A proposed site of action for zinc in DNA synthesis

J. COMP.

PATH.

1976.

VOL.

86.

81

A PROPOSED IN

J. R.

SITE DNA

OF ACTION SYNTHESIS

and I. E.

DUNCAN

FOR

ZINC

DREOSTI

Deportment of Biochemistry, University of Natal Pietermaritzburg, Republic of South Africa

INTRODUCTION

Previous studies have suggested a role for Zn in nucleic acid metabolism (Schneider and Price, 1962 ; Somers and Underwood, 1969 ; Williams and Chesters, 1970) : in particular, Zn deficiency has been shown to inhibit DNA synthesis in rapidly dividing cells (Swenerton, Sharader and Hurley, 1969; Grey and Dreosti, 1972; Duncan and Dreosti, 1974). The proposal has therefore been made that the severe teratogenesis accompanying a maternal Zn depletion in rats may arise as a consequence of impaired DNA synthesis during foetal organogenesis (Swenerton et al., 1969; Grey and Dreosti, 1972). Although the site at which DNA synthesis is affected has not been identified, it has been suggested that it occurs at the level of one or more of the Zn-dependent enzymes directly associated with DNA synthesis (Slater, Mildvan and Loeb, 1971; Nelbach, Pigniet and Gerhart, 1972 ; Prasad and Oberleas, 1974). The present investigation concerns the effect of a dietary Zn deficiency on DNA synthesis in the nuclei and the mitochondria from regenerating rat liver. Both systems were studied since they involve certain distinctly different enzymes (Kalf and Ch’ih, 1968; Berk and Clayton, 1973) and may, therefore, have afforded some additional information concerning the role of Zn at this level. Initially, an examination was made of the effect of added deoxyribonucleosides on DNA synthesis to establish whether Zn deficiency exerted an effect on the synthesis of the precursors required for the subsequent phosphorylation reactions. Thereafter, attention was focused on the thymidine kinase “salvage” pathway since it serves as the major route for the synthesis of thymidine monophosphate in rapidly dividing cells (Weissman, Smellie and Paul, 1960) and possessesall the attributes of the rate determining enzyme in DNA synthesis during cell proliferation (Davidson, 1972). MATERIALS

AND

METHODS

Reagents. 3H-thymidine (methyl-T) 5 mc./mM and sH-thymidine-5’-triphosphate (methyl-T) 47 mc./mM were purchased from the Radiochemical Centre, Amersham. Deoxyribonucleosides were purchased from Sigma Chemicals and DEAE-cellulose paper from Whatman Biochemicals. Animals and diets. Female rats (Wistar strain, 100 to 120 g.) were housed individually in stainless steel cages and fed for 3 days on a ration consisting of, in per cent., sucrose

82

J. R. DUNCAN

AND

I. E. DREOSTI

50, soyabean meal (44 per cent. protein) 38.5, maize oil 6.1, salt mix (Huriey and Swenerton, 1966) 3.0, cod liver oil 0.7, or.-methionine, 0.5 and choline chloride 0.2. The soyabean meal was treated with ethylene diamine-tetraacetic acid to reduce its Zn content (Davis, Norris and Kratzer, 1962) and the entire ration was found to contain less than 0.5 l.tg.Zn/g. Control animals received the same ration supplemented with 60 pg.Zn/g as ZnSO, . 7HsO. Surgical procedure. Partial hepatectomy (70 per cent.) was performed according to the method of Higgins and Anderson ( 193 1) . Plasma
and Randall ( 1951). RESULTS

Additional Deoxyribonucleosides The results indicated that although the relative incorporation rates of 3H-thymidine into DNA differed depending on the deoxyribonucleoside injected, the addition of free deoxythymidine, deoxyadenosine, deoxycytidine or deoxyguanosine either singly or in combination had no significant effect on the inhibition of DNA synthesis accompanying zinc deficiency. In all cases

incorporation was significantly reduced (P < 0.01) in the zinc-deficient rats compared with the control animals. The specific activity of DNA isolated from animals which had been injected with unlabelled thymidine was lower than that in animals receiving no additional deoxyribonucleosides. The effect is

SITE

OF ACTION

FOR

probably due mainly to a dilution of unlabelled deoxynucleoside.

ZN

IN

DNA

of the labelled

SYNTHESIS

thymidine

83

with large amounts

Thtymidine Kinase

The activity of thymidine kinase was significantly reduced (P < 0.01) in the Zn-deficient animals in all 3 enzyme fractions (nuclear, mitochondrial and postmitochondrial supernatant) between 10 and 30 h. after operation (Table 1). The addition of O-1 PM Zn to the Zn-deficient enzyme fractions 60 h. prior to assay restored enzyme activity to the control levels. Addition of other metal ions had no effect (Table 2). TABLE1 ACTIVITY

OF THYMIDINE

Status of rat

KINASE

Time after partial hepatectomy (h.)

Controi Zinc-deficient

0 0

Control Zinc-deficient

::

Control Zinc-deficient

20 20

Control Zinc-deficient

30 30

IN ZINC-DEFIUENT

(ctjmin Nuclear

REGENERATING

RAT

LIVERS

Thymidine kinase acti@ thyidine phosphorylated/mg fiotcin/h.) Post-mitochandrial Mitochondrial supernatunt

221*19 208 + 28

1142593 751+45*

356k27 347 + 24*

402 + 36 255 f 24t

1656k 175 1503+57*

599 f 42 400& 1st

1 148k46 752+52t 997 + 57 695 + 54t

2 904+70 1997+83t

1896& 166 1 252 f 78t

2 871+92 1995+91t

1719k63 1 150+58t

Between 8 and 10 animals were used in each group. Thymidine kinase activity was estimated enzyme fractions isolated from livers at various times (0 to 30 h.) after partial hepatectomy. * P < 0.05 as determined by Student’s t-test. tP < 0.01. TARLE EFFECT

OF METAL

IONS ON THE

Six animals

were

2

ACTIVITY OF THYMIDlNE KXNASE FROM ZINC-DEFICIENT LIVERS 20 h. AFTER PARTIAL HEPATECTOMY

Zn2+ ZnZ+ Znl+ Mg2c Mn2+ Ca2+ cu2+ Fez+

0.05 0.10 0.50 0.10 0.10 0.10 0.10 0.10

used for each treatment.

752+52 1 103+58 1 124+92 688 + 84 820+ 77 781+ 79 755+84 810562 798k81 Metal 60 h. prior

in

ions were to assay.

1997k 1872+ 2 639+ 1213+ 1506+ 1439+ 1337+ 1 307& 1408k added

83 88 129 96 109 83 91 101 74

REGENERATING

RAT

1252+ 78 2 864k122 2 639+ 129 1 950* 151 2 101+ 89 1986+ 108 2008+ 74 2 079& 112 1998& 75

to the enzyme

preparations

for

84

J.

R.

DUNCAN

AND

I.

E.

DREOSTI

DISCUSSION

Studies concerning the injection of deoxyribonucleosides during liver regeneration indicated that the effect of Zn deficiency on DNA synthesis did not occur because of reduced levels of these precursors and suggested that Zn acted at some point beyond deoxynucleoside synthesis. Accordingly, further studies focused on the effect of dietary Zn restriction on the activity of the enzyme thymidine kinase. The reduction in activity of thymidine kinase in regenerating livers from zinc-deficient (3 day) rats confirmed and extended the findings of Prasad and Oberleas (1974) with zinc-deficient (6 to 17 day) rat connective tissue, and indicated a requirement for zinc by thymidine kinase during DNA synthesis in a number of subcellular locations. In addition, the restoration of activity of thymidine by the addition of zinc indicated for the first time that the reduced activity was due to a direct effect of zinc on the enzyme rather than to an overall effect on protein biosynthesis. The inability of other metal ions to effect activation of thymidine kinase suggests a specific requirement for Zn by this enzyme. Although it must be considered that the effect of Zn on DNA synthesis and cell division probably reflects the combined effects of a number of enzyme systems, evidence from these and earlier studies (Duncan and Dreosti, 1973) points to a prime locus of action associated with the activity of thymidine kinase which, it appears, is reduced before any other event in the cycle of cell division is affected. The suggestion affords a biochemical explanation for the rapid cessation of growth in Zn-deficient animals as well as the rapid teratogenicity associated with maternal Zn depletion during pregnancy in rats. SUMMARY

The activity of thymidine kinase was significantly reduced (P < O-01) in nuclear, mitochondrial and post-mitochondrial fractions from regenerating liver between 10 and 30 hours after partial hepatectomy in Zn-deficient rats. Activity was restored by addition of 0.10 mM Zn to the Zn-deficient fractions for 60 hours prior to assay, but not by a variety of other divalent metal ions. The findings point to a specific requirement for Zn by thymidine kinase and afford a possible explanation for the effect of a dietary Zn restriction on DNA synthesis in rapidly dividing cells. ACKNOWLEDGMENT

The authors wish to thank Research fop. the postgraduate

the South African bursary awarded

Council to JRD

for Scientific and Industrial during 1974-75.

REFERENCES

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SITE

OF ACTION

FOR

ZN

IN

DNA

SYNTHESIS

85

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123, 692-696. Kalf,

G. F., and Ch’ih, J. J. (1968). Purification and properties of DNA polymerase from rat liver mitochondria. Journal of Biological Chemistry, 234, 49044916. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193, 265-275. Neibach, M. E., Pigniet, W. P., and Gerhart, J. C. (1972). A role for zinc in the quarternary structure of aspartate transcarbamylase from E. coli. Biochemistry,

11, 315-327. Prasad, A. S., and Oberleas, 0. (1974). Thymidine kinase activity and incorporation of thymidine into DNA in zinc-deficient tissue. Journal of Laboratory and Clinical Medicine, 83, 634-639. Schneider, W. C., and Hogeboom, G. H., (1950). Intracellular distribution of enzymes. Journal of Biological Chemistry, 183, 122-l 28. acid level,: a possible Schneider, E., and Price, C. A. (1962). D ecreased ribonucleic cause of growth inhibition in zinc-deficiency. Biochimica et biophysics acta, 55, 406407. Slater, J. P., Mildvan, A. S., and Loeb, L. A, ( 1971). Zinc in DNA polymerases. Biochemistry and Biophysics Research Communications, 44, 37-43. Somers, M., and Underwood, E. J. (1969). R i b onuclease activity and nucleic acid protein metabolism in the testes of zinc-deficient rats. Australian .yournal of Biological Sciences, 22, 1277-1282. Swenerton, H., Shrader, R., and Hurley, L. S. (1969). Zinc-deficient embryo: Reduced thymidine incorporation. Science, 166, 1014-1015. Volkin, E., and Cohn, W. E. (1954). Estimation of nucleic acids. In Methods of Biochemical Analysis, Vol. I. 0. Glick, Ed. Interscience Publishers, London. Weissman, S. M., Smellie, R. M. S., and Paul, J. (1960). Studies on the biosynthesis of DNA by extracts of mammalian cells. Biochemica et biophysics acta 45, 101-I 10. Wilkins, P. J., Grey, P. C., and Dreosti, I. E. (1972). Plasma zinc as an indicator of zinc status in rats. British Journal of Nutrition, 27, 113-120. Williams, R. M., and Chesters, J. K. (1970). The effect of early zinc deficiency on DNA and protein synthesis in the rat. British Journal of Nutrition, 24, 1053-1059. Witschi, H. P. (1970). Effects of berylium on DNA-synthesizing enzymes in regenerating rat liver. Biochemical Journal, 120, 623-634. [Received for publication,

June 16th, 19751