DNA Polymerase, DNA Ligase, and Thymidine Kinase Activity in Chicken Lens, Related to DNA X-Ray Lesion Repair

Differentiation

Differentiation (1981)20: 188-195

0 Springer-Verlag 1981

DNA Polymerase, DNA Ligase, and Thymidine Kinase Activity in Chicken Lens, Related to DNA X-Ray Lesion Repair M. F. COUNIS', J. C. DAVID2, E. CHAUDUN', and D. CARREz U.118 INSERM, CNRS ERA 842, D6veloppement et Senescence Cellulaire, 29, Rue Wilhem, 75016 Pans, France Laboratoire de Biochimie du Developpement, I.R.B.M. Universitt Pans VII, 2, Place Jussieu, 75221 Paris, Cedex 05, France

The activity of DNA polymerases a, /?,and y ; DNA ligases I and 11; and thymidine kinase in chicken lenses is determined. These enzymes are present in embryonic intact lenses freshly isolated at 6 days and 11 days of development and in lenses isolated at 11days of development and cultured for three days. They are also found in both epithelium and fibers when separated at 10 days of embryonic development and in the epithelium of 14'/,-month-old hen lenses. In the anucleate mature hen lens fibers, the only detected enzyme is thymidine kinase. Previous results showed that repair of X-ray irradiated DNA was total in 11-day-old embryonic lenses while repair was not detected at six days. On the other hand, repair was very much impaired when the 11-day-old lenses were cultured for three days. Enzymic analyses suggest that the defect in DNA repair and the spontaneous DNA degradation observed in chick lenses at certain embryonic stages, are not due to the absence of any of the above enzymic activities. Alternative explanations are discussed.

Introduction Three major DNA polymerases a, /?, and y have been described in chick and other higher vertebrates [6,9] and such work has been reviewed [22, 531. They are immunologically distinct. Several attempts have been made to identify the specific roles played by these enzymes in DNA synthesis and repair. In general, it has been found that the level of activity of DNA polymerase a fluctuates markedly, maximal activity being associated with rapid cellular proliferation, whereas DNA polymerase /? presents a more constant level [8, 10, 19, 46,471. DNA polymerase y seems to be the enzyme acting in mitochondria, but it has also been found in nuclei [6, 231. If DNA replication (initiation and elongation) really occurs under DNA polymerase a action [5, 541, DNA repair is not well understood, and all three polymerases sometimes have been associated with repair mechanisms [5, 11, 24, 511. Thymidine kinase acts in the pathway of d?Tp synthesis, reducing the loss of dTh4P. It has been suggested that a major function of this enzyme is to provide a balanced pool of deoxyribonucleoside triphosphates for DNA synthesis [26]. DNA ligases have been described in many eukaryotic systems (review by SBderhall and Lindahl [45] and [15, 25]), and several different enzymes are known, but their functions are less defined than those of DNA polymerases. DNA ligase I [15,43] is mostly cytoplasmic, seems to be abundant in tissues with a high proportion of dividing cells, and could be associated with the replication process [17, 421. This enzyme may act together with DNA polymerase a because it shows a similar correlation with cell proliferation. DNA ligaseII is found in the nuclei as DNA polymerase B [25, 551. The relationship between DNA ligase I and I1 is still controversial [39, 441. The level of DNA ligaseII does not appear to correlate with cell mitotic activity [16-181. A mitochondria1 enzyme whose relationship to DNA IigaseII is not well established, has also been described [31].

For lens cells in vivo, one of the terminal events in differentiation is the degeneration of deep cortical cell nuclei, followed by their disappearance in the fibers [37, 401. In embryonic chicken lens cells, DNA breaks can be observed in an alkaline sucrose gradient at the 6- and 11-day stages [12,13, 411, while nuclei are still observed in the fibers [35]. In adult chick lens epithelium, the DNA degradation process is still observed, since this phenomenon occurs across the lifespan, whereas the nuclei have completely disappeared in the fibers [30]. It is possible to modulate the process of DNA breakage; cycloheximide has been shown to delay the phenomenon at 11 days [14]. This accumulation of breaks in DNA strands could be the result of either an increase in DNase activity or an impairment in DNA repair. At six days, active repair of X-ray-induced breaks is never detected in these cells, while at 11days, under the same experimental conditions, the cells are able to repair X-ray-induced breaks in DNA within one hour. However, lenses from 11-day-old embryos incubated for three days in a culture medium reveal progressive damage to the X-ray repair system, while the DNA is breaking apart [12,13]. We hypothesize that 6-day-old lens cells or 11-day-old cells after three days in culture, might have a defect either in DNA polymerases or in DNA ligases specifically involved in DNA repair. For this reason, activity of DNA polymerases a, /?,and y ; DNA ligases (forms1 and 11); and thymidine kinase was determined both in the whole lens and in separated epithelium and fibers at different stages of chicken embryonic development and in adults.

Methods Lenses were excised from eyes of 6- lo-, 11-day-old Leghorn chicken embryos and from 141/2-month-oldhens. For each experiment, 1,100 lenses from 6-day-old chick embryos and 350-400 lenses from 11-day-old embryos were prepared. 0301-4681/81/0020/0188/$01.60

M.F.Counis

et al.:

Lens DNA Polymerases and Ligases

These latter were used either immediately or after three days of in vitro culture as previously described [12, 131. Three hundred lenses from 10-day-old chick embryos and 20 from mature hens were dissected into epithelium and fibers. Experiments on 11-day-old chicken embryo lenses and on mature hens were made in duplicate. After isolation, each embryonic lens was cleaned on filter paper. Embryonic lens epithelium and fibers were separated under a binocular lens. The contamination of each fraction by the other one does not appear very important as seen by histologic observations. It was more difficult to obtain clean mature hen lenses since pigmented tissue, probably the pigmented leaflet of ciliary epithelium, was adhering strongly to the equatorial lens capsule. Thus, the epithelium was isolated by a circular cut just inside the zone of attachment of the ciliary processes. The fibers do not appear to be contaminated by the epithelial fraction.

Preparation of the Cellular Extracts. The lens samples were stored at -70" C in phosphate buffered saline (PBS) containing 10% glycerol. They were washed with PBS immediately before use and homogenized with a Dounce homogenizer in 3-10 volumes of extraction buffer (0.5M KCl, 20mM Tris-HCl pH 7.4, 2mM dithioerythritol, and 0.2% NP40) made in 0.8 mM phenylmethylsulfonylfluoride (Sigma) immediately before use [lo]. The extract was sonicated (Alcatel sonicator, position 2.2) for three periods of 20s at 1min intervals, and centrifuged at 140,OOOg for 60 min at 4" C. Samples (250-300 pl) of the supernatants were layered on 5 2 0 % or 10-25% sucrose gradients in 0.5 M NaC1, 50 mM Tris-HC1 pH 7.4, 1mM EDTA, 2 mM dithioerythritol, and centrifuged for 10 or 16 h at 47,000 rpm in a SW 50-1Beckman rotor at 4" C. Fractions of 240 pl were collected and assayed for enzyme activities. Enzymatic Assays DNA Polymerase a. The standard reaction mixture (0.1 ml) contained 50mM Tris-HCI pH 7.4, 8mM MgC12, 16mM dithiothreitol, 0.1mM each of dATP, dCTP, and dGTP, 3.9 pM pH] d l l T (5.2 Ci/mmol), 200 pg/ml bovine serum albumin, 120 pg/ml activated calf thymus DNA (prepared by pancreatic DNase digestion, according to the method of Aposhian [3], as previously described by Carre and Pieau [lo]), and 10 pl of enzyme fraction. The reaction mixture was incubated for 90 min at 37" C. The radioactivity of trichloracetic acid insoluble material was measured on glass fiber filters (Whatman GF/C). DNA Polymerase b. The enzyme fraction (30 pl) was preincubated at 0" C for 30 min with 5 mM N-ethylmaleimide. The reaction mixture (0.1 ml) contained 50 mM Tris-HCl pH 8.2, 8 mM MgC12,75 mM NaCl, 1 mM 2-mercaptoethanol, 0.1 mM each of dATP, dCTP, and dGTP, 3.9pM rH]-dTTP (5.2 Ci/mmol), 200 pg/ml bovine serum albumin, and 120 pg/d activated calf thymus DNA. The reaction mixture was incubated for 90 min at 37" C and acid insoluble radioactivity was measured. DNA Polymerase y. The reaction mixture (0.1 ml) contained 25 mM Tris-HC1 pH 8.2, 50 mM potassium phosphate buffer pH 8.4, 0.5 mM MnC12, 2 mM dithiothreitol, 75 mM KCI, 3.9pM [3HJ-dTl'P (5.2 Ci/mmol), 200pg/d bovine serum

189

albumin, 8 pg/ml poly rA : dT12.18 (Poly rA from Miles, and oligo (dT)lz.ls from PL-Biochemicals, were hybridized at 70" C), and 5 pl of enzyme fraction. The reaction mixture was incubated for 90 min at 37" C and acid insoluble radioactivity was measured.

Thymidine Kinuse. The reaction mixture (0.1 ml) contained 55 1+4 Tris-HC1 pH 8.2, 8 mM MgC12, 4.5 mM Zmercaptoethahol, 7mM ATP, 0 . 9 m [14C]-thymidine (55.3 mCi/mmol), and 4Opl of enzyme fraction. Incubation was performed at 37" C for 120 min. The incubation mixture was then spotted on DE-81 Whatman paper (2.5 x 2.5 cm), dried for 10min at room temperature, and washed four times for 10 min each wash with 10 ml of cold 96% ethanol per paper. The radioactivity retained on the dried papers was measured in a scintillation mixture. DNA Ligases. DNA ligases were assayed after a modification of the method of Olivera [36]. The substrate was prepared as follows: 1mM 01igo(dT)~~.~~ in 50 mM Tris-HC1 pH 7.6 was incubated at 37" C for 30 min in the presence of 10 unitdml of E. coli alkaline phosphatase (Sigma). The reaction was stopped by addition of 0.1 volume of 20mM KH2P04. The solution was then extracted with two volumes of chloroform : isoamyl alcohol (24 : 1) and the aqueous phase kept in a boiling water bath for 15min. The 5' ["PI labeling was performed in the presence of 60 mM Tris-HC1 pH 7.6, 6 mM MgC12, 6 mM 2-mercaptoethanol, 300 pM Y[~~P]-ATP (200 mCi/mmol), 200 pM dephosphorylated oligo (dT)12.18, and 15 unitdml of T4 infected E. coli 5'-polynucleotide kinase (PL-Biochemicals) at 37"C, until reaching the plateau of incorporation. The resulting 5'-[32P] oligo (dT)lL18was stored at -20" C until use. For the DNA ligase activity determination, each assay (0.3 ml) contained 6 pM [32P] oligo(dT)12.18, 6 pM poly(dA) (PL-Biochemicals), 25 mM Tris-HC1 pH 7.6, 6 mM MgC12, 1mM ATP, 2.5 mM dithioerythritol, 31 pg/ml bovine serum albumin, and 75 pl of enzyme solution. The assays were incubated at 37" C for 30 min, then diluted to twice its volume with water, and incubated at 80" C for 30 min. At time 0, 0.5 unit of alkaline phosphatase was added, and 0.25 unit after 10 and 20 min. At the end of the incubation, the assay samples were trichloracetic acid precipitated (5% of final concentration), filtered through Millipore filters, and counted. Chromatography on Phosphocellulose of 140, OOO g Supernatant Containing DNA Ligases. Five ml of the supernatant (45 mg proteins) were dialyzed against buffer containing 50 mM Tris HCl pH 7.4, 0.1 mM EDTA, 1mM 2-mercap toethanol, 20% glycerol, and 50mM KCl (TEMG buffer). After a 5-fold dilution with TEMG, the dialyzed extract was adsorbed onto a phosphocellulose (P 11 Whatman) column (0.5 x 8 cm) previously equilibrated with TEMG. The column was washed with the same buffer. The enzyme was eluted by a linear gradient of 0.1-0.7 M KC1 in TEMG buffer. Fractions of 1ml were collected and 100 pl samples of each fraction were assayed for DNA ligase activity. Others. The protein content was determined on total cellular extracts according to the method of Lowry et al. [32]. One unit of DNA polymerase corresponds to the amount of enzyme catalysing the incorporation of lnmol of total nucleotides in 1h under standard conditions. In the case of DNA polymerases a and @, the nmol of dTMP incorporated

M. F. Counis et al.: Lens DNA Polymerases and Ligases

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with calf thymus DNA are multiplied by 3.5 to account for the base composition of the DNA. One unit of thymidine kinase corresponds to the amount of enzyme catalysing the phosphorylation of 1nmol of thymidine in 1h under standard conditions. One unit of DNA ligase corresponds to the amount of enzyme which renders 1 nmol of 5'-[32P]resistant to alkaline phosphatase in 30 min under standard conditions.

Results In the different experiments presented in this report, the enzymes were partially purified by sedimentation on sucrose gradients, since with crude extracts, variations in activity determinationsare usually observed [lo]. This purification step results in the separation of the different activities studied and provides a useful parameter for characterizing them. The activities in crude extracts and in 140,OOOg supernatants were determined in order to estimate losses. These assays did not reveal any discrepancy with the results of sucrose gradient analysis. Samples were tested quickly after preparation to avoid activity losses. DNA was not used as a denominator of relative enzyme activity, since at older ages, the maturing lens fibers lose their nuclei with a concomitant degradation and loss of DNA. Rather, protein and tissue weight were used to compute specific activities, since these increase quantitatively during the process of lens cell elongation.

I. DNA Polymerases a,

B, and

y and Thymidine Kinase

Whole Embryonic Lenses. The sedimentation profiles of DNA polymerase a,B, y and thymidine kinase activities in extracts of 6- and 11-day-old chick embryonic lenses, either immediately or after 3 days in culture, are presented in Fig. 1. Since the different extracts were prepared with volumes of extraction buffer proportional to the weight of wet tissue, and since equal samples of the 140,OOOg supernatants were layered on the

gradients, these profiles permit direct comparisons of activities per unit weight of tissue in different samples. Two independant experiments performed on 11-day-old chick embryo whole lenses gave enzymic activities which were in agreement within k 10%. Table 1summarizes these results and also presents the enzyme activity per unit of total protein. DNA polymerases a, B, and y and thymidine kinase are detected in the different samples. Their position in the gradients are identical to the position of the enzymes extracted from chick neural retina and used as markers in a parallel gradient. The activity of the DNA polymerases in the lens is much lower than in other chick embryonic tissues as, for example, the neural retina, when determined under identical extraction and assay conditions [lo]. In the lens, DNA polymerase a is, in all cases, the main DNA polymerase activity. The enzymes are all detectable in 6-day-old embryonic lenses (Fig. lA), in 11-day-old lenses (Fig. lB), or in 11-day-old samples after 3 days in culture (Fig. 1C). Between 6 and 11 days, a small increase in the activity of the three DNA polymerases (expressed per weight of tissue) can be seen. No such increase is seen when DNA polymerase activity is related to the lens protein content, since a large accumulation of proteins occurs in the lens between 6and 11-day-old embryonic lenses. There was a loss of proteins both during culture and PBS washing prior to homogenization. This may account for the lower DNA polymerase activity level per unit wet weight observed in the extracts of cultivated 11-day-old embryonic lenses, relative to the extracts of intact lenses of the same age, since this difference vanishes for activity expressed per mg protein. The effect is not as large for thymidine kinase, which does not vary significantly at these embryonic stages.

Embryonic and Mature Lens Separated into Epithelium and Fibers. To determine whether there'were differences in the enzymic content of different regions of the lens, DNA

B

A

t

C

I(

II

II I ,

I

I

LI '1

10

20

fraction w m k r Fii. 1. Sedimentation analysis of DNA polymerases a,/3, and y and thymidine kinase from extracts of total embryonic chick lenses. A: 6-day-old

embryonic lenses. B: 11-day-old embryonic lenses. C:ll-day-old embryonic lenses cultivated for 3 days. The extraction was made with three volumes of extraction buffer. Samples 250 p l were used for the sedimentation which was performed in 5 2 0 % sucrose gradients for 10 h at 47,000 rpm in rotor SW 50-1. Other conditions are described under Methods. A- - -A DNA polymerase a; A A DNA polymerase 8; DNA polymerase y; 0- - -0 thymidine kinase

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Table 1.DNA polymerase and thymidine kinase activity in chick embryo lenses at 6 and 11days of development. Conditions for the sedimentation analysis are described under Fig. 2. Total activities were determined by summing up the activity measured in the different fractions of the gradients Age of embryonic chick lenses

mg of proteindg of tissue

DNA polymerases

Thymidine kinase

B

a Unitdg of tissue

Unitdmg of proteins

Unitdg of tissue

Unitdmg of proteins

Unitdg of tissue

Unitdmg of proteins

UniWg of tissue

Unitdmg of proteins

0.037

0.21

6.9

0.26

0.034

0.29

0.048

0.17

0.008 0.006 0.008

27

7.5

0.28

11 days

54

10.1

0.19

1.oo 1.81

11 days + 3 days in culture

21

3.7

0.17

1.01

6 days

Y

6.7

0.12

5.7

0.27

Table 2. DNA polymerase and thymidine kinase activity in isolated epithelium and fibers from 10-day-old-embryoand adult lenses. Conditions of analysis of the samples are indicated under Fig. 2. DNA polymerase y activity was not assayed in 10-day-old lenses and was below the limits of detection (0.03 unitlg of tissue) in hen lens fractions

Tissue

DNA polymerases

Thymidine kinase

~

B

a mg of proteindg of tissue

Unitdg of tissue

Unitdmg of proteins

10-day-old embryonic chick lenses

Epithelium Fibers

40 41

2.5 0.6

0.062 0.015

14'/* month-old hen lenses

Epithelium Fibers

13 274

1.o 0.1

0.077

O.ooo4

polymerase and thymidine kinase activities were assayed on extracts of isolated epithelium and fibers from 10-day-old embryos and 141/2-month-oldhens (Table 2 and Fig. 2).Fiber and epithelial fractions may be slightly contaminated by each other. The hen epithelial fraction was essentially free from adhering pigmented tissues. Moreover, since there was little hen epithelium, it was more diluted for extraction than were the corresponding lens fibers. A non-purified preparation showed that the activities of DNA polymerases and thymidine kinase were not affected by the dilution. Ten to twenty five per cent sucrose gradients were necessary to analyse the hen extracts, because the protein concentration in the fibers was so high that it resulted in a sinking of the extract when 520% sucrose gradients were used. In lens epithelium (Table 2,Fig. 2A,C),a decline in DNA polymerases a and B per gram of tissue was observed from the embryonic to the adult stage. However, when related to the total protein content, the decrease in DNA polymerase a activity was not so obvious. In the embryonic fibers, (Fig. 2B), DNA polymerase a is low, while DNA polymerase B is at the same level as in the epithelium (Fig. 2A). In the mature hen fibers, all of the DNA polymerase activity was at the lower limit of detection (Fig. 2D).It is not likely that the weak activity in hen lens fibers was due to the presence of any diffusible inhibitor, since no inhibition was observed when these fractions were added to active DNA polymerases. The thymidine kinase level is in the same range for the embryonic 10-day-oldlens epithelium and fibers as for the 6-or

Unitdg of tissue

Unitslmg of proteins

2.7 2.4

0.068 0.059

4.0 5.1

0.10 0.12

0.15

0.0115

< 0.08

< 0.0003

0.6 11.9

0.046 0.043

Units/g of tissue

Unitdmg of proteins

11-day-oldintact embryonic lenses. When this enzymic activity is expressed in units per gram of tissue, it is sharply decreased in the mature epithelium whereas it increases two fold in the mature fibers as compared to the embryonic tissues. However, when it is expressed per unit protein content, the thymidine kinase level is nearly identical in mature epithelium and fibers but it is lower than it was in the embryonic lenses. The sedimentation peak of thymidine kinase activity in hen fibers is asymmetrical (Fig. 2D). The same observation can be made for 11-day-old embryonic lenses (Fig. 1B) while it was not the case for retinal enzyme [lo]. It is likely that this asymmetry results from interactions between the emme and other proteins since proteins are very concentrated in these fractions as seen by the ODm profile. II. DNA Ligases Analysis by Sedimentation on Sucrose Gradients. The results of

the DNA ligase assays performed on the same lens extracts used for DNA polymerases are presented in Fig. 3 and Table 3. Two separate peaks of DNA ligase activity can be observed at the embryonic 6-day stage (Fig. 3A)and in the lens epithelium of 141/2-month-oldhens (Fig. 3D).They correspond to forms I (8.2S) and I1 (6 S) of DNA ligase shown in other chick tissues [15-17,251. In thesucrose gradients, form I of DNA ligase is not observed at the embryonic ll-day-old stage, either immediately after collecting the lenses (Fig. 3B) or after 3 days of culture (Fig. 3C). The only DNA ligase

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the gradient position of DNA ligase I in 11 day with hen epithelium samples. A marked inhibition of hen DNA ligase I was observed. This inhibition was not specific since DNA ligase 11, as well, could be inhibited by these 11-day sucrose gradient fractions. The nature of the inhibitor is not yet established. It is not a phosphatase, since it does not hydrolyse the 5'-[32P] label from oligo (dT). In hen lens fiber extracts, where DNA IigaseI and I1 activities are also very low, no evidence for such an inhibitor has been found.

A

f?

Phosphocellulose Chromatography. To further characterize the DNA ligases of 11-day-old embryonic lenses, chromatographic profiles on a phosphocellulose column were determined (Fig. 4). After this step, the factor inhibiting DNA ligase I was eliminated, and two peaks of DNA ligase activity, eluted with 0.37 and 0.54 M KC1 and corresponding respectively to DNA ligase I1 and I [17], were observed. The two forms of DNA ligases are present at 11days as well as at 6 days in the embryonic lens, and in 141/2-month-old hen lens epithelium, but they are nearly absent in the mature fibers. The phosphocellulose column chromatography of an extract of hen lens fibers did not reveal any more DNA ligase I or I1 activity than did the sucrose gradients.

C

Discussion

7.0 6.0 $0 4.0

30

1

8

8

20 1.0

0 traction wmbw

Fq.2. Sedimentation analysis of the DNA polymerase and thymidine b a s e activities from extracts of l0-day-old embryonic lenses and 141/2-month-oldhen lenses separated in epithelium and fibers. 10-day-old embryonic lens epithelium (A)and fibers (B).Hen lens epithelium (C) and fibers (D).A---A DNA polymerase a; A---A DNA polymerase 8; 0---0thymidine kinase. For chick embryo lens fractions the extraction was made with 10 volumes of extraction buffer. Samples of 300 pl of high speed supernatant were used for the centrifugation performed on 5 2 0 % sucrose gradients for 16 h at 47,000 rpm. For the hen lenses, the extraction was made with 4.8 volumes of buffer for the fibers and 8.3 volumes for the epithelium. Samples of 250 pl of supernatants were layered on 10-25% sucrose gradients centrifuged for 16 h at 47,000 rpm. The activities were corrected for a dilution of the extracts identical to the dilution used in the experiment of Fig. 1

detected in these extracts is form 11.Its activity increases in the embryonic lens from 6-11 days. After 3 days in culture, DNA ligase I1 is still present in 11-day-old embryonic lenses and its proportion among the other proteins is not sigdicantly modified. A high activity level of both DNA ligase I and I1 is found in hen lens epithelium, whereas both enzymes are at trace levels in the mature fibers (Fig. 3D). The absence of DNA ligase I in 11-day-old embryonic lenses, and its presence at 6 days and in adult epithelium suggests the possible presence of an inhibitory factor in the sucrose gradient fractions containing DNA ligaseI. This possibility was checked by mixing fractions corresponding to

Regulation of eukaryotic DNA synthesis and repair could occur at several different levels in the DNA synthesispathway. Thus a defect in DNA repair may be occasioned either by a structural modification of the genome which would not be recognized by repair enzymes, leading to an accumulation of transcriptionally inactive genes, or by a lack of any enzymes or factors involved in the repair systems. Three general types of DNA repair (excision, strand breaks, and postreplication) have been described in mammalian cells after chemical and physical injuries [21, 481. X-irradiation induces single strand breaks in DNA which are repaired by several components including DNA polymerase(s) and DNA ligase(s), but the mechanism is not yet clearly understood [33,50]. DNA polymerase B has been shown to be involved in DNA repair in some particular tissues. For example, rat neuronal nuclei, which contain no DNA polymerase a, only 0.8% of DNA polymerase y and 99.2% of DNA polymerase /?,are able to perform UV-induced DNA repair synthesis in vitro, while synaptosomes, containing only DNA polymerase y are not [24,51]. Repair of X-ray induced single strand breaks in pulse labeled DNA is not detectable in Way-old embryo lenses, while under the same experimental conditions, it is efficient at 11 days. After 3 days in culture, the repair capacity of the 11-day-old lens cells is considerably reduced, although other specific functional activities of the cells are retained, such as, the capacity to synthesize B crystallins, protein characteristic of lens differentiation [13]. We have compared the activity of DNA polymerases a, /?,and y , the DNA ligases I and 11, and thymidine kinase in the embryonic chick lens at 6 and 11days. In the latter case, the lenses were used either immediately or after 3 days in culture. These different enzymes are present at all the stages studied. Differences in activity are not very large. Therefore, the absence of X-ray repair observed in 6-day-old chick embryo lenses cannot be explained by the absence of any of these enzymes in the lens at this stage. Similarly the impairment of the repair capacity in 11-day-old lenses after 3 days of culture is not due to the loss of the studied enzymes.

M. F. Counis et al.: Lens DNA Polymerases and Ligases

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A

C

Fig. 3. Sedimentation analysis of DNA ligase activities from extracts of embryonic and adult chick lenses. A:6day-old embryonic intact lenses. B:

11-day-old embryonic intact lenses. C: llday-old embryonic lenses cultivated for 3 days. Experimental conditions are as for the experiment reported in Fig. 1.D:141/2-month-oldhen lenses. A -A lens epithelium; A A lens fibers. Experimental conditions are described under Fig. 2. The activities were corrected for a dilution factor identical to the factor used in the experiments 3 A, B, C

Table 3. DNA ligase activity in sucrose gradient fractions from extracts of embryonic and adult chick lenses. The experimental procedure is described under Fig. 1 for embryonic extracts and under Fig. 2 for the isolated hen lens epithelium and fibers

DNA ligases

Tissue

Form II

Form I mg of proteindg of tissue

Chick embryonic lenses

141/2-month-oldhen lenses

6 days 11 days 11 days + 3 days in culture Epithelium Fibers

21

Unitdg of tissue 1.8

21

< 0.03 < 0.03

13 274

11.9 0.2

54

Q. 4. Phosphocellulose column chromatography of DNA ligase activities from extracts of llday-old embryonic lens

Wicker et al. [52] also found no variation in the level of DNA polymerases a,B, and y that could be related to repair phenomena in monkey-kidney CVl cells irradiated by W, while Mezzina and Nocentini [34] showed an important de novo synthesis of DNA ligase in the same UV irradiated cells.

Unitdmg of proteins 0.066

c o.Ooo5 < 0.0015 0.918 O.OOO9

Unitdg of tissue

Unitdmg of proteins

2.0 4.5 2.4

0.074 0.084 0.117

9.4 0.3

0.727 0.001

Parker and Lieberman [38] and Bertazzoni et al. [7]found no significant alteration in the activity and sedimentation properties of the polymerases a, /I,and y in human inherited diseases such as xeroderma pigmentosum, Franconi's anemia, Bloom's syndrome, or ataxia telangiectasia, known to involve a reduced or altered DNA repair process. If no gross variations in DNA polymerase or in DNA ligase activities can account for the differences in X-ray repair activity found between embryonic lens tissues at different stages, other variables must be examined. The repair defect could be due to the absence of other factors essential for DNA synthesis or to the presence of in vivo inhibitory factors not detected in the in vitro assay. The presence, at 11days, of an inhibitor of DNA ligase I in sucrose gradients, eliminated by the phosphocellulose chromatography, does not seem to have any relationship to repair mechanisms, since at the ll-day stage, X-ray irradiated DNA can be repaired within one hour, while at 6 days repair does not occur. The distribution of the different enzymes involved in the repair process and the accessibility of the DNA lesions to their activity should also be considered. The detection procedure used to identlfy X-ray lesion repair [13], is limited to the detection of the properties of those cells which were replicating

194

their DNA at the time of labeling. DNA repair capacity of quiescent cells is not monitored by this technique. Therefore, the observed differences in repair capacity between 6- and 11-day-old embryonic lenses are for cells which have just replicated their DNA and will ultimately differentiate for most of them into fiber cells [13]. After 3 days in culture, the cells which replicated their DNA 3 days earlier and are now differentiating have lost most of their repair capacity. This repair capacity is present at 11days and absent at 6 days. The minor differences in enzymic activities among extracts of lenses at the different stages, may hide important differences in activity among cells at different stages of mitotic division and differentiation within a given lens. However, when epithelium was separated from fibers at 10 days, a 4-fold decrease of DNA polymerase a and no variation of DNA polymerase /3 were found. It seems that, at least for DNA polymerase 8, no major difference exists between the differentiated fibers and the epithelial cells at this stage, making it unlikely that important enzymic differences exist among cells at different stages of differentiation in the same tissue. A differential accessibility of DNA lesions to repair enzymes may be due to the compartmentalization of enzymes inside the cells at various stages of differentiation. Variations in the intracellular distribution of some DNA polymerases have been reported, .for example, during the early develop ment of the sea urchin egg[20]. One must also consider genome integrity. In 11-day-old lenses, the differentiating cells may have naturally accumulated so many DNA lesions in 3 days of culture, that the tridimensional structure of chromatin could have changed. Accordingly, enzymes might be unable to repair this overly degraded DNA. The only case where the DNA metabolism enzymes are missing is the 141/2-month-oldhen lens fibers. It is known that mature lens fibers do not possess any nuclei [30]. Ten-day-old embryonic chicken lens fibers still contain nuclei as shown by Modak and Perdue (351, while in the 1V2 week post-hatching chick lens, only a small zone of fibers contains pycnotic nuclei. Thus the presence of non-pycnotic nuclei in the fiber cells can be correlated with the presence of DNA polymerase activities and, as soon as the nuclei become missing, the enzymes disappear. On the other hand, there was no decrease in thymidine kinase activity as a function of lens aging and differentiation. The hen lens fibers present the highest thymidine kinase activity (per weight of tissue). The enzyme found in the lenses has properties analogous to the thymidine kinase A [26-291, also called adult form [2, 491 or mitochondrial form [l]. This enzyme uses ATP as the preferential energy donor and, with a lower efficiency, dATP and CTP. It is inhibited by addition of dCTP. It has an electrophoretic mobility of 0.6, as does the A enzyme, on polyacrylamide gel electrophoresis (unpublished data) under the condition used by Kit et al. [27]. The other form of thymidine kinase, called F form (or fetal or cytoplasmic) was not detected under our conditions of extraction. An important thymidine kinase activity (of the same type) was also found in the mature and aging chick neural retina [lo]. However though this neural tissue does not synthesize nuclear DNA at the adult age, it is rich in mitochondria and contains intact nuclei. In contrast, mature lens fibers no longer contain any nuclei or mitochondria [4]. The presence of thymidine kinase in the lens is therefore puzzling. One might look for a role of thymidine derivatives in processes other than DNA synthesis.

M. F.Counis et al.: Lens DNA Polymerases and Ligases Acknowledgements. We thank Dr. C. Pieau for critical reading of the manuscript and Prof. F. Chapeville and Dr. Y. Courtois in whose

laboratories this work was camed out.

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