Volume 9 Number i1 1981
Nucleic Acids Research
Effect of several metal ions on misincorporatdon during tiption
Sail K.Niyogi and Rose P.Feldman
Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA
Received 16 March 1981
ABSTRACT
By use of poly(dA-dT) as template and Escherichia coli RNA polymerase,
several metal ions were tested for their effect on the efficiency of
transcription and on the misincorporation of CMP into the poly(rA-rU)
product. In the presence of 10 nM MgC12, Mn2+ has a stimulatory effect on
the transcription, Co2+ has very little effect on the reaction, Cu2+ and
Zn2+ are strongly inhibitory, and Cd2+ and Ni2+ are less inhibitory. The
background misincorporation of CMP in the presence of MgC12 is about
1 nucleotide per 2000 correct nucleotides incorporated and is independent
of Mg2+ concentration. Zn2+, Ca2+, Sr2+, Li+, Na+, and K+- all nonmutagenic and noncarcinogenic - do not increase misincorporation. Mn2+
causes a concentration-dependent threefold increase in the misincorporation
that can be slightly reversed at higher MgC12 concentrations. Cd2+ causes
a dramatic increase in the misincorporation with increasing CdC12
concentration that can be substantially overcome by higher concentrations
of Mg2+. Cu2+ also increases the misincorporation, Ni slightly increases
it, and Co2+ does not increase it at all. Several control experiments
indicate that the misincorporation of CMP is dependent on the templatedirected synthesis of poly(rA-rU). Nearest-neighbor analysis indicates
that CMP is incorporated in place of UMP into the poly(rA-rU) product.
The increase in misincorporation appears to be related both to the
'hard-soft" character of the metal ions and to their carcinogenic potential.
+
INTRODUCTION
Divalent metal ions play a crucial role in enzymatic reactions
involving phosphate transfer. In almost all cases, Mg2+ is the preferred
cation serving as a cofactor of the reaction. Mn2+ can replace Mg2+ in
most reactions and in some cases Co2+ and Zn2+ can only partially replace
Mg2+. The ability of metal ions to react with various electron donor sites
on polynucleotides as well as to provide optimal conditions for the RNA
polymerase reaction has received considerable attention (1-8). Certain
metals have been identified as potential environmental carcinogens through
occupational exposure as well as in the laboratory (9,_10). Metal ions in
this category as well as metal atagens have been shown to decrease the
©) IRL Pres Umitod, 1 Falconberg Court, London W1V 5FG, U.K.
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Nucleic Acids Research
fidelity of DNA synthesis in vitro (11). Such metal ions also stimulate
RNA chain initiation in vitro at concentrations that inhibit overall RNA
synthesis, whereas metal ions not in this category inhibit initiation at
concentrations that inhibit overall synthesis (12).
Investigators from various laboratories have revealed that RNA
polymerase can insert noncomplementary nucleotides into RNA during
transcription of polydeoxynucleotides (13-17). For example, Paetkau et al.
(15) demonstrated T-G base pairing during transcription of well-defined
polydeoxypyrimidines by Escherichia coli RNA polymerase in vitro;
specifically, poly(rG) (the only homopolymer made) was synthesized in the
presence of defined repeating DNAs having only T and C residues on one
strand. Mn was a better (by about threefold) divalent cation than
Mg for poly(rG) synthesis, indicating the promotion of base mispairing by
2+
Mn . Using E. coli RNA polymerase and synthetic polydeoxynucleotide
templates, Springgate and Loeb (17) found that the highest ratio of
noncomplementary to complementary nucleotides incorporated followed the
order CMP > AMP > UMP > GMP. The frequency of misincorporation was not
significantly affected by the nature of the template, but primarily by the
type of noncomplementary nucleotide. Rather surprisingly, Mn2+, a known
mutagen that has been shown to decrease the fidelity of DNA synthesis (11),
increased the fidelity of transcription of poly(dA-dT) by E. coli RNA
polymerase (17).
Studies of replication and transcription are usually conducted at an
optimal alkaline pH (7.8-8.1) and in the presence of reducing agents like
2-mercaptoethanol and dithiothreitrol, conditions that cause precipitation
of some of the test metals, particularly at higher metal ion concentration,
thereby causing artifacts and difficulty in assessing the effective metal
ion concentration (Niyogi and Feldman, unpublished observations).
Accordingly, we undertook an examination of the effects of several metal
ions (Mn , Cd , Cu , Ni2+, Co 2+, Sr 2+, Ca 2+ Mg2+ ,z 2+ , Na+ K, and
Li ) on misincorporation during transcription of the well-defined template
poly(dA-dT) with E. coli RNA polymerase under conditions of lower pH,
namely 7.1, and absence of reducing agents so that the metal ions remained
in solution. These investigations comprise the subject of this manuscript.
MATERIALS AND METHODS
Unlabeled ribonucleoside triphosphates were products of P-L Biochemicals.
[3HICTP, [a- 32P]ATP, and [a- P]CTP were purchased from either Amersham/Searle
2616
Nucleic Acids Research
or New England Nuclear. The purity of each substrate was determined by
chromatography on BioRad Aminex A-28, following the procedure of Khym (18).
Only those batches of labeled CTP were used that were free of UTP. Poly(dA-dT)
was purchased from Miles Laboratories and dialyzed against 10 mM Tris-HCl
(pH 7.1) before use.
RNA polymerase from E. coli B was purified according to the method of
Stevens (19), except that the final sucrose density gradient centrifugation
step was replaced by the glycerol gradient centrifugation procedure of Burgess
(20). The preparation was over 90% pure, as judged by electrophoresis on
polyacrylamide gels in the presence of sodium dodecyl sulfate. It was free
of contaminating activities such as polynucleotide phosphorylase and DNA
polymerase and of detectable nuclease activity, as measured by use of
radioactively labeled RNA and DNA preparations as substrates for the release
of acid-soluble radioactive material. The preparation did not introduce
single-strand nicks in DNA, as detected by alkaline sucrose density gradient
centrifugation.
The reaction mixture (0.1 ml) for measurement of the efficiency of
transcription (reacton mixture A) contained 20 mM Tris-HCl (pH 7.1), 20 mM
MgCl2, 5 pg poly(dA-dT), 50 pM UTP, 50 pM [ 4CATP (4000 cpm/nmol), and
varying concentrations of the test metal chloride. The reaction mixture
(0.1 ml) for measurement of the incorporation of complementary and
noncomplementary ribonucleotides (reaction mixture B) contained 20 mM
Tris-HCl (pH 7.1), 5 pg poly(dA-dT), 50 pM UTP, 50 pM [a- 32P]ATP
(10-20 cpm/pmol), 50 pM [ H]CTP (1000-2000 cpm/pmol), and varying
concentrations of MgCl2 and the test metal chloride, as indicated in the
figures and tables. RNA synthesis was initiated by the addition of 5 pg
of E. coli RNA polymerase holoenzyme, and the isotope incorporated into
acid-insoluble material was measured after a 10-min incubation period at
37°C. The reaction was stopped by the addition of cold 5% trichloroacetic
acid containing 10 mM sodium pyrophosphate, and the solution was filtered
through a Whatman glass paper (GF/C) disc. The disc was then washed
extensively with the same solvent, followed by cold ethanol, then dried
under an infrared heat lamp, placed in a scintillation vial, and counted
with BBOT-toluene solution (4 g of BBOT in a liter of toluene) in a Packard
Tri-Carb liquid scintillation spectrometer.
The 2',(3') ribonucleotides produced by alkaline hydrolysis of the
polyribonucleotide products were separated by Biogel DM2 column chromatography
similar to that described by Fujimura (21).
2617
Nucleic Acids Research
RESULTS
Control experiments
A. Effect of reaction conditions on efficiency of transcription. Studies
in our laboratory have shown that alkaline pH (7.5 and above) and the presence
of sulfhydryl reducing agents caused precipitation of some of the test
metals (Niyogi and Feldman, unpublished observations). We decided to rectify
the problem by eliminating such reducing agents and lowering the pH of the
transcription reaction. With poly(dA-dT) as template, omission of
2-mercaptoethanol in the assay mixture had no appreciable effect on the
reaction. Between pH 7.1 and 8.1 there is very little change in the rate of
transcription with poly(dA-dT) template. These results testify to the high
efficiency of poly(dA-dT) as a template for E. coli RNA polymerase. With the
choice of pH 7.1 for the poly(dA-dT)-directed reaction, it was desirable to
study the effect of MgCl2 concentration at that pH. The reaction is totally
dependent on added divalent cation, as expected. The reaction rate increases
with MgCl2 concentration, reaching a maximum at 10 mM, with no appreciable
increase at MgCl2 concentrations of 15 and 20 mM.
B. Requirements for incorporation of complementary and noncomplementary
nucleotides. Control experiments to check whether the template was indeed
poly(dA-dT) and whether poly(rA-rU) was the product produced the following
results. (1) There is no incorporation of labeled ATP in the absence of UTP.
(2) There is no incorporation of labeled UTP in the absence of ATP. (3)
Nearest-neighbor analysis with [a-3 P]ATP + [3H]UTP or [a-3 PIUTP + [ 3HIATP
confirms that the product is indeed poly(rA-rU). (4) The synthesis of
poly(rA-rU) depends on the presence of both poly(dA-dT) and Mg2 besides
ATP + UTP.
C. Purity of CTP . The labeled CTP used was analyzed by chromatography
on BioRad Aminex A-28 (18), was found to be > 99%, and more importantly,
had no detectable UTP. Nearest-neighbor analysis of the RNA product confirmed
that the labeled CTP was indeed incorporated as CMP and not as UMP (as
described under "Nearest-neighbor analysis of the polyribonucleotide product").
D. Dependence of the incorporation of CMP on the template-directed
synthesis of poly(rA-rU). (1) There is no incorporation of CMP in the absence
of either poly(dA-dT) or Mg or both, even in the presence of both ATP and
UTP. (2) The poly(dA-dT)- and Mg -dependent incorporation of CMP requires
the presence of both ATP and UTP; neither ATP nor UTP alone is sufficient.
Effect of test metal ion concentration on poly(dA-dT)-directed
transcription. Increasing concentrations of each metal chloride were tested
2618
Nucleic Acids Research
in the presence of 10 mM MgC12 - the optimum concentration of Mg
for the
poly(dA-dT)-directed reaction. As shown in Fig. 1, Mn2+ has a concentrationdependent stimulatory effect up to a concentration of 0.5 mM, with no further
stimulation up to 2.0 mM. Co2+ has very little effect on the reaction. Ni2 ,
Cd , and Cu show a concentration-dependent inhibition of transcription.
Cu
shows the greatest inhibition, at CuCl2 concentrations of 0.02, 0.05,
and 0.07 mM, the residual reaction is 85, 14, and 0.5%, respectively. Zn2+
is also strongly inhibitory, at ZnCl concentrations of 0.02, 0.07, 0.10,
0.15, and 0.20 mM, the residual reaction is 90, 65, 25, 13, and 5%,
respectively. Zn has a similar inhibitory effect with calf thymus DNA
or phage T4 DNA as template (Niyogi, Feldman, and Hoffman, manuscript in
preparation). This inhibition by Zn is somewhat surprising, because E. coli
RNA polymerase, and in fact all the RNA polymerases studied thus far, appear
to have very tightly bound Zn as an integral part of the enzyme (22-27).
Cd and Ni have a more moderate inhibitory effect on the reaction, with
Cd being more inhibitory. The reaction is inhibited about 50% at a CdCl2
concentration of 0.1 mM and at a NiCl2 concentration of 0.5 mM. It should
2+~~~~~~~~~~~~~~~~~~~~~~~
140-
Mn2+
o
Co 2+
!p
0
a:
a:
z
a.
.2+
0
n
METAL ION CONCN. (mM)
Fig. 1. RNA synthesis was measured with reaction mixture A as described
under Materials and Methods.
2619
Nucleic Acids Research
be pointed out that both the stimulatory and inhibitory effects are observed
at much lower concentrations of the metal ions at pH 7.1 and in the absence
of 2-mercaptoethanol than at pH 8.1 in the presence of 2-mercaptoethanol.
The latter conditions lead to precipitation of the metals, thereby reducing
the effective metal ion concentration in solution. This point will be
discussed in detail in a subsequent publication (Niyogi, Feldman, and Hoffman,
manuscript in preparation).
Effect of MnCl on misincorporation of CMP during poly(rA-rU) synthesis.
As shown in Table I, the background misincorporation of CMP in the presence
(the required metal ion) and in the absence of any test metal ion is
of
about 1 pmol/2000 pmols of AMP + UMP incorporated. This value, which
does not change appreciably at different MgCl2 concentrations (Table I),
agrees quite well with the value obtained by Springgate and Loeb (17), namely,
1 CMP per 2400 complementary nucleotides incorporated during poly(rA-rU)
synthesis.
In the presence of MnCl2 alone there is a concentration-dependent
increase in the misincorporation of CMP, reaching a value of about 3 per
2000 complementary nucleotides incorporated at 2.0-10.0 mM MnCl2' It
should be noted that Mn and Co were the only divalent metal ions able
2+
to support RNA synthesis without the addition of Mg . It was of interest
to see whether MgCl2 would be able to overcome the effect of MnCl2 in
promoting misincorporation. As shown in Table I, there is a suggestion that
the Mn effect is overcome with increasing concentrations of MgCl2, particularly
at very low (0.1 and 0.2 mM) MnC12 concentrations. MgCl2 has no effect on
the misincorporation at higher concentrations of MnC12.
Effect of CdCl on misincorporation of CNP during poly(rA-rU) synthesis.
As shown in Table II, there is a dramatic increase in the misincorporation of
Because of the
CMP in the presence of increasing concentrations of CdCl 2
great inhibition of the reaction at CdCl2 concentrations > 1.0 mM, the exact
values of the misincorporation of CMP at these higher concentrations should
be accepted with some caution, although these values are still considerably
higher than the zero-time background incorporation of CMP, and there is a
definite CdCl2 concentration-dependent increase in the misincorporation
(Table II). Unfortunately, the misincorporation could not be studied at
CdCl concentrations beyond 2.0 mM because of the drastic inhibition of
transcription at these higher concentrations and points out the limitations
of such studies with highly toxic metal ions. The Cd 2+-induced misincorporation,
2+
unlike that in the presence of Mn , can be substantially overcome by higher
Mg2+
2620
Nucleic Acids Research
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Nucleic Acids Research
concentrations of Mg ; the degree of recovery is particularly apparent at
the lower Cd concentrations.
on misincorporation of CMP during poly(rA-rU) synthesis.
Effect of CuCl, 2
Because of the extremely inhibitory effect of Cu on the transcription
reaction (Fig. 1), its effect could be studied at only a few concentrations.
At CuCl2 concentrations of 0.0, 0.02, 0.05, and 0.07 mM (the overall
transcription being 100, 85, 14, and 0.5%, respectively), the misincorporation
of CM? is 0.90, 0.92, 1.65, and 2.52, respectively, per 2000 residues of
complementary nucleotides incorporated.
Effect of NiCl, and CoCl on misincorporation of CMP during poly(rA-rU)
synthesis. In the presence of 10 mM MgCl2 and at NiCl2 concentrations of 0.0,
0.1, 0.2, 0.3, 0.5, 1.0, 1.5, and 2.0 mM (the overall transcription being 100,
85, 65, 60, 52, 36, 25, and 17%, respectively), the misincorporation of CMP
is 1.2, 1.3, 1.4, 1.4, 1.5, 1.5, 1.6, and 1.7 pmol, respectively, per 2000 pmol
of correct complementary nucleotides incorporated. Because of this small
increase, the effect of MgCl2 concentration was not studied in this case.
CoCl2, at the above concentrations and in the presence of 10 mM MgCl2, has
virtually no effect on the misincorporation of CMP. Since Co 2+, like Mn
is able to support RNA synthesis without the addition of Mg 2+, its effect was
studied in the absence of MgC12. Even up to a concentration of 10 mM, CoC12
has practically no effect on the misincorporation of CMP.
Nearest-neighbor analysis of the polyribonucleotide product. As a test
of the manner of incorporation of CMP, the polyribonucleotide product
synthesized with poly(dA-dT) as a template and with [a- 32P]CTP as the
noncomplementary nucleotide and UTP + ATP as the complementary nucleotides
was hydrolyzed with alkali so that the 3 P was transferred to the
5'-adjacent nucleotide. After separation of the nucleotides on a Biogel
DM2 column (21), practically all (,. 99%) of the 32p was found in 2',(3')AMP
and virtually none in UMP or CMP (Table III). When the experiment was
performed with [a-3 P]CTP and UTP + [3H]ATP, both the 32p and 3H co-migrated
almost exclusively with 2',(3')AMP. With [ 3H]CTP and [a- 32P]ATP, the 3H
and 32p co-migrated as 2',(3')CMP. Thus, CMP is incorporated in place of
UMP and opposite to dAMP on the poly(dA-dT) template. The same results
were obtained whether Mg2+ or Mn2+ was the only divalent metal ion in the
reaction (Table III) or whether a test divalent metal ion was added along
with Mg 2
~~~~~~2+
2623
Nucleic Acids Research
TABLE III.
Nearest-neighbor analysis of the RNA product obtained
with poly(dA-dT) template*
-Nearest-neighbor
sequence found
*
Cpm in product synthesized in
10 m MgC 2
|
5
! MnC2l
ApC
9,757
31,839
CpC
65
237
UpC
73
273
Two reaction mixtures (0.1 ml each) were made containing 20 aM
Tris-HCl (pH 7.1), 5 pg poly(dA-dT), 50 pM UTP, 50 pM ATP, and 50
uM [a-32PCTP (10,000 cpm/pmol). One reaction contained 10 mM
MgCl2 and the other 5 mM MnCl2. RNA synthesis was initiated by
the addition of 5 pg of E. coli RNA polymerase. After a 10-min
incubation at 37°C, each RNA product was collected and washed on
a glass paper disc, as described in Materials and Methods. The
disc was transferred to a scintillation vial and incubated with
0.5 ml of 0.3 N KOH at 37°C for 18 hr. The solution was
transferred to a tube. The disc was washed with several small
volume of 0.3 N KOH, and the washings were pooled with the above.
The pooled sample was neutralized to pH 4.0 with 1 N HCIO4, and
the resulting KC104 precipitate was removed by centrifugation.
The nucleotides in the superniatant were then separated on a
Biogel DM2 column according to the procedure of Fujimura (21).
DISCUSSION
The background error rate during transcription of poly(dA-dT) with
2+
E. coli RNA polymerase in the presence of Mg - the obligatory
physiological metal ion for transcription - is about 1 CMP incoroporated
per 2000 complementary nucleotides incorporated into poly(rA-rU), in good
agreement with the value obtained by Springgate and Loeb (17), namely,
1 per 2400. This error rate, although infrequent, is 200 times higher than
that reported for replication of the same template with E. coli DNA
polymerase I - the error rate, although for dGMP incorporation, being 1 per
400,000 (28). This increased fidelity cannot be explained solely on the
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basis of the presence of the 3' exonuclease, associated with DNA
polymerase I as the editing enzyme (29), since eukaryotic DNA polymerases
have no such error-correcting activity and yet copy polynucleotides with
high accuracy (30, 31).
The error rate of CMP incorporated into poly(rA-rU) remains unaffected
2+
2+
at different concentrations of Mg . Mn , on the other hand, causes a
concentration-dependent increase in the misincorporation of CMP, reaching a
level that is about threefold more than the control value. This agrees
with the results of Paetkau et al. (15), who showed that Mn2+ was a better
(by about threefold) cation than Mg2+ for poly(rG) synthesis with
poly(T-C)-poly(G-A) template. Our results on promotion of misincorporation
of CMP by Mn differ from those of Springgate and Loeb (17), who found
about a twofold increase in fidelity during transcription of poly(dA-dT).
The latter result is somewhat surprising in view of the fact that Mn, a
known mutagenic and carcinogenic metal (9, 10), promotes misincorporation
during DNA replication (11, 32, 33) and increases initiation during
transcription (12).
Cd dramatically increases the misincorporation of CMP into
poly(rA-rU) even at very low concentrations of the metal ion. The
Cd 2+-induced misincorporation could be partially overcome by higher
2+
2+
concentrations of Mg , particularly at lower concentrations of Cd . It
should be noted that on a concentration basis Cd2+ was the most efficient
metal ion in increasing chain initiation while decreasing overall
transcription (Niyogi, Feldman, and Hoffman, manuscript in preparation).
It is interesting that higher concentrations of MgCl2 are able to
partially overcome the misincorporation caused by Mn and Cd2+,
particularly at the lower concentrations of these metal ions. It would
indeed be useful to study whether Mg has a similar effect with other
error-causing metal ions. Among other metal ions tested, Zn 2+, Ca2+, Sr+
Li , Na , and K - all nonmutagenic and noncarcinogenic metals - did not
increase misincorporation of CMP during poly(dA-dT) transcription (Niyogi
and Feldman, unpublished observations). It was, however, rather surprising
that Ni increased misincorporation of CMP to only a small extent and
2+
had practically no effect on the misincorporation, particularly since
Co
both metals are known mutagens and carcinogens (10).
In view of the above, the carcinogenic potential of different metal
ions cannot be used as the sole rationale for the increase in
misincorporation caused by the ions. Our results could be partially
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Nucleic Acids Research
explained on the basis of the "hard-soft" character of the metal ions (34).
Ahrland et al. (35) classified electron donor and acceptors into two types.
Class (a) ions and molecules form their most stable complexes with ligands
that have electron donor atoms from the first short period (N, 0, F), whereas
class (b) species form their most stable complexes with donor atoms from the
second (P, S, Cl) or later period. Pearson (36) introduced the terminology
"hard" and "soft" for the classes (a) and (b). For example, among metals,
and Zn2+ as borderline; and
Na, Mg , and Ca are classified as hard; Co
Cd , Pb , and Hg2 as soft. In general, hard ions are less polarizable
and prefer to bind with hard acceptors, whereas soft ions are more polarizable
and prefer to bind with soft acceptors. Thus, predominantly covalent bonding
occurs between soft ligands, in contrast to the ionic bonding that characterizes
the association of hard donor and acceptor partners. The basic physicochemical
concepts relating hard/soft character to metal ion biochemistry and toxicity
are discussed in recent reviews (37, 38). The interaction of metal ions with
nucleic acids can be characterized according to the preference of metal ions
for hard sites like phosphate or soft sites that are electron-rich, like the
bases. On this basis, the affinities of metal ions for nucleic acid bases
compared to phosphate are as follows: Pb2+ > Cd2+ > Cu2+ > Mn2+ > Zn2+ >
Ni
-Co
> Mg
> Ca
> Li , Na , K
(1, 2).
Our results could be partially
explained on the above basis. Obviously, many more metal ions need to be tested
before a definitive conclusion can be reached.
ACKNOWLEDGMENT
Research sponsored by the Office of Health and Environmental Research,
U. S. Department of Energy, under contract W-7405-eng-26 with the Union
Carbide Corporation.
REFERENCES
1.
2.
3.
4.
5.
6.
2626
Eichhorn, G. L. and Shin, A. Y. (1968) J. Am. Chem. Soc. 90, 73237328.
Izatt, R. M., Christensen, J. J. and Rytting, J. H. (1971) Chem. Rev.
71, 439-481.
Fuchs, E., Millette, R. L., Zillig, W., and Walter, G. (1967) Eur. J.
Biochem. 3, 183-193.
So, A. G., Davie, E. W., Epstein, R. and Tissieres, A. (1967) Proc.
Natl. Acad. Sci. USA 58, 1739-1746.
Maitra, U. and Barash, F. (1969) Proc. Natl. Acad. Sci. USA 64,
779-786.
Hoffman, D. J. and Niyogi, S. K. (1973) Biochim. Biophys. Acta 299,
588-595.
Nucleic Acids Research
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
Mangel, W. F. and Chamberlin, M. J. (1974) J. Biol. Chem. 249,
3002-3006.
Domingo, E., Escarmis, R. C. and Warner, R. C. (1975) J. Biol. Chem.
250, 2872-2877.
Sunderman, F. W., Jr. (1977) in Advances in Modern Toxicoloty,
Goyer, R. A. and Mehiman, M. A., Eds., Vol. 2, pp. 257-295,
Hemisphere, Washington, D.C.
Sunderman, F. W., Jr. (1978) Fed. Proc. 37, 40-46.
Sirover, M. A. and Loeb, L. A. (1976) Science 194, 1434-1436.
Hoffman, D. J. and Niyogi, S. K. (1977) Science 198, 513-514.
Chamberlin, M., Baldwin, R. and Berg, P. (1963) J. Mol. Biol. 7,
334-340.
BuJard, H. and Heidelberger, C.
(1966) Biochemistry 5, 3339-3344.
Paetkau, V., Coulter, M. B., Flintoff, W. F. and Morgan, R. A. (1972)
J. Mol. Biol. 71, 293-306.
Strniste, G. F., Smith, D. A. and Hayes, F. N. (1973) Biochemistry 12,
603-608.
Springgate, C. F. and Loeb, L. A. (1975) J. Mol. Biol. 97, 577-591.
Khym, J. X. (1975) Clin. Chem. 21, 1245-1252.
Stevens, A. (1969) J. Biol. Chem. 244, 425-429.
Burgess, R. R. (1969) J. Biol. Chem. 244, 6160-6167.
Fujimura, R. K. (1970) Anal. Biochem. 36, 62-71.
Scrutton, M. C., Wu, C.-W. and Goldthwait, D. A. (1971) Proc. Natl.
Acad. Sci. USA 68, 2497-2501.
Coleman, J. E. (1974) Biochem. Biophys. Res. Commun. 60, 641-648.
Falchuk, K. H., Mazus, B., Ulpino, L. and Vallee, B. L. (1976)
Biochemistry 15, 4468-4475.
Auld, D. S., Atsuya, I., Capino, C. and Valenzuela, P. (1976) Biochem.
Biophys. Res. Commun. 69, 548-554.
Lattke, H. and Weser, U. (1976) FEBS Lett. 65, 288-292.
Wandzilak, T. M. and Benson, R. W. (1977) Biochem. Biophys. Res.
Commun. 76, 247-252.
Trautner, T. A., Swartz, M. N. and Kornberg, A. (1962) Proc. Natl.
Acad. Sci. USA 48, 449-455.
Brutlag, D. and Kornberg, A. (1972) J. Biol. Chem. 247, 241-248.
Chang, L. M. (1974) J. Biol. Chem. 249, 7441-7446.
Loeb, L. A. (1974) in The Enzymes, Boyer, P., Ed., Vol. 10,
pp. 174-208, Academic Press, New York.
Hall, Z. W. and Lehman, I. R. (1968) J. Mol. Biol. 36, 321-333.
Van de Sande, J. H., Loewen, P. C. and Khorana, H. G. (1972) J. Biol.
Chem. 247, 6140-6148.
Ahrland, S. (1968) in Structure and Bonding, Vol. 5, pp. 118-149,
Springer-Verlag, New York.
Ahrland, S., Chatt, J. and Davies, N. R. (1958) Quart. Revs. (Lond.) 12,
265-276.
Pearson, R. G. (1963) J. Am. Chem. Soc. 85, 3533-3539.
Nieboer, E. and Richardson, D. H. S. (1980) Environ. Pollut. (Ser. B) 1,
3-26.
Jacobson, K. B. and Turner, J. E. (1980) Toxicology 16, 1-37.
2627