethanol giving a stock concentration of 2.2 mM [14]. Aliquots of stock were added to the culture medium to give a final concentration of psoralen of 22 PM. Following each exposure of [3H]TdR, nuclei were lysed on subbed microscope slides and the DNA was spread [19]. The slides were dipped in Ilford K2 photographic emulsion, dried and subsequently exposed for 2-4 months at 5”C, in the presence of a desiccant. The slides were developed using Ilford phenisol and prepared as permanent autoradiographs. Replicon size was determined following each pulse using the midpoint-to-midpoint (M-M) method [5, 201. Thus estimates were made of the distance between one symmetrical pair of silver grain arrays to the corresponding gap in an adjacent pair. Rates of replication of single replication forks were determined using published methods [19]. Over 500 measurements were recorded from ten replicate tibre autoradiographs for each pulse time following each treatment. Lengths of individual tracks of silver grains from the tandem arrays were also plotted against pulse time.

DNA Polymerase Assays Assays of DNA polymerase-a were carried out as described previously [21].

RESULTS AND DISCUSSION Synchrony Procedure Following culture in White’s medium for 48 h, cells of the pea root meristem accumulated in G2 (4C) and to a lesser extent in Gl (2C) of the cell cycle with a complete absence of nuclei with the 3C amount of DNA (fig. 1 a). The distribution of nuclei at this stage conformed to a stationary phase meristem [22]. Incubation with FUdR resulted in a gradual increase in the proportion of cells in Gl at the expense of those in G2 (fig. 1 b-d). However, synchrony was incomplete, particularly since there were always nuclei at the 3C level (fig. 1 tid). Thus, FUdR did not completely block the transition of cells from Gl to S phase. Consistent with this view was a labelling index of 10% at the end of the final incubation in White’s medium (Iig. 2). However, between 15 and 45 min following E.xp Cell Res 158 (1985)

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Fig. 1. Nuclear DNA contents (C values) of interphase nuclei in squashes of the apical 1 mm of 5 mmlong root tips of 4-day-old seedlings of Pisum sariuum cultured successively in (a) White’s medium (48 h) (20°C); (b) White’s + FUdR (2 h) (5°C); (c) White’s + FUdR + sucrose (24 h) (20°C); (d) White’s (24 h) (20°C). 150 nuclei were measured for each histogram. (c) is defined as the amount of DNA in a gamete. Fig. 2. The relationship between mean labelling index (% cells undergoing S phase f SE and time) in squashes of the apical 1 mm of 5 mm-long root tips cultured successively as described in fig. 1 and pulse-labelled with [3H]TdR (15 min) at the end of the interval in 0, final White’s (fw), and 0, labelled with [3H]TdR for 15-60 min following subculture to White’s +2% sucrose. Fig. 3. The relationship between mean length (SE) of labelled DNA segments which conformed to specific criteria (see Materials and Methods) and duration of high sp. act. pulse with [3H]TdR in l , control; 0, UV-treated; and A, psoralen-UV-treated root tips following the synchronization procedures.

transfer to White’s + sucrose, a 5-fold increase in the labelling index was observed (fig. 2). Thus, a substantial population of cells moved into S phase over this interval. Additional comment is necessary on the use of FUdR as a synchronizing agent. First, the extent of synchrony was not as complete as that obtained previously [171. Earlier procedures consisted of 12 h rather than 24 h exposures to White’s EXP Ceil

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Fig. 4. Percentage frequency distribution of individual labelled segments (urn) of tandem arrays of silver grains on autoradiographs for root tips pulsed with high sp. act. f3H]TdR: Controls (a) 30; (b) 60 min; UV controls (c) 30; (d) 60 min; UV-psoralen (e) 30; fl 60 min. Fig. 5. Frequency distribution of midpoint-to-midpoint distances Qun) between adjacent tandem pairs of labelled DNA segments on autoradiographs for root tips pulsed with high sp. act. r3H]TdR: Controls (a) 30; (b) 60 min; UV controls (c) 30; (d) 60 min; UV psoralen (e) 30; cf) 60 min; N, No. of measurements.

+ FUdR, White’s + FUdR + sucrose and White’s at 23°C [17]. Using 12 h exposures at 2O”C, we could only obtain 20-25% of cells in early S phase. By prolonging the FUdR incubations to 24 h, the proportion of nuclei in early S phase was increased (fig. 2). Second, the labelling index tended to plateau following a 60 min exposure to [3H]TdR, whereas theoretically the percent of labelled cells should have increased. There is no explanation for this, although this type of response has been obtained before with cultured pea roots exposed to FUdR (J. Van’t Hof, personal communication). However, for our purposes, the synchrony procedure sufficed to provide a synchronized population of cells in early S phase. Eflects of Psoralen on DNA Replication Following the synchrony procedure, the rate of replication (per single replication fork) at 20°C was 3.8 pm h-r (fig. 3). This measurement compares well with 4.5 urn h-r at 23°C recorded for early-S nuclei of pea root tips [20]. Exposure to psoralen in combination with long-wavelength UV light, resulted in a threefold E*p Cell Res IS8 (1985)

Effects of psoralen on DNA replication

505

reduction in the rate of fork extension compared with the controls, following a 30min pulse with [3H]TdR. Moreover, further replication during the next 30 min exposure to isotope was largely prevented (fig. 3). Exposure to UV alone also affected the rate of fork extension, although not to the same extent as that in the psoralen treatment. In the controls, changes in the percentage frequency of fibre length between 30-60 min exposures to [3H]TdR (fig. 4a, b) were consistent with a slow but steady rate of replication (fig. 3). Adding psoralen to the incubation medium resulted in a skewed distribution of fibre length following both 30- and 60-min exposures to [3H]TdR (fig. 4e,f). Thus, following both exposures, 80% of tibres were 1 urn in length; UV alone gave an intermediate distribution of fibres relative to control and psoralen-treated roots (fig. 4 c, 6). Taken together, data on rates of fork extension and tibre length (% frequency) were consistent in showing that psoralen slowed down the rate of replication over a 30-min interval and prevented any further fork extension over the next 30 min relative to control, and UV control samples. Midpoint-to-midpoint measurements gave a replicon size in the controls of l&15 urn following the 30-min pulse (fig. 5 a), and 15-25 urn following the 60-min pulse (fig. Sb). If 10-15 urn represents replicon size at 30 min, measurements at 60 min should, in theory, be multiples of this value peaking at l&15 and 20-30 urn. However, our data indicate that these distances increased with labelling time. Conceivably, the synchrony procedure activated additional origins of replication represented by the 10-15 urn peak. However, the subsequent 15-25 urn peak would be consistent with these shorter replicons becoming quiescent 60 min following the addition of sucrose to the medium (fig. 5). The use of FUdR as a synchronizing agent may have resulted in this response. These measurements for the controls compare with the average replicon size in the pea of 18 urn [20]. From fibre autoradiographs of psoralen-treated nuclei, the distribution of replicon size was skewed to the left and gave a modal size of 5-10 ym following both 30and 60-min exposures to [3H]TdR (fig. 5 e,j). Moreover, fibre autoradiographs from psoralen-treated roots were characterized by short tracks of silver grains, compared with a gradual lengthening of fibres in the controls (fig. 6). UV alone resulted in a replicon size of 5-15 pm, a value intermediate between that for control and psoralen-treated nuclei (fig. 5 c, d). It has been shown that short-wavelength (265 nm) UV does induce DNA damage and subsequent repair in pea root tips (J. A. Bryant, D. Francis, J. E. Grey & P. P. Daniel, unpublished data). In the current experiment using long-wavelength (365 nm) UV, the action spectrum of the DNA damage response is such that the amount of UV-induced damage would be minimal. Furthermore, repair of UV-induced damage would not produce regular spacing of tracks of silver grains but instead sporadically spaced small patches of repair. Our autoradiographs do not show this pattern (fig. 6b). Thus long-wavelength UV does affect the rate of replication and replicon spacing (fig. 5) rather than inducing DNA damage. Clearly these effects become Exp Cell Res 158 (1985)

506 Francis et al.

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Fig. 6. Autoradiographs of isolated DNA tibres of synchronized pea root tips, pulse-labelled with high sp. act. [3H]TdR for 60 min. (a) Controls; (b) psoralen-treated. Arrows locate putative replicon origins. Bar, 10 km.

more pronounced when the cells were exposed to UV latter induces extensive cross-linking. In order to check whether inhibition of fork extension linking and not to inhibition of the deoxyribonucleotide assays of DNA polymerase-a were performed. In UV treated tissue, there was an apparent 35% inhibition

and psoralen, since the was in fact due to crosspolymerization reaction, control and in psoralenof DNA polymerase-a

Table 1. DNA polymerase-a activity in synchronized root tips Enzyme activity: pmol dTMP min-’ g-’ fresh weight Control UV control Psoralen-treated Exp Cell Res 158 (1985)

15.4k2.1 9.95kO.85

10.02+0.06

mg-’ soluble protein 18.4k2.4 19.4k3.8 19.2f 1.8

Effects of psoralen on DNA replication

507

activity when the data were expressed on a fresh-weight basis (table 1). However, this apparent inhibition may be ascribed to a greater difficulty in extracting ‘soluble’ proteins from the tissue following exposure to UV, since when activity is expressed on a protein basis, the values for control, UV control and psoralentreated tissues are very similar (table 1). The basis for the greater difftculty in extracting the enzyme from tissue previously exposed to UV is not clear, but the addition of abrasives (e.g., fine sand) to the homogenization medium does not help. Overall, therefore, it is most unlikely that the experimental procedures led to an inhibition of enzyme activity great enough to explain the observed decline in fork rate. In fig. 6 b, the length of unlabelled regions between labelled segments ranges between 1.3-4.6 urn. Assuming a fork rate of approx. 4 urn h-i (fig. 3), initiation at these origins possibly took place 5-20 min before psoralen cross-linking. However if so, spacing of origins in the controls (fig. 6a) should be similar to the psoralen-treated tissues (fig. 6 b). In fact, the modal replicon size in the presence of psoralen (5-10 urn) (fig. 5 e,f) is approximately half the modal size (15-25 urn) in the untreated controls (fig. 5 a, b). Thus, in response to psoralen, replication was apparently initiated at more sites along the DNA molecule than in the controls. This shift towards more closely spaced initiation points following psoralen cross-linking in vivo suggests that the frequency of initiations within a given region of DNA can be modulated by factors which impede fork progression. The 30- and 60-min pulses used, however, do not enable us to determine whether initiation is simultaneous at all start points in psoralen-treated DNA. Therefore, it is unclear whether hierarchies of ‘strong’ and ‘weak’ (primary and secondary) initiation points exist, although our data suggest that the replication machinery possesses flexibility in the deployment of initiation sites. Our aim is now to isolate and characterize the newly replicated sequences which are trapped by psoralen cross-linking and to answer the questions of whether there are yet more closely spaced sites at which DNA replication can be initiated and whether such sites are defined by DNA sequence or by some feature of chromatin structure. Observations of tracts of closely spaced initiations (fig. 6) suggest that specific regions of DNA become available for replication at specific times during S phase and that once a given replicon is first initiated then the replicative machinery will adjust itself to complete replication of that region by inserting extra initiation points if necessary. We thank the SERC Biotechnology Secretariat for a cooperative research grant (CZUB99071)and the Nuffield Foundation for support under the Small Grants Scheme.

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Van? Hof, J & Bjerknes, C A, Exp cell res 136 (1981) 46. Francis, D & Bennett, M D, Chromosoma 86 (1982) 115. Callan, H G, Proc r sot Lond B 181 (1972) 19. Francis, D, Kidd, A D C Bennett, M D, The cell division cycle (ed J A Bryant & D Francis) p. 61. Cambridge University Press, UK (1985). 8. Stinchcombe, D T, Thomas, M, Kelly, J, Selker, E L Davis, R W, Proc natl acad sci US 77 (1980) 4559. 9. Beach, D, Piper, M & Shall, S, Nature 284 (1980) 185. 10. Monteil, J F, Norbury, C J, Tuite, M F, Dobson, M J, Kingsman, A J & Kingsman, S M, Nucleic acids res 12 (1984) 1049. 11. Waterborg, J & Shall, S, The cell division cycle in plants (ed J A Bryant & D Francis) p. 15. Cambridge University Press, Cambridge, UK (1985). 12. Sibson, D R, Hughes, S G & Bryant, J A. Unpublished data. 13. Broach, J R, Li, Y Y, Feldman, J, Jayaram, M, Abraham, J, Nasmyth, K A & Hicks, J B, Cold Spring Harb symp quant bio167 (1982) 1165. 14. Wiesenhahn, G P, Hyde, J E & Hearst, J E, Biochemistry 16 (1977) 925. 15. Cech, T R & Karrer, K M, J mol biol 136 (1980) 395. 16. Russev, G & Vassilev, L, J mol biol 161 (1982) 77. 17. Kovacs, C J & Van? Hof, J, J cell bio147 (1970) 536. 18. Francis, D & MacLeod, R D, Ann bot 41 (1977) 1149. 19. Van’t Hof, J & Bjerknes, C A, Chromosoma 64 (1977) 287. 20. Van’t Hof, J, Exp cell res 103 (1976) 395. 21. Stevens, C & Bryant, J A, Planta 138 (1978) 127. 22. Van? Hof, J, Brookhaven symp bio1254 (1973) 152. Received October 3 1, 1984

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