self-complementary oligodeoxynucleotides (1-8) containing at the 3' terminus either a ribose, 2'-deoxyribose, arabinose, or 2'-deoxyxylose nucleoside.
Volume 15 Number 10 1987
Volume 15 Number 10 1987
Nucleic Acids Research Nucleic Acids
Research
Template-primer analogs as substrates for DNA polymerase
Thomas R.Webb*, Parkash Jhurani and Peter G.Ng
Department of Molecular Biology, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080, USA Received January 28, 1987; Revised and Accepted April 21, 1987
ABSTRACT In order to gain more understanding about the mode of action of DNA polymerase, eight related partially self-complementary "hairpin" shaped oligodeoxynucleotides were prepared. Four of the oligomers contained either
1-o-D-arabinofuranosyluracil (ara-U) or 1-o-D-2'deoxyxylofuranosylthymine (dxTT nucleoside analogs at their 3' termini. We investigated the ability of the oligomers to prime DNA synthesis in relationship to the stability of the hybridized region, the nature of the sugar terminus and the DNA polymerase used (reverse transcriptase, or polymerase a). The results are discussed in relation to the mode of action of some nucleoside analog inhibitors of DNA polymerase. An understanding of the mechanism of DNA polymerase action was used to design template-primer analogs as polymerase inhibitors. Two of the oligodeoxynucleotide analogs prepared were found to be potent inhibitors of polymerase a.
INTRODUCTION There has been significant interest in delineating the mechanism of inhibition of DNA polymerase by polynucleotidela or nucleoside analogs lb . While some of these studies have used homo-oligonucleotides, none have so far used oligonucleotides of defined length and sequence containing all four deoxynucleotides. Recently there have been considerable advances in the synthesis of oligodeoxynucleotides which make the synthesis of deoxyoligomers as long as 70-80mer relatively routine2. We have taken advantage of this progress and have synthesized a number of self-complementary oligodeoxynucleotides (1-8) containing at the 3' terminus either a ribose, 2'-deoxyribose, arabinose, or 2'-deoxyxylose nucleoside. We report here results of a study of the template-primer requirements of reverse transcriptase and a-DNA polymerase using these synthetic oligonucleotides. These results are of significance in the elucidation of the mechanism of action of certain antiviral and antitumor nucleoside analogs, and the rational design of new DNA polymerase inhibitors. For the purposes of this study we desired a simple oligodeoxynucleotide © I R L Press Limited, Oxford, England.
3997
Nucleic Acids Research SCHEME 1 HO\o
T
O
HO
HO
OH
dT
U
0
OH
rU
TT T T AT
ara-U
dxT
TT T T TA GC GC CG AT TA GC TA CG AX 3'
GC GC TA TA GC AX 3' G C C G C
C
T A T
A T
T 5' A
G C T A C G 5' C
1; X
-
dT
HO-OT
(1-B-D-2-deoxyribofuranosylthymine)
5; X
-
dT
7; X - rU (1-B-D-ribofuranosyluracil) 6; X - ru 7; X = aU (1-o-D-arabinofuranosyluracil) 7; X - aU 1; X = dxT (1-o-D-2 -deoxyxylofuranosylthymine) B; X . dxT
TT T T AT GC GC TA TA GC AX GC CG CG GC CG AT TA TA AT 5' 36
9
which would serve as a template-primer. Oligodeoxynucleotides having a self-complementary sequence have been studied and have been shown to adopt a "hairpin" conformation.3 We, therefore, prepared a series of oligodeoxynucleotides (1-8) having the secondary structure shown in Scheme 1. These oligomers have all of the characteristics of a primer-template on a single molecule and should give a single full-length product (such as 9, Scheme 1). These properties make the analysis and interpretation of primer-template experiments relatively simple. The oligomer 1 (a 27mer) was found to be a substrate for calf thymus DNA polymerase a (Pol a)5, and both Moloney murine leukemia virus (MVRT) and avian myeloblastosis virus reverse transcriptase (AMVRT) (data not shown) at 37 C under identical conditions (except for enzyme concentration, see "Experimental") when analyzed by polyacrylamide gel electrophoresis/ autoradiography (see Fig. 1). AMVRT gives only partial conversion of 1 to the full-length (36mer) product 9 (-5 percent). The results are the same with 100-fold the AMVRT concentration shown in Fig. 1 (data not shown).
3998
Nucleic Acids Research 1 2 3 4 5 6 7 8
mo
wproducts
.~~~~~~~~~~ 0. -
1-4
Figure 1.
Autoradiogram of denaturing polyacrylamide gel (20%). Reaction of each 10 pl reaction) using the kinase Lane 1: 32P-labelled 1 (1*), Lane 2: 1* plus equimolar unlabeled 2, Lane 3: 1* plus equimolar 3,jLane 4: 1* plus equimolar 4, Lane 5: 3*, Lane 6: 1* with 0.05 unit AMVRT, Lane 7: 2*, Lane 8: 4*. -
oligomeir 1-4 with AMVRT (0.1 unit in assay, 1 h,737 C (see Experimental).
Under the same conditions (except for enzyme concentration), Pol a gives 31 percent conversion of 1 to 9 (a product having the mobility of the chemically synthesized full-length product), using conditions that are near optimal for AMVRT4,6 (see Table 1). The oligomer 5 (a 35mer), which contains three more base pairs than 1, gives much more efficient conversion compared to 1, with both MVRT and Pol a (see Table 1). Free oligomer 1 3a should be near its Tm under these conditions , and would be expected to be relatively inefficient at priming synthesis, as observed (see Table 1). In contrast, oligomer 5 should adopt the stable hybridized structure shown 3999
Nucleic Acids Research Table I.
Relative Efficiency of Priming of Transcription by
Oligodeoxynucleotides 1-8. Oligomer Enzyme
1
2
3
4
MVRTd,e
.5%a
oc
-5%a
oc
5
6
47%b
.5%a
7
8
traceb
oc
oc Pol af 1Q%a oc 31%b 83%b 100%b 6g%b 8%b These are relative numbers for a 15 minute reaction. The reaction of 5 with Pol a is arbitrarily assigned the value of 100%. The values shown represent percentage of counts incorporated relative to the counts incorporated with 5 using Pol a. For conditions, see "Experimental" under polymerase assays. a) Kinase assay (*2%). b) Incorporation assay (*5%). c) No synthesis detected by method a or b. d) 1-4 gave similar conversion with 0.5 units of AMVRT per 5OX reaction. e) 20 units of MVRT per SOx reaction. f) 0.5 units of Pol a per 50x reaction.
in Scheme 13a. [Longer incubation (1 hour) of 1 and 5 with Pol a gives near complete conversion to the corresponding full length products.] These observations are in agreement with results that have been interpreted as demonstrating the ability of Pol a to stabilize a template-primer 5 structure5. Our data would suggest that AMVRT lacks this ability. As can be seen in Figs. 1 and 2, intermediate length products are not seen. This result is consistent with previous reports that these enzymes are processive ' , that is, all nine (or in the case of 5, twelve) deoxynucleotide units are added before the enzyme primer-template complex dissociates. When the oligomer 2 (containing a 3'-terminal ribose) was subjected to the standard conditions (see experimental) with MVRT, no product was observed (Fig. 1). This is consistent with the observation that .6 reverse transcriptase prefers an oligodeoxynucleotide primer . On the other hand, oligomer 2 was more efficient at priming transcription than 1 in the presence of Pol a (in 15 min. at 37 C; see Table I). This is in agreement with the observation that Pol a can efficiently utilize an oligoribonucleotide primer 7,8 . The more stable hybrid 6, however, is less efficiently utilized than 5 by this enzyme (see Table I). There has been considerable controversy concerning the mechanism of action of antitumor nucleosides containing o-D-arabinoselb' This 4000
.:.,
~ ~ ~ ~ ~ .:
Nucleic Acids Research
1 t2...4.1 ~ .C
: :: >:.:
015::. S
_. .16
........X .......~~~~~~~~~~... . . . ..
.... .....
...
.....
.. .. ....
....
...
....
*-products
..
::
.: .. .:
..
....
. .: ~ ~~~~~ ~ ~~~~~ ~~~~~~~ ..::
.
...
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.
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of denaturing polyacrylamide gel (20%). Reaction of 1T with Pol a (0.5 unit in each 50 ul reaction, lanes 1-4 and 9-12) and MVRT T2U units in each 50 ul reaction, lanes 5-8 and 13-16) using the incorporation assay, 15 min. reaction at 370C (see Experimentai7.7IThe results of this and other experiments are summarized in Table 1.] Lane 1: 1, Lane 2: 2, Lane 3: , Lane 4: 4, Lane 5: 1, Lane 6: 2, Lane 7: 3, Lane 8: 4, Lane 9: 5, LaneClo: 6, Lanie 11: 7, Laine 12: 8, Lane 13: 5, Lane 14: 6, Lane 15: 7, Lane 16: U.
Figure 2. Autoradiogram
mechanism appears to be multifaceted; for example, ara-CTP selectively inhibits the incorporation of dCTP into a poly dG template-oligo dC primer when purified Pol a is used 10 11 .It has been reported that the 4001
Nucleic Acids Research incorporated nucleoside terminates further synthesis in this system10. Other investigations have shown that ara-C serves as a poor primer terminus for DNA synthesis, but is incorporated in full-length DNA of leukemic cells11. The ara-C DNA produced was found to be structurally abnormal. Since all four 8-D-arabinofuranosyl nucleoside triphosphates have been found to inhibit DNA synthesis , we decided to prepare the 1-a-D-arabinofuranosyluracil (ara-U) containing oligomers, 3 (27mer) and 7 (35mer). We found that oligomers 3 and 7 acted as template-primers for MVRT and Pol a, though at a much reduced rate compared to 1 or 5 (see Fig. 2 and Table I). Oligomers 3 and 7 were prepared from ara-U which was greater than 99.5 percent pure. The use of solid phase synthesis assures that all oligimers derived from the ara-U support must terminate in >99.5 percent ara-U. It is not unusual to have small amounts (10 4 M) 21 , since these characteristics are required for a processive mode of polymerization. These facts probably explain the potent inhibition observed with 4 and 8, since these oligomers must inactivate Pol a by tight binding. No inhibition of Pol a by oligomers 2, 3, 6, or 7 is detected at concentrations as high as -6 4x1O M under these conditions (see Figure 3). Likewise, oligomers 2, 3, 4, 6, 7, or 8 do not cause any detectable inhibition of AMVRT (0.5 units per 50 ul reaction) or MVRT (20 units per 50 ul reaction) at 4x1O M (see Fig. 1) under the same conditions.
~~~~~~~~~~~~15
CONCLUSION It can be seen that synthetic oligodeoxynucleotides such as 1-8 are useful probes in elucidating structure-activity relationships in the priming of DNA synthesis. Some insights have been obtained which may be related to at least part of the mechanism of action of anti-cancer drugs such as ara-C, or antiviral drugs such as acyclovir. Template-primer analogs such as 1-8 have several advantages over the standard heterogenous template-primers that have been used in the past. These analogs are of defined sequence and 4003
Nucleic Acids Research length, they possess a single 3' terminus, and a single full-length product is obtained. These properties reduce the number of variables in a template-primer experiment. Analogs 4 and 8 are over fifty times more potent than aphidicolin17 or its synthetic analog18 (on a molar basis) in inhibiting Pol a in vitro. Further investigations using this type of oligodeoxynucleotide may allow us to understand more precisely the mechanism of action of inhibitors of enzymes involved in the replication of DNA.
EXPERIMENTAL Chemistry.
The synthesis of 1 and 5 was accomplished19'20 using the
standard 5'-(4,4'-dimethoxytrityl)-2'-deoxythymidine-3'-hemisuccinate attached to control pore glass (CPG) (Pierce, aminopropyl-500 A/125-177
mu).
The oligomers 2-8 were prepared using the appropriately functionalized
5'-(4,4'-dimethoxytrityl) (5'-DMT) nucleoside hemisuccinate CPG, prepared using the same procedure 22 . Unreacted hydroxyl groups were capped with acetic anhydride/4-dimethylaminopyridine treatment of the functionalized CPG. The loading of support was quantitated in the usual manner; all supports had loadings of 20-40 uM/g. Uridine and ara-U (>99.5 percent pure) were purchased from Sigma Chemical Co. and converted to the 5'-DMT 16 DTxwa derivatives according to the published procedure . 5'-DMTdxT was prepared via the anhydrothymidine nucleoside according to the method of Miller and Fox14. The synthetic cycles, materials and methods of the
phosphoramidite16
or
hydrogen-phosphonate chemistry used here
are
the same
as those published 21922 . All oligodeoxynucleotides were >95 percent pure as judged by gel electrophoresis and autoradiography (see below).
Polymerase assays. DNA polymerase a (calf thymus) was purchased from Pharmacia. This enzyme is stored at 250 units/ml in 60 mM potassium phosphate (pH 7.6), 500 mM KCl, 50 mM 2-mercaptoethanol, 50 percent glycerol. AMV reverse transcriptase was purchased from Promega Biotec. AMVRT is stored at 10,000 units/ml in 100 mM potassium phosphate (pH 7.2), 0.2 percent Triton X-100, 2 mM dithiothreitol (DTT), 50 percent glycerol. MVRT was purchased from Bethesda Research Laboratories. This enzyme was stored at 200,000 units/ml in 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 0.01 percent NP-40, 0.1 mM EDTA, 100 mM NaCl, 50 percent glycerol. All reactions were performed in an assay buffer consisting of 50 mM Tris-HCl (pH 8.3), 1 mM DTT, 6 mM MgCl2, 40 mM KCl, 0.1 mg/ml BSA and 0.085 mM in each dNTP. The unit definition used is that of the enzyme supplier. One unit of Pol a catalyzes the incorporation of a nmole of dAMP into acid-insoluble form in 1 4004
Nucleic Acids Research hour at 37 0C using "activated"7 DNA as the template-primer. The same unit definition is used for AMVRT and MVRT except that the reaction is run for 10 minutes at 37 C using polyA-oligo dT as the template-primer. Experiments were performed to determine the relative number of units of enzyme needed to give efficient conversion of 1, 2, 5 or 6 into full-length transcripts. It was found that ca. 30-100 fold more units of MVRT was needed to give results comparable to Pol a. In the case of 1-4, use of larger amounts of AMVRT or MVRT did not give more conversion to product. Kinase assay. The most sensitive method used for determining whether an oligomer is a substrate for a polymerase is the following; the potential substrate (1-8) was 5'-phosphorylated using y_32 ATP (Amersham, >5000 Ci/mMol), and polynucleotide kinase (PL Biochemicals). A solution of 150 ng of oligomer (1-8) in 10 ul of assay buffer was allowed to react with 0.1 to 200 units of each enzyme at 37 C for 1 hour, unless otherwise indicated. After this period of time 2 ul of the reaction mixture was diluted with 8 il of formamide and run on a denaturing polyacrylamide gel (20 percent for 1-4 and 15 percent for 5-8). The commercial Pol a used in this study contained phosphatase activity so reactions using this enzyme were heated to 95gC for three minutes, then P 5' phosphorylated and analyzed as above. The full-length transcript of 5 was synthesized chemically as above and comigrated with the product of the enzyme catalyzed reaction of 5. The gels were exposed and the resulting autoradiograms were integrated using a LKB Bromma 2202 Ultrascan laser densitometer equipped with a Hewlett-Packard 3390A integrator. Incorporation assay. For quantitation of results, it was convenient to use a procedure like that of Tamblyn and Wells23, where the incorporation of a-32P-dCTP is measured. A 50x reaction contained 1.0 ug (1-8), 5uC of a-32P-dCTP and assay buffer (see above). The inhibition experiments also contain the indicated amounts of inhibitor obtained by serial dilution. After the indicated periods of time at 37 C, 3 ul of the reaction mixture (plus 7 ul formamide) was run on a denaturing gel (20 percent) and analyzed as above.
ACKNOWLEDGEMENTS We wish to thank Mitchell A. Avery, Mark D. Matteucci and Norbert Bischofberger for helpful discussions, and also Mark Vasser for assistance in the synthesis of some of the oligodeoxynucleotides used in this study.
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Nucleic Acids Research *To whom
correspondence should be addressed
REFERENCES 1. a) For an example of a template analog inhibitor of a DNA polymerase see: De Clercq, E., Fukui, T., Kakiuchi, N., Ikehara, M., Hattori, M., Pfleiderer, W. (1979) Cancer Lett. 7, 27, b) Suhadolnik, R.J. (1979) "Nucleosides as Biological Probes", John Wiley and Sons, New York. 2. For a review see: Caruthers, M.H. (1985) Science 230, 281. 3. (a) Haasnoot, C.A.G., den Hartog, J.H.J., de RooiT,7J.F.M., van Boom, J.H., Altona, C. (1980) Nuc. Acids Res. 8, 169. (b) Ikuta, S., Chattopadhyaya, R., Ito,-.T, Dickerson, T.E., Kearns, D.R. (1986) Biochemistry 25, 4840-. 4. Verma, J.M.. (IT81) Boyer, P.D. (ed.), "The Enzymes", Academic Press, Inc., New York, Vol. XIV, pp. 87. 5. Fisher, P.A., Korn, DT1979) J. Biol. Chem. 254, 11040. 6. Verma, J.M. (1977) BBA 473, 1. 7. Wilson, S.H., Matsukage, A., Bohn, E.W., Chen, Y.C., Sivarajan, M. (1977) Nuc. Acids Res. 4, 3981. 8. Weissbach, W7(1781) Boyer, P.D. (ed.), "The Enzymes", Academic Press, Inc., New York, Vol. XIV, pp. 67. 9. Fry, M. (1983) Jacob, S.T. (ed.), "Enzymes of Nucleic Acid Synthesis and Modification", CRC Press. Boca Raton, Vol. I, p. 57. 10. Momparler, R. (1972) Mol. Pharmacol. 8, 362. 11. Kufe, D.W., Major, P.P (1982) Med. Ped. Onc. Supp. 1, 49. 12. Derse, D., Cheng, Y., Furman, P-T, CTair,WM.H.,jElion, G.B. (1981) J. Biol. Chem. 256, 11447. 13. Atkinson, M.R., Deutscher, M.P., Kornberg, A., Russell, A.F., Moffatt, J.G. (1969) Biochemistry 8, 4897. 14. Fox, J.J., Miller, N.C. (1962) J. Tr Chem. 27, 936. 15. Fisher, P.A., Wang, T.A., Korn, D.71979) J. Biol. Chem. 254, 6128. 16. Fisher, P.A., Korn, D. (1979) J. Biol. Chedm. 754, 11033. 17. Ikegami, S., Taguchi, T., Ohashi, N., Oguro`, FW7 Nagano, H., Mano, Y. (1978) Nature 275, 458. 18. McMurry,7J.E.,Wbb, T.R. (1984) J. Med. Chem. 27, 1367. 19. Adams, S.P., Kavka, K.S., Wykes, E.J., Holder, 3B., Gallupi, G.R. (1983) J. Am. Chem. Soc. 105, 661. 20. Fro-eiler, T.C., Ng7.G., Matteucci, M.D. (1986) Nuc. Acids Res. 14, 5399. 21. Webb, T.R., Matteucci, M.D. (1986) Nuc. Acids ResT4,[7T6T. 22. Chow, F., Kempe, T., Palm, G., (198TJNuc. Acids Res. 9, 2807. 23. Tamblyn, T.M., Wells, R.D. (1975) BiocHemistry 141417.
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