A MOLECULAR MECHANISM OF MITOMYCIN ACTION: LINKING OF COMPLEMENTARY DNA STRANDS*,t BY V. N. IYER AND W. SZYBALSKI - PNAS
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VOL. 50, 1963 MICROBIOLOGY: IYER AND SZYBALSKI 355
15 The absence of a preheating effect in PRi is to be contrasted with the marked preheating
effect observed by Horiuchi et al., J. Mol. Biol., 3, 703 (1961), in a mutant showing thermolabile
repression of f-galactosidase synthesis.
16 Garen, A., and H. Echols, these PROCEEDINGS, 48, 1398 (1962).
17 Gallant, J., and R. Stapleton, Bacteriol. Proc., p. 124 (1963).
18 Gallant, J., and R. Stapleton, manuscript in preparation.
19 By "temperature-sensitive," we mean responding differently to temperature than growth
rate. The use of the differential plot automatically normalizes to the growth rate.
A MOLECULAR MECHANISM OF MITOMYCIN ACTION: LINKING OF
COMPLEMENTARY DNA STRANDS*,t
BY V. N. IYER AND W. SZYBALSKI
MICROBIOLOGY RESEARCH INSTITUTE, CANADA DEPARTMENT OF AGRICULTURE, OTTAWA, AND MCARDLE
MEMORIAL LABORATORY, UNIVERSITY OF WISCONSIN, MADISON
Communicated by Kenneth B. Raper, June 7, 1963
The selective action of the antibiotic mitomycin C (MC)' on deoxyribonucleic
acid (DNA),2-6 together with its reported antineoplastic," 7 mutagenic,8 and phage-
inducing9 activities, has stimulated several investigations on the mechanism of its
action. The preferential inhibition of bacterial DNA synthesis by MC, accom-
panied by progressive and extensive breakdown of the DNA, indicates that DNA is
the principal target. However, the rapidity of MC-induced "death" seemed to
be out of step with the relatively much slower process of DNA breakdown. This
suggested that the effects hitherto observed might be secondary to an earlier action
of the antibiotic on DNA. Such a primary lesion is described here and interpreted
as in vivo MC-induced linking ("cross-linking") of the complementary strands of
the DNA molecule.
Materials and Methods.-Samples of mitomycin C were kindly provided by Dr. J. Lein, Bristol
Laboratories, Syracuse, N. Y., by Dr. R. B. Ross, Cancer Chemotherapy National Service Center,
NIH, Bethesda, Md., and by the Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan. The bacterial
strains used included Escherichia coli strain B, Sarcina lutea strain ATCC-272, and the following
Bacillus subtilis mutant lines:'0' " wild-type, the indole-requiring, the linked indole- and histidine-
deficient (I-, H-), and a prototrophic derivative of the indole-requiring strain 168, the latter bear-
ing in addition a marker (mac-rl) conferring resistance to the macrolide group of antibiotics.
Bacteria which were growing exponentially in Difcoos antibiotic medium 3 (Penassay broth) were
exposed to MC under the conditions specified, and their survival determined by plating on nutrient
agar. To terminate MC exposure the cells were chilled, washed twice with cold SSC (0.15 M
NaCl + 0.015 M Na3* citrate), and frozen with 100 jg lysozyme/ml. The procedures used for the
isolation of protein- and RNA-free DNA and for the determination of its buoyant density and
transforming activity were outlined earlier." Thermal transition ("melting") curves were ob-
tained with a recording thermospectrophotometer.'2 Thermal denaturation was carried out by
exposing the DNA in 0.015 M NaCl + 0.0015 M trisodium citrate at pH 7.7 (DSC) to 100'C (or
other specified temperatures) for 6 min followed by rapid cooling in an ice bath."
Results.-The exposure of an exponentially growing culture of B. subtilis to an
inhibitory concentration of MC resulted in very rapid cell death, colony-forming
capacity dropping by several powers of ten in a matter of minutes (Fig. 1, Table 1).
Native DNA extracted at this early period from MC-exposed cells and examined
Downloaded by guest on September 24, 2021356 MICROBIOLOGY: IYER AND SZYBALSKI PROC. N. A. S.
TABLE 1
EFFECT OF MITOMYCIN C (MC) ON CELL SURVIVAL (%) AND TRANSFORMING
ACTIVITY (% T.A.)*
Exposure to MC MC concentration (Ag/mi)
(min) 0.1 0.5 1.0 2.5 12.5
1 (B. subtilis) 100 0.1
5 " - 100 - 4 X 10-5
15 " 100 10 0.8 0.6 10-
15 (E. coli) 5 10-2 2 X 10-7
15 (% T.A.) 100 54 45 21
30 (B. subtilis) 4 X 10-3VOL. 50, 1963 MICROBIOLOGY: IYER AND SZYBALSKI 357
FIG. 2.-Microdensitometer tracings of photographs taken after 40 hr of
TN JN IN CsCR82SO4 equilibrium density-gradient centrifugation (31,410 rpm; 250C)
DofNA extracted from B. subtilis cells, never exposed to mitomycin (A),
or grown for 15 min in the presence of 12.5 ;sg mitomycin (B,C,D), dena-
1!1
.'. s tured (6 min 100°C; 0.02 M Na+; pH = 7.8), and rapidly chilled at O'C.
In experiment C, 1% HCHO was present during denaturation, and 0.2%
HCHO during centrifugation. The DNA used in experiment D was
mechanically sheared prior to denaturation (forced 3 times through a
'j gauge 25 needle (I.D. = 0.254 mm), using a spring-loaded constant rate
i ,A ®@ syringe CR 700, Hamilton Company, Inc., Whittier, Calif., at a concen-
X t| |i tration of 15 sg/ml). The shaded areas roughly correspond to the mito-
0
- -l
tmycin-linked DNA. The NN peaks (native B. subtilis DNA-dotted
lines) and the dN-T6 peaks (denatured, rapidly cooled coliphage T6
DNA-broken lines) were obtained by repeating the centrifugation after
! (MCHO), addition, directly to the centrifuge cell, of 5 Ml (1 M&g) of the respective
DNA's, serving as reference density markers. Buoyant densities:
B. subtilis native DNA (NN)- 1.424, denatured (dN)-1.446. Sedi-
©: | @ mentation constants S0o,w (in SSC) equal to 37 (A), 33 (B,C), and 19
A / )(D),
i\ which
j correspond to mol. wts. of approximately 32, 23, and 5 X 106,
al i respectively (J. Eigner, personal communication). The proportion of de-
1A5 natured, nonlinked DNA, determined by comparing the areas under the
1OYANDkENSlTy (r,_3) peaks, increases upon shearing from 15% (PO = 0.15) (B) to 65% (P0 =
0.65) (D).
the native cross-linked DNA should result in separation of the linked from the non-
linked DNA fragments and a decrease in the relative amount of native-like "mole-
cules" upon subsequent denaturation (Fig. 7, third line), as revealed by density-
gradient centrifugation. This prediction was verified in the experiment illustrated
in Figures 2B and 2D. When shearing reduced the sedimentation constant of
the DNA from S02o,w = 30-40 to 15-20 S, the amount of cross-linked molecules de-
creased from 85 to 35 per cent.
Dependence of the cross-linking on MC concentration, period of exposure, and tem-
perature: When exponentially growing B. subtilis cells were exposed to a concen-
tration of 0.1 ,ug/ml MC at 370C, multiplication of the cells was arrested, but there
dN NN 44770 RPM
0
18A xz
0
FIG. 3.-Microdensitometer tracings of photographs taken after
22 hr of CsCl equilibrium density-gradient centrifugation (44,770
rpm; 250C) of DNA extracted from B. subtilis cells grown (nutri-
12 ent broth) in the presence of 0 (A), 12 (B-E), or 1 (a-H) ug of
t12 mitomycin (MC) for the indicated periods of time (min). To
prepare the DNA represented by tracing F, the cells were first
frozen with lysozyme (100 jug/ml of SSC), thawed, and incubated
J ir . 12 IS for 30 min at 370C, and the resulting lysate was exposed to 12
Jug of mitomycin/ml. With the exception of tracing D (70C), ex-
rc/I-07
t posure to mitomycin was carried out at 370C. All the DNA
2 15samples were denatured under the conditions outlined in the
18 The broken (B. subtilis) and dotted (Clostrid-
legend to Fig. 2. lines
LYSATE
12
,um perfringers) represent native, density-reference DNA's,
added directly to the cells prior to repeating the centrifugation.
I2 ISO Buoyant densities: C. perfringens native DNA-1.691 g/cm3; B.
subtilis native DNA (NN)-1.703, denatured DNA (dN)-1.718
g/cm3. Frequencies of MC links: 1 link per mol. wt. of 10 X 106
AJi s (C), 34 X 106 (D), 135 X 105 (G), 30 X 106 (H).
)
PA 120
1.73 l"d (0/CM
BUOYANT LCN*ITY
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was no observable reduction in viability during the 20-hr period of observation (Ta-
ble 1). By the experimental criteria employed, DNA extracted from these cells
behaved like normal DNA. The MC-imposed anomaly in the banding pattern of
the denatured DNA became apparent at higher concentrations of MC. The rela-
tive amount of the DNA that resists denaturation (shaded areas) increased with
increasing concentrations of MC (1-12 tug MC/ml; Figs. 3G and 3B or 3C), or in-
creasing periods of exposure (5-120 min; Figs. 3G and 3H); it was also a function
of the temperature (70 versus 370C; Figs. 3D and 3C). For intermediate degrees
of reaction with MC, the heated and rapidly cooled DNA comprised two distinct
molecular classes, one of which had the buoyant density of denatured normal mole-
cules, and the other (spontaneously renatured cross-linked molecules; shaded areas
in Figs. 2 and 3) banding at a buoyant density slightly greater than that of the na-
tive normal molecules. On further reaction, the fraction banding in the renatured
region increased until there was no detectable banding in the denatured region,
unless hydrolytic or mechanical breakdown of the DNA intervened. Spreading of
the bands (Fig. 3E) and a decrease in the alcohol precipitability of the DNA heralded
the onset of its enzymatic destruction.
Cross-linking in the absence of DNA and protein synthesis, and lack of in vitro in-
teraction between purified DNA and MC: The question of whether the changes in-
duced by MC in DNA molecules in vivo are dependent on active synthesis of DNA or
protein was examined by studying the effect of MC (12 ,ug/ml, 15 min, 370C) under
conditions of inhibition of these syntheses by 5-fluorodeoxyuridine (10 ,g/ml added
5 min ahead of MC) and chloramphenicol (50 ,g/ml added 15 min ahead of MC),
respectively. Notwithstanding the previous exposure to and continued presence
of these inhibitors, MC induced the molecular changes in the DNA to the same de-
gree as in the absence of the inhibitors.
The implication that no concomitant DNA or protein synthesis is necessary for
the MC-induced linking to occur was further strengthened by the observation that
MC effectively cross-links purified B. subtilis DNA in the presence of either homol-
ogous or heterologous (Sarcina lutea) cell-free lysates prepared by lysozyme treat-
ment and osmotic rupture (cf. Fig. 3F and legend). Nevertheless, MC failed to
induce such changes in deproteinized DNA or DNA plus RNA extracts even after
prolonged periods of in vitro exposure to 100 ,ug MC/ml (banding profile identical
to that in Fig. 3A). Likewise, the transforming activity of purified DNA was un-
affected by MC in vitro.
Stability of the effect: The continued presence of MC in the culture was not a re-
quirement for persistence of the observed effects on the DNA. In fact, 2 hr in-
cubation (37°C) of MC-treated (15 min, 12 ,ug MC/ml), washed cells in the ab-
sence of the antibiotic failed to reverse the MC effects.
Generality of the effect: In the light of recent reports", 15 that a fraction of the
DNA molecules of B. subtilis normally fails to undergo strand separation on thermal
denaturation, it was important to determine whether the observed effects of MC
were peculiar to this bacterial species or could be extended to others. DNA ex-
tracted from Escherichia coli strain B cells exposed to MC under similar conditions
behaved in a manner analogous to the B. subtilis DNA on heating and rapid cooling.
A mutant strain of E. coli, partially resistant to MC, required 10 times higher MC
concentrations to exhibit comparable bactericidal and DNA cross-linking effects.
Downloaded by guest on September 24, 2021VOL. 50, 1963 MICROBIOLOGY: IYER AND SZYBALSKI 359
100 -*8
MITOMYCN
LCOU DNA
piA 7i2M Nai
04
CONTROL 5
fo2 7ell 0 MAC
I T
20 0 40 60
TEMPERATURE0) (M 70 80 90
FoliG.lines)
FIG. 4.-Thermal The
denaturation
.-Themal dnaturtion 0.03mlsampleofDTEMPERATURE
5.-Thermal
FIG. (Cnof
inactivation
profiles in 7.2 M NaClO4 obtained transforming DNA extracted from B.
with the "recording thermospectro- rubtilis cells grown in nutrient broth
photometer"'12 for DNA's isolated
from E. coli cells grown in nutrient sanldeinse fortoa15 ndoni (33')t
adepsdfr1
ted s) to0
(cooint epotedine)for (MC-
et 10 mitomycin/mi. Each point corre-
(s5ol l tine)orlg
m himci The
sar-ponds to an assay performed on an
soli
nsindaThed
first heating (0.5C/min) is indicadcDSC
by the thicker lines. Absorbance
0.03 ml sample
(0.015 M NaCl, 0.0015dissolved
of DNA, M Nacit- in
rate, pH = 7.8) at 10 ug DNA/ml,
changes (O.Dsi60) during subsequent sealed in a glass capillary, heated for
cooling (50C/min) and heating 10 mnm at the indicated temperature,
(0.50C/min) cycles, which were car- anrpilchleincewtr(C)
ried
tecc nte omofpent arerepre- Transforming activity(IND)
was assayed for
sentedb thetompannerlnes.
pointedbtransthionne
vlunes, The
The
(meling" midr-
indole
agar)
independence
and for resistance
(minimal
to the macro-
poin trasitinvauest. (meltng" lide group of antibiotics (MAC) (0.5 uig
temperature), correspond to 48.500 erythromycin/ml), by plating 0.05 ml
(control) and 48.00C (mitomycin- of a transforming mixture containing
linked DNA). 0.002 ug of DNA and approximately
2 X 107 recipient cells.
In preliminary studies, we have also observed similar effects of MC on the DNA
of cultured human cells and of replicating coliphage T4 or B. subtilis phage PBS 2,
although 5-10 times higher MC concentrations were required.
The thermal transition characteristics of "MC-linked" DNA: The changes ob-
served in the absorbance of E. coli DNA, both normal and MC-linked, slowly heated
in 7.2 M NaClO4,12' 14 indicate that most of the hydrogen bonds "melted out"
(Fig. 4, heavy lines). Differences were observed only in subsequent cooling and
heating cycles, which were carried out in the thermospectrophotometer cuvette
compartment. The temperature-absorbance profiles indicate that a high propor-
tion of the MC-linked DNA returns to the native-like (hypochromic) state (Fig.
4, thin solid line) unlike the normal DNA (thin dotted line). Analogous results
were obtained with B. subtilis DNA.
Transforming activity of "MC-linked" DNA: While MC-induced loss of cell
viability and DNA cross-linking are rapid processes, the transforming activity of the
DNA, tested for three different characters, is only gradually lost (Fig. 1). Even
when cross-linking was quite extensive (Figs. 2B and 3B), as much as 20 per cent of
the original transforming activity was still retained by the molecules. Under these
conditions, the ability of two genetically linked markers (I+, H+) to be cotransferred
was also retained, suggesting that the region concerned had not yet suffered any
damage. Furthermore, the transforming activity of MC-linked DNA, unlike
the activity of normal DNA, was not critically and abruptly destroyed over a narrow
temperature range (Fig. 5). This remarkable heat stability of DNA isolated from
MC-treated cells indicates that its residual transforming activity is primarily
associated with the cross-linked DNA molecules.
Downloaded by guest on September 24, 2021360 MICROBIOLOGY: IYER AND SZYBALSKI PROC. N. A. S.
On prolonged exposure to MC (over 2 hr), the ability of the I +H+ region to be
cotransferred to the doubly deficient strain fell more rapidly than that of the com-
ponent markers. A similar genetic dissociation of associated markers under the
influence of A1\C has recently been reported by Yukil6 in the Escherichia coli 1K12
system. Prolonged exposure to MC resulted in eventual loss of all detectable trans-
forming activity and recoverability of polymerized DNA.
Discussion.-The most obvious interpretation of the foregoing results is the for-
mation of rare links between complementary DNA strands by metabolically ac-
tivated MC. The high chemical reactivity of this antibiotic could be inferred from
its structure (Fig. 6): aziridine ring 1, 2, la, methyl-
urethane
CH2CNH2 substitutions position 9, quinone structure 5, 8, and
N I at
H2N 8 at positions 7 and 9a are all highly re-
63C 9 OCH3 active groups. The polycyclic structure might favor
INH
intercalation of this compound into DNA,"8 as a step
prior to selective cross-linking. The density shift in
MITOMYCIN C
the presence of HCHO (Fig. 2C) and the increase in
FIG. 6.-Structure of mito- absorbance of the MC-linked DNA (Fig. 4, solid heavy
mycin C according to Webb
et al.'7 line) attest to almost complete collapse of the hydro-
gen-bonded structure during thermal denaturation,
as schematically indicated in Figure 7 (column B), although the MC-induced link
does not permit complete separation of the complementary strands. This link,
maintaining the original alignment, permits very rapid return of the DNA mol-
ecule to the original native, hydrogen-bonded structure, a process kinetically im-
probable for denatured normal DNA (column C). Assuming a Poisson's distribu-
tion of the links, and further, that one of these links is sufficient to permit the
spontaneous renaturation of the molecule, it is possible to make a very rough esti-
mate of the average number of cross-links per molecule, m = In (1/Po) where Po
is the proportion of the molecules unlinked. Based on the numerical data derived
from Figures 2B and 2C, the average frequency of MC-induced links corresponds
to one per 20,000 nucleotide pairs, i. e., per mol. wt. 12 X 106 (1.8 links (B) per
mol. wt. 23 X 106, or 0.43 link (D) per mol. wt. 5 X 106). Similar estimates for
various conditions of MC treatment are listed in the legend of Figure 3.
1000C
DENATURATION
00oc
QUENCHING
NORMAL D FIG. 7.-Molecular events follow-
ing the denaturation (A to B) and
rapid cooling (B to C) of normal and
MC-linked DNA, the latter before
and after subjection to hydrody-
MC namic shear. The possibility of hy-
LINKED drolytic breakage of the phosphate
ester bonds and its consequences are
indicated for MC-linked DNA (mid-
DSH ,AKn dle row).
MC
LINKED
SHEARED
~ ~ ~ r
Downloaded by guest on September 24, 2021VOL. 50, 1963 MICROBIOLOGY: IYER AND SZYBALSKI 361
From our present findings and those of others,2-6 it is suggested that the "cross-
links" imposed by MC (approximately 50 per bacterial DNA complement for 5 min
exposure to 1 ,gg MC/ml; Fig. 3F) (1) interfere with DNA replication; (2) rapidly
lead to cell death;"9 (3) nevertheless have little effect on the ability of the DNA to
transform for a given marker2 or direct the synthesis of one or a few proteins,21
since these two activities reflect a function of a relatively small fraction of the total
DNA complement. The reported partial resistance of viral DNA replication to
the action of MC3 9, 22 remains to be explained, but may reflect the relatively
smaller size of the viral DNA molecules and/or their fragmentation2 during the
replication process.
The observation that some natural bacterial and viral DNA's appear to be cross-
linked,11' 15, 24 together with the results here obtained with mitomycin, which is a
natural product, suggests some biological role for this cross-linking process. If
reversible, this process would permit turning on or off DNA synthesis, and thus
would assume a regulatory significance. Although the determination of the exact
chemical nature of the cross-links might be difficult because of their low frequency,
the presently acquired knowledge of the mechanism of MC action permits applica-
tion of this antibiotic in a variety of experiments, both with intact cells25 and trans-
forming DNA.
Summary.-Exposure of Bacillus subtilis or Escherichia coli cells to inhibitory
concentrations of mitomycin C for periods of 1-60 min results in covalent linking
of the complementary DNA strands. This cross-linked DNA denatures upon
thermal treatment, but renatures spontaneously even with rapid chilling, as revealed
by its return to the native-like buoyant density in CsCl or Cs2SO4 gradients and by
the reversibility of the optical density changes during heating and cooling cycles.
Hydrodynamic shearing of the cross-linked DNA permits separation of the linked
and nonlinked regions.
Only 10-4 per cent of B. subtilis cells survive 15 min exposure to 12 ,g of mito-
mycin per ml. Transforming DNA isolated from cells so treated (one cross-
link per mol. wt. of 10-12 million) retains 20-30 per cent of its activity, which be-
comes resistant to thermal denaturation. Higher concentrations of mitomycin
are required to effect a comparable degree of cross-linking of vegetative T4 or PBS 2
phage DNA and the DNA of human cells or mitomycin-resistant bacterial mutants.
Mitomycin has no in vitro effect on purified DNA unless a cell extract is added.
We are indebted to Dr. Elizabeth H. Szybalska for her editorial help, to Dr. I. Takahashi for
the B. subtilis strain carrying the mac-rl marker and for the phage PBS 2, to Dr. H. Gottschling
for the phage T4(N24), to Mrs. Z. Opara-Kubinska for her counsel with the transformation ex-
periments, and to Mr. Larry Fenton, Mr. Hans Kvinlaug, and Miss Gwen Nettles for their able
technical assistance.
*
These studies were supported by grant G-18165 from the National Science Foundation and by
grant CY-5215 from the National Cancer Institute, USPHS. Contribution No. 559 from the
Microbiology Research Institute, Canada Department of Agriculture, Ottawa.
tThe following abbreviations are used in this paper: DNA, deoxyribonucleic acid; RNA,
ribonucleic acid; RNase, ribonuclease; MC, mitomycin C; UV, ultraviolet light.
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19 The degree of cross-linking observed for short-term MC exposures might be somewhat exag-
gerated, since cell-absorbed MC could have been active during cell washing (4°C) and during
subsequent lysozyme treatment (370C). This delayed effect of MC might explain the high via-
bility of cells exposed for 5 min to 1 ug MC/ml (Table 1), although on the average their DNA
showed one cross-link per mol. wt. 135 X 106 (Fig. 3F).
20 The effect of subinhibitory concentrations of MC on pneumococcal receptor cells was re-
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25 The rapid and presumably selective effect on DNA exhibited by MC might suggest the use
of this agent in antineoplastic perfusion techniques (Cohen, D. H., Med. J. Australia, 2, 807
(1960)), where other less selective cross-linking agents, e.g., nitrogen mustards, are now principally
employed. See Research in Radiotherapy, ed. E. T. Krementz, NAS-NRC Publ. 888 (1961), p.220.
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