Volume 4 Number 6 June 1977

Nucleic Acids Research

Levels of DNA polymeraseso,/S, andyin control and repair-deficient human diploid fibroblasts1

Vann P.

Parker2 and Michael W. Lieberman3

Environmental Mutagenesis Branch, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Received 29 March 1977

ABSTRACT

The activities of DNA polymerases a, 0, and y were determined in control and repair-deficient human fibroblasts (xeroderma pigmentosum complementation groups A, C, and D; Fanconi's Anemia; and Bloom's syndrome). Assays were done on 103,OOOXG supernatants which had been chromatographed on DEAE cellulose to remove nucleic acids and on fractions containing polymerase activities which had been separated from one another on a second DEAE cellulose column. All repair-deficient cell types contained all three DNA polymerase activities. Caffeine, which has been observed to inhibit some DNA-repair processes in intact cells, had no effect on DNA polymerase activities from XP-A, XP-C, XP-D or XP-variant cells. These data indicate that all three polymerases are present in cells which have reduced or absent repair functions and that the caffeine effects observed in living cells are probably not due to the direct action of caffeine on DNA polymerases.

INTRODUCTION Several pathways for DNA repair in mammalian cells appear to involve a step requiring DNA polymerase(s). The best characterized DNA repair process in mammalian cells, excision repair, involves the insertion of approximately 100 nucleotides into a gap left after the removal of damage. A second type of repair process, post replication repair, appears to involve the insertion of nucleotides on the strand opposite a damaged site during the process of replication. In addition, other processes involved with maintenance of chromosomal integrity may require DNA polymerases. A number of disease syndromes in humans are associated with deficiencies in DNA repair: These include xeroderma pigmentosum, Fanconi's anemia, and possibly Bloom's syndrome. It would be of interest to evaluate these cell types for the presence of known DNA polymerases for several reasons: 1) Although one class of repairdeficient cells (excision-repair-deficient XP cells) has a defect in the ability to remove thymine dimers 495 and acetylaminofluorene residues from DNA6, presumably resulting from altered or absent endonuclease function(s), C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England

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Nucleic Acids Research it is not clear whether the defect is limited to this function alone or if it also includes other repair functions, such as gap-filling. 2) Another class of repair-deficient human cells (XP variants)7 appears to have reduced or absent post-replication repair (insertion of nucleotides opposite the region of damage), and this process is reported to be especially sensitive to caffeine. 3) Other cell types (e.g. FA and BS) are unable to repair chromosomal aberrations, a process which, like gap-filling and post replication repair, may require DNA polymerase activity 8,9 . Recent advances in our understanding of at least some of the DNA polymerases present in mammalian cells allow a relatively straightforward approach to assessing levels of polymerases in small samples of cultured human cells and thus for evaluating levels of polymerase activities in control and repair-deficient cells.

MATERIALS AND METHODS Cell Culture: All fibroblasts were obtained from the American Type Culture Collection (Table 1) except for FU (started from a foreskin by Dr. S.L. Huang), WI-38 (obtained from L. Hayflick, Stanford University), and XP-25 and 1492 (obtained from the Human Genetic Mutant Cell Repository, Camden, NJ). Cells were grown to confluence in 150 mrn Petri dishes (Falcon #3025) in Dulbecco's Modified Eagle's Medium and 10% fetal calf serum except for WI-38 cells which were grown in Diploid Growth Medium and 10% fetal calf serum. Cells were maintained in a 370 incubator at 10% CO2. They were harvested just prior to confluence, washed four times with cold 0.15 N saline and stored as a pellet (8-15 dishes, 0.3-0.6 ml packed cell volume) in liquid N2 until assayed. No loss of enzyme activity was detected in samples stored in liquid N2 over an 8 mo period. Densely confluent cells were not used because in preliminary experiments we obtained spuriously low values from cultures which were left at confluence for extended periods. Instead semi-confluent cells (X90% G ) were used since polymerases a and y are known to vary with the cell cyclel°. Although an attempt was made to prepare all cell pellets uniformly, differences in growth rate, nuclear to cytoplasmic ratio, and cell density at confluence suggested that preparations from different cell types might be only approximately equivalent.

DNA Polymerase Assays: Assays for polymerases

370

and a were carried out at for 30 min in a volume of 0.1 ml containing 50mM Tris HC1 pH 7.5, 50pg/ml a

poly d(A-T)-poly d(A-T) (Collaborative Research), 5pM ( 3H-methyl)-TTP (1818.6 Ci/mnol, New England Nuclear), 8OpM dATP (Sigma), 2.5mM MgCl2, 1mM DTT (Sigma) and 50mM KC1. Assays for polymerase y were carried out at 300 for 2030

Nucleic Acids Research 30 min in a volume of 0.1 ml containing 50mM Tris HC1 pH 7.5, 50-pg/ml oligo(dT)10*poly(rA) (Collaborative Research), 5pM 3H-TTP, 0.25mM MnCl2' 1mM DTT and 50mM KCl. Reactions were stopped by placing the tubes on ice and imnediately applying 0.08 ml of the reaction mix to 2.3 cm DE 81 filter discs (Whatman). The discs were subsequently washed six times in 0.5M Na2HPO4 to remove any unincorporated activity 11 . Discs were dried by rinsing them with ethanol and dipping them into ether. They were counted in toluene-spectrofluor (Amersham-Searle) scintillation fluid, and the number of picomoles of thymidine monophosphate incorporated was calculated as described by Roeder11 Analyses were run in duplicate and are expressed as the average of the two values after subtraction of the reagent blank value (usually less than 5% of enzyme activity). Duplicates usually were within 10-15% of one another. NEM sensitivity was examined by incubating the enzyme preparations with 1OmM NEM (Sigma) at 00 for 40 min before assay. Caffeine sensitivity was assessed by adding caffeine (Baker) (0.1 to lOmM final concentration) to the reaction mixture before addition of the polymerase preparation. Assays for a and 6 were linear over the 30 minute time course and with increasing amounts of enzyme preparation (protein concentration). Assays for polymerase y were linear for about 20 min and showed a small reduction in rate over the last 10 min; however, estimates of relative amounts of enzymes from different cell types were unaffected by the small deviation from linearity. Analysis of 3H TTP showed it to be at least 96% radiochemically pure as judged by thin layer chromatography.

Preparation of Cell Extracts: Cell pellets were thawed and extracted using a modification of the procedures of Lewis et al.12 The pellets were homogenized at 00 in 2.5 ml of 50mM Tris HCl, pH 7.8, lmM EDTA, lmM DTT, 0.3M KCI, 10% Glycerol, and 0.3% triton-X 100 (buffer A) using 20 strokes with the loose-fitting pestle and 10 strokes with the tight-fitting pestle of a 7 ml Dounce homogenizer (Kontes). The samples were then stirred for 15 minutes at 00 prior to being spun at 18,000g for 15 minutes in a Sorvall RC-2B centrifuge. The pellet was re-extracted as above using 1.2 ml buffer A. The two extracts were combined and spun in a Beckman 5OTi rotor at 35,000 RPM (103,000g) for 1 hr (40). To remove remaining nucleic acids, samples were loaded onto a 5 cm x 0.5 cm (diam) DEAE-cellulose (Whatman DE-52) column equilibrated with 50mM Tris HCI pH 7.8, lmM DTT, 10% Glycerol, and 0.3M KC1 (buffer B). The column was washed with 2.0 ml buffer B. The effluent and the wash were collected and dialysed overnight against 6 Tris HC1 pH 7.8, lmM DTT, 0.02% triton-X 100, and 20% Glycerol (buffer C) and 0.01M KC1

(40).

This post 2031

Nucleic Acids Research dialysis fraction above.

used for analysis of polymerase levels as described

was

Separation of Polymerase Activities: DNA polymerase assays were also run on polymerase fractions in which polymerase a, and y activities had been separated by chromatography of the post dialysis samples on a second set of DEAE columns (modified from Bolden et al.13). Samples (3.3 ml) were loaded onto a 4 cm by 0.5 cm (diam) DEAE cellulose column equilibrated with buffer C containing 0.025M KC1. The effluent was collected and the column was washed with 6.5 ml of buffer C containing 0.025M KCl. The effluent and the first 1.3 ml of wash contained polymerase 6 (see below). Little or no activity was recovered in the additional 5.2 ml of wash. The column was then washed with 8.0 ml of buffer C containing 0.07M KCl. The first 3.8 ml contained most (Xt70-80%) of the polymerase y activity and some (X20-30%) of the polymerase a activity. Most of the polymerase a activity was then eluted with 4 ml of buffer C containing 0.12M KC1. This procedure resulted in separation of polymerases a and a as judged by sensitivity to NEM and sedimentation velocity in sucrose gradients (see below) and partial separation of polymerase a from polymerase y as judged by template preferences. Protein content was determined as described by Loebl4. S,

Velocity Sedimentation Analysis of Fractions Containing Polymerases a or a: Fractions were prepared for velocity sedimentation by dialysing 2-3 ml sample against 50 vol 50mM Tris HCl pH 7.8 for 90 min (40) and subsequent concentration by lyophilization. For each gradient 0.2 ml of redissolved sample was layered onto a linear 5-25% sucrose gradient (w:w) containing 10% glycerol (v:v), 50mM Tris HCO pH 7.8, and 50mM KC1. Centrifugation was for 16 hours at 50,000 RPM in a Beckman SW65Ti rotor at 40 as described by Lazarus and Kitron15 Ovalbumin (m.w. 45,000) was centrifuged in a parallel tube for comparative purposes. RESULTS

Assays of DNA polymerases a, a and y were carried out on the post-dialysis samples from 12 different cell types in two separate experiments (Table 1). Values for polymerases a and a were obtained by taking advantage of the reported sensitivity of polymerase a to NEM and the insensitivity of polymer16 ase a to this agent Values for polymerase 6 represent enzyme activity after incubation with NEM while those for a represent activity of untreated preparations minus treated preparations. Polymerase y was assayed for using poly(rA)-oligo(dT) with Mn2+ as the divalent cation (Materials and Methods). .

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Nucleic Acids Research TABLE 1 Activities of DNA Polymerases a, a and y in Control and Repair-Deficient Human Cells

Source

Cel 1 Type

FU El San WI-38 Ba Del Jay Tim XP-25 Gor Doa Ge Ar Cay Wena 1492 Ce Rela On Ser

Control Control Control Control

XP-Ae XP-A XP-C XP-C XP-D Bloom's Fanconi 's Lesh-Nyan

Pol a Actc,d

Exp 1

Exp 2

1.00 0.17 0.92 0.56 0.31 0.39 0.43 0.42 0.64 1.21 0.42 0.54

1.00 0.34 1.05 _ 0.11

0.19 0.21 ----

0.11 0.25

Pol

Actb

Expi1 Exp 1.00 0.81 1.28 1.22 1.04 1.09 1.31 1.52 0.90 2.02 1.61 1.37

2

1.00 0.84 0.76 0.60

1.04 0.68 0.92 0.68

Pol a Actc Exp 1 Exp 2 1.00 0.08 0.66 0.29 0.14 0.29 0.16 0.177 0.45 0.39 0.23 0.37

1.00 0.24 0.63 _ 0.39

0.34

0.47 _

0.17 0.55

aSamples used for the two experiments with these cells were harvested and at the time. bfrozen To facilitate comparisons results are expressed as the fractional activity of FU preparations. Actual values (in pmol TMP incorporated per mg protein per assay) for FU were a, 1983; ,, 309; y, 742 (experiment 1) and a, 1340; 467; y, 208 (experiment 2). c6,Polymerase a and a activities were calculated from their NEM sensitivities outlined in the text. das The order of presentation (s y, a) corresponds to elution order from DEAE and is meant to facilitate comparisons with Table 2. ecellulose A, C, D refer to the complementation group of these cells20.

Control fibroblasts from four sources (FU, neonatal foreskin; El San, 8 year old skin; WI-38, embryonic lung; Ba Del, 3 year old skin) showed considerable variation in levels of DNA polymerase a and a activity while polymerase y levels showed less variation. Examination of Table 1 suggests that five excision repair-deficient XP cell types (Jay Tim, XP-25, Gor Do, Ge Ar, and Cay Wen), BS fibroblasts, FA fibroblasts and Lesh-Nyan fibroblasts (hypoxanthine-guanine-phosphoribosyl transferase-deficient fibroblasts included as an additional control) all have DNA polymerase activities which approximate values for control cells. To confirm these results we separated the post dialysis fraction from 2033

Nucleic Acids Research these 12 cell types into polymerase a, f3, and y activities on a second DEAE column as described in Materials and Methods. This procedure was especially important in the case of polymerases a and 6 which have similar template preferences and which were distinguished from each other in the above-described experiments by the presence of an inhibitor (NEM). To verify that polymerases a and 0 were actually separated from one another, the DNA dependent DNA polymerase activity from the 0.025M KCl and the 0.12M KC1 eluents were sedimented in sucrose-glycerol gradients (Figure 1). It is clear that the 0.025M Kcl eluent contains the low molecular weight polymerase (polymerase a), while the high molecular weight polymerase (polymerase a) is contained in the 0.12M KC1 eluent. This assignment was confirmed by assaying each peak after incubation with NEM: As expected, the polymerase activity yielding the fast sedimenting peak was >99% inhibited by a 40 min exposure to 10mM NEM, while the polymerase activity in the slow sedimenting peak was relatively resistant (30% inhibition under similar conditions). The results of DNA polymerase assays of the separated fractions from the second DEAE cellulose column are presented in Table 2 (no NEM present). These

25

y

OVA

20

-

15

-

0

10

FRACTION NO

Figurye 1: Velocity sedimentation of DNA dependent DNA polymerases in sucrose-

glyceroT gradients (see text for details). Polymerase activity from the 0.025M KCl wash of the DEAE cellulose column (o---.-o), polymerase activity from the 0.12M KCl wash (.-.o), and ovalbumin (OVA) were centrifuged in separate tubes and plotted on the same axes for comparison. Fraction 1 represents the bottom of the gradients. 2034

Nucleic Acids Research TABLE 2 DNA Polymerase Activities from the Second DEAE Cellulose Column Fractions Pol 6 Actc Exp 1 Exp 2

FUa,b El San WI-38 Ba Del Jay Tim XP-25 Gor Doa Ge Ar Cay Wena 1492 Ce Rela On Ser

1.00 0.25 1.08 0.37 0.32 0.23 0.27 0.33 0.26 0.39 0.21 1.02

1.00 0.48

1.00 ----

0.22 ----

0.23 ----

0.23 ----

0.36 0.52

Pol y Act E

Pol a Act

1.00 0.92 1.27 0.45 0.53 0.47 0.85 0.39 0.39 0.71 0.88 0.67

1.00 0.08 0.28 0.27 0.19 0.29 0.23 0.38 ----

0.38 0.21 0.12

aExperiments

1 and 2 are from cells harvested at the same time. Results are expressed as the fractional activity of FU preparations to facilitate comparisons. Actual values (in pmol TMP incorporated per mg protein per assay) for FU were a, 599; 6, 1500; y, 11,400 1) and $, 960 (experiment 2). c(experiment Order of elution from the second DEAE cellulose column (see Materials and Methods for details).

results are similar to those presented in Table 1 and support the observation that all cell types have normal or near normal levels of DNA polymerase activity. A series of experiments was conducted to assess the effects of caffeine on the three DNA polymerase activities. One of these is presented in Table 3. Caffeine was added (0.1, 1.0 or 1O.OmM final concentration) to the assay mixture before the addition of the enzyme preparation from the second DEAE column, and the assays were carried out as described in Materials and Methods. Polymerases a and a showed little inhibition in either control or XP cells, and the addition of even 1O.OmM caffeine did not result in a further reduction of enzyme activity. Polymerase y showed slightly more inhibition with l.OmM caffeine. Addition of 10mM caffeine did not increase the extent of inhibition, and preparations from control and XP cells were about equally inhibited.

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Nucleic Acids Research TABLE 3 Caffeine Sensitivities of DNA Polymerases from Control and Xeroderma Pigmentosum Fibroblasts

FU Jay Tim Gor Do Cay Wen Wo Mec

Pol 8

Pol y

Pol a

95 92 87 84 88

80 61 71 63 61

101 92 71 100 105

Effect of lmM caffeine on DNA polymerases a, y, and a from control and XP fibroblasts. Values are expressed as percentage of enzyme activity of each cell type without caffeine. Assays were run on fractions separated on a second DEAE column as described in Materials and Methods. Wo Mec (ATCC-1162) is an XP variant line20.

DISCUSSION It is clear that cells from individuals with classical XP, BS and FA have levels of polymerases a, B, and y comparable to control values. Thus it appears that deficiencies in DNA repair (XP) and related phenomena (FA, BS) are not accompanied by absent DNA polymerases. In the case of the classical XP cells, at least, the defect seems limited to the recognition and/or the removal of damage and does not extend to DNA polymerase activity and repair synthesis. This conclusion is supported by the data of Tanaka et al.17 who found that unscheduled DNA synthesis in ultraviolet-irradiated XP cells occurred to the same extent as in normal cells, if cells were fused bacteriophage T4 UV endonuclease. Bertazzoni et al. 8 report normal levels of polymerases at and 8 in classical XP, an XP variant and FA cells. These findings are in agreement with ours. In addition Cook et al. 19 found that extracts of XP cells were able to excise damaged thymine dimers which had been incised with bacteriophage T4UV endonuclease as well as those from control cells. Our data suggest that absence of DNA polymerasea, 8 or y does not accompany deficiencies in DNA repair; however, they do not rule out the possibility that some as yet unidentified polymerase is absent from these cells or that important quantitative differences exist between the polymerases of normal and repairdeficient cells. DNA polymerases from control fibroblasts were relatively insensitive to caffeine even at relatively high concentrations. Of interest is the observa2036

Nucleic Acids Research tion that, like polymerases from control cells, neither classical-XP- nor XPvariant-fibroblast polymerases were especially sensitive to caffeine. These data suggest that, whatever the mode of action of caffeine is on DNA repair processes, it probably does not act directly on DNA polymerases. Thus it appears that inhibition of post-replication repair in XP variant cells is not due to increased sensitivity of XP variant polymerases to caffeine. Additional experiments are needed to confirm this finding especially in view of the fact that the polymerase preparations used to assess caffeine sensitivity were not highly purified. Cells from individuals with FA and BS have normal levels of DNA polymerases. This result suggests that the induced and spontaneous chromosomal anomalies and rearrangements seen in these syndromes is not due to a quantitative reduction of DNA polymerases. REFERENCES

Abbreviations used are: xeroderma pigmentosum, XP; Fanconi's Anemia, FA; Bloom's Syndrome, BS; dithiothreitol, DTT; N-ethyl maleimide, NEM. 2Present address: Department of Chemistry, Duke University, Durham, North Carolina, 27706. 3Address reprint requests to: Michael W. Lieberman, Department of Pathology, Washington University School of Medicine, St. Louis, MO, 63110. 4Setlow, R.B., Regan, J.D., German, J. and Carrier, W.L. (1969) Proc. Nat. Acad. Sci. USA 64, 1035-1041. 5Takebe, H., Nii, S., Ishii, M.I. and Utsumi, H. (1974) Mutat. Res. 25, 383390. 6Amacher, D.A. and Lieberman, M.W. (1977) Biochem. Biophys. Res. Commun. 74, 285-290. 7Lehmann, A.R., Kirk-Bell, S., Arlett, C.F., Paterson, M.C., Lohman, P.H.M., De Weerd-Kastelein, E.A. and Bootsma, D. (1975) Proc. Nat. Acad. Sci. USA 72, 219-223. 8Latt, S.A., Stetten, G., Juergens, L.A., Buchanan, G.R. and Gerald, P.S. (1975) Proc. Nat. Acad. Sci. USA 72, 4066-4070. 9Chaganti, R.S.K., Schonberg, S. aii7 German, J. (1974) Proc. Nat. Acad. Sci. USA 71, 4508-4512. 10Spadari, S. and Weissbach, A. (1974) J. Mol. Biol. 86, 11-20. 11Roeder, R.G. (1974) J. Biol. Chem. 249, 241-248. 12Lewis, B.J., Abrell, J.W., Smith, R.G. and Gallo, R.C. (1974) Biochim. Biophys. Acta 349, 148-160. 13Bolden, A., Fry, M., Muller, R., Citarella, R. and Weissbach, A. (1972) Arch. Biochem. Biophys. 153, 26-33. 4Loeb, L.A. (1969) J. Biol. Chem. 244, 1672-1681. 15Lazarus, L.H. and Kitron, N. (197TFJ. Mol. Biol. 81, 529-534. 16Weissbach, A. (1975) Cell 5, 101-108. 7Tanaka, K., Sekiguchi, M. and Okada, Y. (1975) Proc. Nat. Acad. Sci. USA 72, 4071-4075. ertazzoni, U., Stefanini, M., Pedrali-Noy, G., Nuzzo, F. and Falaschi, A. (1977) Nucleic Acid Res. 4, 141-148. 19Cook, K., Friedberg, E.C. and Cleaver, J.E. (1975) Nature 256, 235-236. 20Kraemer, K.H., Coon, H.G., Petinga, R.A., Barrett, S.F., Rahe, A.E. and Robbins, J.H. (1975) Proc. Nat. Acad. Sci. USA 72, 59-63. 2037