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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS - 1963
Orm -8-15%,
J. Mol. Biol.
JUN 24 1965
Elfects of UV-irradiation on macromolecular
synthesis in Escherichia coli*
CFSTI PRICES
H.C. $ 2.00; MN 50
P. A. Swenson and R. B. Setlow
Biology Division, Oak Ridge National laboratory,
Oak Ridge, Tennessee, U.S.A.

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RENDRASED FOR ANNOUNCEMENT
IN NUCLEAR SCIENCE ABSTRACTS
*Research sponsored by the U.S. Atomic Energy Commission under
contract with the Union Carbide Corporation.
* Present address: Zoology Department, University of Massachusetts,
Amherst, Massachusetts, U.S.A.
TI
Running head:
-
-
Send proof to:
.
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1. INTRODUCTION
:
untraviolet irrmation kills bacteria and characteristically results
in a lag in DNA synthesis (Kelner, 1953). The inhibition of DNA synthesis
18 temporary for radiation resistant strains but is permanent for sensitive
strains
It is obvious that many factors contribute to the sensitivity of
microorganisms to ultraviolet radiation. For ezample, the base composition
would be expected to determine the numbers and proportions of photoproducts
formed by irradiation. In general, the higher the thymine content of DNA,
the greater the sensitivity for colony inhibition (Haynes, 1965). Dimers
formed be cween adjacent pyrimidine residues are important photoproducts
in cells and can account for a large fraction of lethal biological damage
that is produced by ultraviolet irradiation (Setlow, 1964a, b; Smith, 1964;
Wacker, 1963). Probably more important than the number of dimers or other
photoproducts is the capacity of the cells to repair the damage (reviews
in Hollaender, 1960; Sobels, 1963). In both radiation sensitive and
resistant strains of E. coli, pyrimidine dimers seem to act as blocks to
DNA synthesis (Setlow, Swenson & Carrier, 1963). It is felt that the
sensitive strains lack the ability to repair this damage and a few dimers,
formed by very low doses, are lethal whereas the radiation resistant strains
are able to excise dimers from their DNA (Setlow & Carrier, 19642; Boyce
& Howard-Flanders, 1964) as part of a repair process -leading to resumption
of DNA synthesis -- a necessary step 10 colony formation (Setlow & Carrier,
1964a).
In this paper we examine, for several sensitive and resistant strains
of E. coli B some of the factors that influence the inhibition of DNA
synthesis by u.v. irradiation and some factors related to the repair of
u•v. lesions. The quantitative analysis of the inhibition of DNA synthesis
is described in terms of a model in which a pyrimidine dimer acts as a
complete block to DNA synthesis. Finally we show the consequence of u.v.
irradiation to RNA and protein synthesis in radiation resistant and sensitive
strains.
2. MATERIALS AND METHODS
Bacterial strains. The strains we have used and some oť their
characteristics (Hill & Simson, 1961; Hill & Feiner, 1964) are listed in
Table 1. All strains, with the exception of B/r (ORNL) were graciously
T-1
supplied by Ruth Hill.
Growth of cells.-E. coli cells were grown at 37°C in M-99 medium
supplemented with 0.25% casamino acids. Cultures growing exponentially
(aivision time 34 $ 2 min) at a concentration of 2 x 200 cells/ml, were
diluted ten-fold into M-9 medium minus glucose for irradiation.
Irradiation
Two milliliter samples of a stirred cell suspension in Becknan quartz
cuvettes were irradiated with ultraviolet radiation from a large quartz
monochromator 11luminated by a 500-watt mercury arc. The incident intensity,
about 10 ergs/mm/sec, was measured with a calibrated photocell and the
average intensity through the sample was calculated according to Morowitz
(1950). Photoreactivating light (4050 A) from the monochromator was passed
through a Corning No. 3850 filter before 12luminating cell suspensions so
as to filter out any scattered light of wavelength less than 3600 Å. The
intensity, 1440 ergs/mm/sec, was measured with a calibrated thermcpile
and breaker amplifier.
Radioisotope incorporation.--Incorporation of high specific activity
PH-labeled thymidine, uridine and leucine into acid insoluble materials was
taken to be a measure of the synthesis of DNA, RNA and protein respectively.
The micromethod of van Tubergen & Setlow (1961) was used. For thymidine
incorporation (Boyce & Setlow, 1962) each reaction tube at 37°c contained
5 w (H)thymidine (6 c/mM; 1 mc/m2), 10 ul adenosine (2.5 mg/mi), ml
0.5% casamino acids, 20 m2 of 4% glucose and 200 ml of cell suspension in
M-9 medium - the latter added at zero time. At intervals duplicate 10 u
samples were removed and gently blown into drops of water on stainless steel
planchets. To each drop was added to drop of 2% formalin to stop incorporation
and the planchet was dried at 65°c on a slide warmer. The acid-soluble
materials were removed by immersing the planchets for 20 minutes in 3:1
alcohol-acetic acid followed by 2 minutes each in 95, 80, 70, 50 and 30%
ethyl alcohol and finally water. They were then dried and counted in a
windowless gas-flow counter. For (H)uriaine and leuci.ne incorporation,
the media were the same as for thymidine incorporation except for a different
tritiated precursor and the omission of adenosine. Specific activities weze
uridine: 6.8 c/mM, .. mc/ml; leucine: c/MM, 0.5 mc/m2. In the (PH)leucine
experiments, the 20 ml samples were blown into a drop of 1 M d-1 leucine
in .001 N HM. After the acid and alcohol washes and drying the planchets
were further processed to remove adsorbed leucine as follows: 10 minutes in
.
Chromatographic assay of thymine dimers. These procedures have been
described elsewhere in detail (Wulff, 1963; Setlow, Swenson & Carrier 1963).
It is important to note that the two-dimensional system used in this work
(Butanol/7,0; (NE_) 50, /sodium acetate (H20) does not resolve the dimers TT
and UT. The latter arises from CT by deamination during acid hydrolysis of
the irradiated DNA. The racioactivity in UT is about logo of that in TT
(Setlow, Carrier & Boillum, 1965a).
3. RESULTS and DISCUSSION
DNA synthesis.-Figure I shows the effects of a low dose of 2650 Å
radiation on DNA synthesis in various strains of E. coli. These strains
fall into three categories with respect to ultraviolet-induced inhibition
of DNA synthesis. The DNA synthesis of the sensitive strains, B., and
S-
1
B y is permanently inhibited. Synthesis in strains Ba-z78849 is markedly
inhibited for a period of time and when synthesis resumes it is at a slower
rate than for unirradiated cultures. On the other hand, in strains B and
B/r one observes only a short delay in DNA synthesis followed by resumption
of synthesis at a near normal rate. The remainder of this paper will be
devoted to strains Banten, and B, B/r -- the extreme cases for ultraviolet
effects on DNA synthesis.
DNA synthesis in sensitive strains.-In a previous paper Setlow,
Swenson & Carrier (1963) showed for strain Ban that a dose of 5 ergs/mm
causes complete inhibition of DNA synthesis, but that a small amount of
synthesis occurs before cessation is complete. As the dose is increased
this residual synthesis becomes smaller and at 200 ergs/mm it is negligible.
Because the inhibition was photoreactivable {: about the same extent to which
****** ****
.
.
w
dimers were monomerized and because dimers inhibit in vitro polymerization
of DNA (Bollum & Setlow, 1963), a model was considered in which a dimer is
assumed to act as a complete block to further polymerization along a DNA
template. Since dimer formation is probably a random process in a DNA
strand, the everage distance over which synthesis can take place before
reaching a block becomes smaller as the dose is increased and more dimers
are formed.
- The model assumes that 1) polymerization takes place at a constant
rate along a template; 2) a dimer in the template stops polymerization at
the position of the dimer; and 3) initiation of polymerization aŭ the origin
takes place only if polymerization has gone to the end of the template.
Figure 2 shows the expected synthesis for particular distribution of dimers
in e population that starts at the template origin at time zero. In any
irradiated, exponentially growing culture a more complicated system exists
than that shown in Fig. 2 because 1) the biücks are distributed randomly
along the template; 2) the templates are not synchronized but are in various
stages of completion at the start of an experiment; and 3) some templates
will receive no blocks because of statistical fluctuations and hence will
complete a round of polymerization and continue to increase exponentially.
Thus, to use the above model to compute the expected polymerization from a
known average number of blocks per template , we must average the simple
picture in Fig. 2 over these factors. The details of the analysis appear
in Appendix 1. In Fig. 3 are shown the curves derived from the model by
assuming a known average number of blocks in a DNA synthetic unit. Also
plotted are data taken from experiments similar to those shown in Fig. 1.
The experimental data fit the model reasonably well if we identify 2 ergs/rom? .
at 2650 Å as representing an average of one block per template. Chromatographic
analysis of hydrolysates of DNA from cells irradiated with higher doses shows
that 2 ergs/mom produces an average of one pyrimidine dimer every 200 u along
a single strand of DNA (Setlow, Carrier & Bollum, 19650). If our assumptions
were correct we would conclude that the unit of DNA synthesis in E. coli Baaz
is 200 u in length. Cairns (1963) used radioautographic data and obtained the
length of the E. coli chromosome as 1000 w. The difference between the two
numbers is reasonable because our initial assumptions are only approximations.
For example (a) only dimers in particular sequences may be blocks, (b) there
may be a very small probability of going around each block, and (c) synthesis
may resume from the chromosome origin even though polymerization has been
blocked further up the template. In any event the mean lethal dose at 2650 Å
(2 ergs/min for k = 1) for cessation of DNA synthesis is only twice that for
colony formation in Bar -- a result indicating the close relationship between
the two properties.
We have compared the relative efficiencies of several wavelengths for
inhibiting DNA synthesis by using the above method of analysis on the
experimental data at these wavelengths. Table 2 shows the results of this
comparison. Wavelength 2650 Å 18 the most efficient; if we assign it a
value of 1.00 per incident quantum, 2390 Å and 2805 Å have values of .41
and .50 respectively.
DNA synthesis in resistant strains - The effects of various wavelengths
in producing delays in DNA synthesis for B/r were also studied. On a
conventional linear plot (see Fig. 1), it is difficult to determine the
M
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o
:
per la commemorgen
10
length of complete inhibition. To obtain a less ambiguous interpretation,
DNA synthesis, in counts per minute, were plotted vs. 2+/+-1 (see Appendix 2)
where t 1s the time at which the measurement was taken and 1 is the division
time. Ca such a plot the data for unirradiated cultures should fall on a
straight line through the origin. If yov. Irradiation inhibits DNA synthesis
in each bacterium for a finite time, t, the data should fall on a straight
line of decreased slope wiiuse intercept on the abscissa is 2'0/" -1, whereas
1f the rate of synthesis per bacterium were decreased or only some bacteria
affected one would observe a straight line through the origin with reduced
slope. Figure 4 shows typical data for the inhibition of DNA synthesis by
various wavelengths of u•v. The points for the non-irradiated sample fit a
straight line fairly well (in most cases the fit is better than that in
Fig. 4). These data and those in Fig. I show that DNA synthesis is stopped
by irradiation of resistant strains, as it is in sensitive strains, and that
synthesis resumes before incorporation equal to a normal division (34 min or
(2%-) = 1] has been attained. Thus resumption of synthesis in B and B/r
but some other type of repair mechanism. This conclusion does not necessarily
hold for strains B5-3°8:12
If the only effect of irradiation were the delay of synthesis, the
slopes of the incorporation curve: in Figure 4 should be related to the
delay time (Appendix 2). It is apparent that this is not true. For example,
incorporation after 200 ergs/rom? is much less for 2805 Å than for 2390 Å even
though the two wavelengths produce about the same delays. The differences
between simple theory and experiment are brought out by comparing the slopes
X
of the incorporation curves of Fig. 4 and slopes calculated from the
observed delay times (Table 3). At low coses synthesis is more rapid than
expected on the model. The same result is apparent in the data shown in
Fig. I since the absolute difference between irradiated and unirradiated
-
.
-
.
-
1
cultures remains constant whereas the simple model would predict a constant
ratio at large times. Thus the irradiated cultures tend to catch up with
the unirradiated ones. The explanation for the phenomenon is not known but
could be that the rate of polymerization increases or that the chromosome
origin reinitiates earlier. Moreover DNA synthesis is aberrant after
irradiation (Pettijohn & Hanawalt, 1964) and new growth points seem to be
initiated when synthesis resumes (Hewitt & Billen, 1964). At high doses
some of the bacterial DNA no longer participates in replication (Hewitt &
Billen, 1964; Doudney, 1965). The above observations emphasize the fact
that DNA synthesis is not a sufficient condition for colony formtion.
The relative sensitivity of the inhibition of DNA synthesis to different
wavelengths may be assessed by comparing the effects of these wavelengths
on the delay in synthesis and on the slopes of the incorporation curves
(Fig. 5). The data, corrected for quantum energies, are summarized in
Table 4. They indicate that the relative sensitivities are similar for both
sensitive and resistant strains, are in between the absorption spectra of
DNA and thymiaine as is the action spectrum for dimerization of thymines in
E. coli DNA (Wulff, 1963). · The action spectrum differs significantly frou
that for synthesis delay in M. radiodurans -- a high GC organism (Setlow &
Boling, 1965).
A further indication that DNA synthesis does not necessarily imply
colony survival comes from a comparison of strains 8 and B/r. Strain B is
2
more sensitive but it has a significantly shorter delay time (Fig. 6). Both
streing excise dimers into the acid-soluble phase of cells but B does so
wore rapidly (Setlow & Carrier, 1964a). The delay times for DNA synthesis
are comparable to the excision times for doses less than 200 ergs/mom but
at higher doses this may not be true. These results indicate that dimers
are blocks to DNA synthesis in resistant as well as sensitive strains .
Preliminary data indicate that during the delay period the DNA of strain B
contains more single chain breaks than does that of B/r (Setlow & Carrier,
19640).
Recovery in non-nutrient medium.-E. coli cells have at least two ways
of repairing DNA danaged by u.v. radiation. One is by direct photoreactivation--
a process in which photoreactivating enzyme with visible or long wave length
u.v. Illumination monomerizes dimers (review by J. K. Setlow, 1965), the other
involves excision of dimers as parts of oligonucleotides. Dimer excision
occurs rapidly in B/r cells suspended in nutrient medium and slowly in
non-nutrient medium but not at all in Be- under the same conditions (Setlow
& Carrier, 1964a). We have tested the ability of B/r to repair its DNA
synthesizing capacity when held at 37°C in a medium containing no energy
source. The Irradiated suspension was divided into 3 parts. One part was
used to measure (H) thymidine incorporation immediately after u.V.-irradiation.
A second part was held for 30 minutes at 37°C and a third at 0°C for 30 minutes
in M9 minus glucose and before addition of (A) thymidine. A similar set of
incorporation experiments was carried out with non-irradiated cells.
Figure 7 shows that repair does occur under non-nutrient conditions at 37°C
and even a slight amount of 0°C and that the holding conditions have no
-
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--
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----
12
significant effect on nonirradiated cells. Similar results are found for
strain B. During the repair time about 30% of the dimers are excised
(Setlow & Carrier, 1964e). Shuster (1964) has found a similar recovery in
E. coli 15 tº in the absence of t.bymine.
Photoreactivation. Monomerization of dimers by photoreactivation
occurs in both Bea and B/r and the u.v. inhibition of DNA synthesis 18
photoreversed to about the same extent as the monomerization (Setlow, Swenson
& Carrier, 1963). We attempted to answer the question: Assuming that
synthesis is blocked by dimers, can the photoreactivating enzyme operate
on a dimer which 18 actually blocking DNA synthesis? Cells of Bali unable
to carry out dark repair, were used. They were irradiated with a dose of
10 ergs/wo? and then allowed to synthesize up to a block, following which
the cells were 11luminated with the photoreactivating light. Figure 8 shows
that 34 minutes of growth after u.v. drastically reduces the ability of
photoreactivation to stimulate additional DNA synthesis. However dimers
are split at near normal rates by photoreactivating light under these
conditions (Setlow & Carrier, 1964), although there is no evidence that all
the dimers are monomerized. The photoreactivability of colony formation is
similar to that found for the inhibition of DNA synthesis in Fig. 8.
Chloramphenicol. everal studies have appeared in which chloramphenicol,
an inhibitor of protein synthesis, has been shown to prevent the resumption
of DNA synthes16 in irradiated cells (Harold & Ziporin, 1958; Drakulic &
: Errera, 1959), and although we have not found this to be the case, it was of
Interest to determine, by use of chloramphenicol, whether protein synthesis
18 necessary for the excision of thymine dimers from the DNA of irradiated
cells. When chloramphenicol 18 used at a concentration of 5 wom/mi,
protein synthesis 16 Inhibited completely in E. coli and DNA synthesis 18
mich reduced (Fig. 9). Figure 9 also shows that 50 ergs/nom? delays DNA
synthes18 for 15 minutes and that in the presence of chloramphenicol
(added immediately after Irradiation) a slightly larger delay occurs
followed by a resumption of synthesis parallel to that of an unirradiated
culture. Table 5 shows that chloramphenicol, at 5 uom/ml, does not affect
the excision of dimers from the DNA of irradiated cells. On the other
hand, acridine orange added after u.V.-Irradiation definitely increases
the lag in DNA synthesis and also slows down the rate of dimer excision
(Setlow, 1964).
In E. coli 15 Tº the absence of thymine from the medium does not
stop dimer excision (Shuster and Boyce, 1964) nor inhibit completely the
recovery of the ability to synthesize DNA when thymine is added back to
the medium (Shuster, 1964). All these data provide further evidence that
the excision of dimers is a necessary step in the rapid resumption of DNA
synthesis in resistant strains.
-
- ..
Effects on RNA and protein synthesis.We now compare effects of uov.
on DNA and RNA and protein synthesis in sensitive and resistant cells.
Figure 10a shows that following a dose of 6 ergs/mm RNA synthesis in Bog
proceeds nearby exponentially for 30 minutes and then continues linearly.
.
The point at which the rate becomes constant is approximately the time at
which DNA synthesis almost ceases. A dose of 25 ergs/mm allows only a
slight amount of DNA synthesis; RNA synthesis proceeds approximately linearly
for 50 minutes or so and then decreases slightly. Even following a dose of
-.-............
...
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...
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200 ergs/com considerable RNA synthesis occurs -- at first linearly for
30 or 40 minutes and then at a somewhat slower rate. Protein synthesis
(718. 10b) 18 less sensitive than RNA synthesis, especially at the low
doses since following 6 and 25 erga/wom the rate of protein synthesis 18 .
constanily increasing albeit at rates considerably less than that of the
control. A dose of 200 ergs/vom reduces protein synthesis by approximately
the same amount as RNA synthesis. The order of increasing resistance to
U.V.-irradiation, DNA-, RNA-, protein synthesis is the expected one on
current views of macromolecular synthesis in vivo and fits the following
interpretation. A few dimers inhibit DNA synthesis but they are so far
apart that the transcription of RNA from DNA templates is not affected.
Since DNA synthesis is inhibited RNA continues at.a constant rate but the
rate of incorporation of label into protein increases with time because
the total amount of RNA, but not necessarily messenger RNA, increases. At
high ultraviolet doses (200 ergs/mm) the average distance between dimers
1s small (2 m) and RNA and protein syntheses are affected.
Data for strain B are presented in Fig. 1).. A dose of 200 ergs/mum?
was used, sufficient to block DNA synthesis completely for about 50 minutes.
RNA synthesis is at a roughly constant rate until 50 minutes, the time at
which DNA synthesis resumes. Then the rate of synthesis increases in an
exponential fashion. The rate of protein synthesis appears to be linked
to the rate of RNA synthesis. The picture for B/r 18 almost identical to
that for B. These data are similar to those obtained with 32p-incorporation
into the nucleic acids and s into the protein of irradiated E. coli B and
15 T" (Hanawalt & Setlow, 1960).
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4. GENERAL DISCUSSION
The effects of uiv. Irradiation on macromolecular syntheses and on
colony survival of various strains and mutants of E. coli depend markedly
on the mutant strain (review by Adler, 1965). As far as DNA synthesis 18
concerned the mutants of E. coll B may be divided into three classes
(Hg. 1). Other strains of E. coli are similar to two of these three
classes. Strain 15T“ (Hanawalt & Setlow, 1960) and the resistant strains
of E. coli K-12 (Howard-Flanders, 1965) are similar to strain B and B/r,
whereas the sensitive strains seem to be 21ke Be-3°8:22. The results of
Rörsch et al. (1962) Indicate no induced lag in DNA synthesis, as measured
by p-uptake, in either resistant or sensitive mutants of E. coli B.
This result is in flagrant disagreement with the above observations.
The mutant Boy seems anomalous because its DNA syuthesis 18 blocked
by low doses yet it excises dimers and shows host cell reactivation. It
was speculated that this mitant is deficient in steps that fix the excised
region of DNA (Setlow & Carrier, 1964a). This mutant seems to have many
properties in common with the recombinationless mutants of K-12 (Clark &
Margulies, 1965; Clark et al., 1965).
Since the experimental data on the numbers of u•Vo-induced dimers in
the various mutants of E. coli Bor K-12 show no significant differences
(Setlow, Swenson & Carrier, 1963; Setlow & Carrier, 1964a; Boyce &
Howard - Flanders, 1964) among them, the big differences in sensitivity do
not seem to arise from different numbers of photoproducts but from differences
in molecular repair mechanisms. This notion 18 strengthened by the following
argument. DNA syathesis in the very sensitive strains 18 blocked by only
resina
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a small number of dimers (~10) in the bacterial chromosome whereas the
resistant cells recover from the effects of 1000 or more dimers. The time
It takes for synthesis to resume 18 approximately the same as the time it
takes for the dimers to be excised from the bacterial DNA, and the various
treatments that effect colony formation -- such as photoreactivation,
liquid holding restoration (see for example Jagger et al., 1964) and
acriflavin (Alper, 1963; Witkan, 1963) also affect DNA synthesis and
excision in similar ways. (In sensitive strains there is no exc1.8ion, DNA
synthesis does not resume, and the cells do not form colonies.)
The data are consistent with the idea that dimers act as blocks
(but not necessarily absolute blocks, to synthesis of DNA and they are
extraordinarily effective because DNA 18 synthesized on very long templates.
Doses that stop DNA synthesis in the sensitive strains have no immediate
effect on RNA or protein synthesis. Such effects are only observed at
large doses when the number of dimers per wit length of DNA becomes large
(200 ergs/cm², 1 dimer per 100 daltons). If DNA synthesis resumes so does
RNA and protein synthesis. It is noteworthy that u.v. irradiation of l-even
phages, with doses similar to those used in this work, results in the
inhibition of the synthesis of phage DNA but does not affect the rate of
appearance of phage-specific enzymes (vidaver & Kozloff, 1957; Flaks et al.,
1959; Dirksen et al. 1960; Delihas, 1961).
The simplicity of the above interpretation should not blind us to the
fact that DNA synthesis is a necessary but not sufficien: condition to
cellular reproduction nor to the fact that there are u.V.-induced photoproducts
other than a imers in DN (Smith, 1964; Setlow, 1964a; Wacker, 1963). However
ELS
none of these products have been shown to be photoreactivable. As we
pointed out above, excision 18 more rapid and DNA synthesis resumes quicker
in strain B than in B/r but the latter shows higher colony survival. The
difference in colony-forming sensitivity between B and B/r seems to depend
on the fact that, after reasonable doses of u.v., B does not divide but
forms long non-septate fllaments, or snakes, and as a result does not form
a colony (van de Putte, et al., 1963). After small u.v. doses the filaments
eventually divide and form macroscopic colonies and at these doses fllament
formation 18 photoreactivable (Deering, 1958). Further evidence that the
overall repair process (in which excision is probably an early step) is not
100 percent efficient 18 that even strain B/r 18 photoreactivable (Kelner,
1949) -- repair tig" monomerization is more efficient than repair by excision.
It has not been demonstrated that DNA synthesis stops at a pyrimidine
dimer. Therefore while it is tempting to suppose that the explanation for
the results in Fig. 5 18 that the growing point of the chromosome is not
.
accessible to the photoreactivating enzyme, we could also imagine that a
random, noncomplementary synthesis took place around a dimer as occurs in
vitro (Bollum & Setlow, 1963) and that synthesis stopped because large parts
of adjacent strands were not complimentary. In the latter case monomerization
of dimers would not lead to further DNA synthesis.
Despite the various complications mentioned above it seems that the most
useful description, at the level of a first approximation, of the lethal
effects of uove on microorganisms 18 that it forms dimers in the DNA, that
such dizers act as blocks to DNA synthesis, and that unless they are removed
.
inte ativities
by some repair mechanism the organism dies. Obviously 1f all the dimers can be
properly repaired, the organism will not die from dimers but from other
ain.com
photoproducts. .
m
F
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Harm, W.
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.......Mama uomos
-
Hanawalt, P. & Setlow, R. (1960). Blochim. biophys. Acta 42, 283.
Haynes, R. H. (1964). In Physical Processes in Radiation Biology, p. 51.
New York: Academic Press.
Hewitt, R. & Billen, D. (1964). Biochem. Biophys. Res. Comm. 25, 588.
F121, R. F. (1964). J. Bacteriol. 88, 1283.
#111, R. F. & Feiner, R. R. (1964). J. gen. Microbiol. 35, 105.
#122, R• F. & Simson, E. (1961). J. gen. Microbiol. 24, 1.
Hollaender, A., Ad (1960). Radiation Protection and Recovery. New York:
Pergamon Press.
Howard-Flanders, P. (1965). Personal comm.
Jagger, J., Wise, W. C. & Stafford, R. S. (1964). Photochem. Photobiol. 2, 11.
Kelner, A. (1949). J. Bacteriol. 58, 522.
Kelner, A. (1953). J. Bacteriol. 65, 252.
Morowitz, H. J. (1950). Science 21, 229.
Pettijohn, D. & Hanawalt, P. (2964). J. Mol. Biol. 2, 395.
Roberts, R. B., Abelson, P. H., Cowie, D. B., Bolton, E. T. & Britten, R. C.
(1955). Studies of Biosynthesis in Escherichia coli. Washington:
Carnegie Inst. Wash.
Rörsch, A., Bdelman, C., Van Der Kamp, C. & Cohen, J. A. (1962). Biochim.
biophys. Acta 81, 279.
Setlow, J. K. (1965). In Current Topics in Radiation Research, ed. by
M. Ebert & A. Howard, in press. Amsterdam: North-Holland Pub. Co.
Setlow, J. K. & Boling, M. E. (1965). Biochim. biophys. Acta, in presse
Setlow, R. B. (1964a). In Mammalian Cytogenetics, and Related Problems in
Radiobiology, ed. by C. Pavan, C. Chagas, D. Frota-Pessca & I. R. Caldas,
p. 291. Oxford: Pergamon Press •
20
.
.
.
.
Setlow, R. B. (1964b). J. Cellular Comp. Physiol. 84, Suppl. 1, 51.
Setlow, R. B. & Carrier, W. L. (1964a). Proc. Nat. Acad. Sci. Wash. 54, 226.
Setlow, R. B. & Carrier, W. L. (1964b). Proc. 4th Int. Photobl.ol. Congress,
· in press . .
Setlow, R. B., Carrier, W. L. & Bollum, F. J. (1965b). Submitted for
publication.
Setlow, R. B., Carrier, W. L. & Bollum, F. J. (1965a). Abstracts, Biophys.
Soc. 9th Ann. Meeting, p 111.
Setlow, R. B., Swenson, P. A. & Carrier, W. L. (1963). Science 142, 1464.
Shuster, R. C. (1964). Nature 292, 614.
Shuster, R. C. & Boyce, R. P. (1964). Biochem. Biophys. Res. Comm. ze, 489.
Smith, K. C. (1964). In Photophysiology, ed. by A. C. Glese, vol. 2, 329.
New York: Academic Press.
Sobels, F. H., ed. (1963). Repair from Genetic Radiation Damage. New York:
Pergamon Press.
Vidaver, G. A. & Kozloff, L. M. (1957). J. Biol. Chem. 225, 335.
Wacker, A. (1963). Progr. Nucl. Acid. Res. 2, 369.
Witkin, E. M. (1963). Proc. Nat. Acad. Sci. Wash. 52, 425.
van de Putte, P., Westenbroek, C. & Rörsch, A. (1963). Biochim. biophys.
Acta 7€, 247
van Tubergen, R. P. & Setlow, R. B. (1961). Biophysical J. Z 589.
Wulff, D. L. (1963). Biophysical J. % 355.
***
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FOOTNOTES
-Pyrimidine dimers are adjacent pyrimidine residues in a polynucleotide
that are joined together at the 5, 6 positions. Thymine dimers are particular
members of the class of pyrimidine dimers. The relative nuniers of dimers
In irradiated E. coll DNA 18 TT: 50%; CT: 40%: CC: 10% (setlow, Carrier &
Bollum, 1965).
29 medium: (Roberts, et al., 1955) NA, C1, 1 gm; Ne.HPO4, 6 gm; KE POLO
3 gm; Naci, 5 gm; MgSO4, 0.1 gm; glucose, 4 gre; 1,0, 1 liter.
.
.
.
.
.
. .
.
.
---...
...
... ...
. ... ...
...
. ciasto...
. V
4
22
Table 1
Some characteristics of the bacterial strains
Host cell*
reactivation
Ability to**
excise dimers
Strain
~ 1/e dose for
killing
(ergs/com?, 254 m)
500*
B/T
high
high
Bool
I + II 1
Bs.3
negligible
negligible
negligible
uw 6
low
B5-11
B5-12
negligible
Data in columns 2 and 3 from Hill & Simson (1961), 6111 & Feiner (1964)
and H11 (1964). Data in column 4 from Setlow & Carrier (1964 & unpublished).
*Host cell reactivation refers to the ability of bacteria to reactivate
4.V.-irradiated TI phage.
**Excision of dimers is measured as the decrease in dimers in the acid
insoluble fraction of cells and their appearance in the acid soluble fraction.
*Not exponential survival.
Table 2
The number of blocks to synthesis from the analysis in
Appardix 1 and data such as shown in Figure 3
Wavelength.
(8)
Dose
(ergs/mm)
Blocks to
synthes16
Blocks
per erg/nm
Relative value for
incident quantum
Strain Bs-1
2805
4
1.5
0.38
0.25
( .32)
.
Avg
0.50
2650
vun
0.5
0.5
ůů
0.5
ota ancora
0.5
20
0.7
1.0
0.5
Avg (0.60)
24.5
1.00
2390
ܟ
ܐ
0.25
0.18
( .22)
Avg
0.42
Strain Bs-11
2650
uno
ino
Avg
(0.63)
...
-
Table 3
A comparison of the observed and calculated relative slopes of the
(R)thymidine incorporation curves in Fig. 4. The calculated slopes
are obtained from a model in which u.v. only stops DNA synthesis
for a finite time but does not affect the rate of synthesis along
a chromosome. The model does not agree with observations.
Observed slope
Calculated slope
Wavelength
(A)
Dose .
(ergs/m?)
2390
0.84
0.54
206
0.69
0.35
2650
0.78
0.51
0.54
0.36
106
2805
100
0.75
0.49
200
0.30
0.33
...
og to
m
mer.
25
Table 4
The relative effects, per incident quantum, of several wavelengths on the inhibition
of DNA synthesis in sensitive and resistant strains of E. coli. (Data from Table 2
and Fig. 5; absorption from Beavan, Holiday, & Johnson, 1955; sensitivity of thymine
dimerization in polyt from Deering and Setlow, 1963).
Relative sensitivity
Relative
Wavelength
Sensitive
(Brand
sensitivity
Relative
absorption
DNA Thymidine
Resistant (B/r)
(delay) (slope)
IT dimerization
0.29
0.52
2390
2650
0.41
1.00
0.50
0.57
1.00
0.60
0.40
1.00
0.58
0.61
1.00
0.56
1.00
1.00
2800
0.64
0.44
-----.. .
.
.
26
Table 5
The effect of chloramphenicol (5 ugm/mi) on the excision of dimers from
the acid insoluble fraction of E. coli B following 200 ergs/mm, 2650 Å.
Activity in dimers
Time
Counts per min
Activity in thymine
(with chloramphenicol) (without chloramphenicol)
(min)
ÚT + TT
T
o
0.129
68.6
12.3
53,200
45,600
0.027
0.132
0.026
0.026
u
40
0.6
43,700
0.002
g
0.000
Cells labeled with (H) thymidine were irradiated, transferred to nonradioactive
medium and grown for the indicated times. Approximately equal volume samples
-
were collected by centrifugation and separated into acid-soluble and insoluble
-
.
'
fractions. Each fraction WELS hydrolised in formic acid, chromatographed and
-
.
the dimer and thymine regions counted.
-
-
*
-
-
women met options porno gay ramonequin mon...!"
Appendix 1
We use an oversimplified model to derive an expression for the effect
of random blocks on DNA synthesis in sensitive strains of E. coli.
Assume: 1) Synthesis takes place on a template of length lo. It
starts at the origin at time zero and procedes at a cons cant rate until
it reaches lo at the division time a at which time the number of templates
doubles.
2) If there is a block, synthesis takes place up to the block and
stops
3) The blocks are randomly distributed among the templates and along
the length of a template.
We use the following terminology
l = length along template
do = length of template
t = time
q- division time
R = average blocks per template
n = actual blocks' along a template.
If there are actually a blocks along lo, the probability of finding
none of them in l 18 (1-6/2.)", and the probability of going a distance
between l' and bread without encountering a block is proportional to the
differential of the above expression.
pleyae - (
; s'blabae 1
thus the amount of synthesis in q 18 given by
o
facerea
The probability of finding n blocks if the average number 18 k 18 the
Poisson expression
P(n) -
-*/ 01
Thus the expected synthesis in r 18
Pla)
srother -
M8
le.2)
(a+2)!
R
o
p
(2+1)!
R
26-337
And te relative syathesis 18 compared to a template with no blocks 18
l-e-R
Rei. Synthter -
-
..
.-
For times tsp
-.--
-
..
Rel. Syathtasy *
.docx
-
.
.-
..
-
For times t 2q we may take, with negligible loss in accuracy, the
wirradiated templates as having synthesized 2%-1 templates. The only
irradiated templates that continue growing are those with no blocks, i.e.
Plo) - R. After 't = 9 they synthesize 23/1-2. Thus
••..-
postpay na
.
memper
my per
to
= (1-e By + 8 +R(2+/-2)
Rel. Synthezo
-
(5)
2
-1
We gain no further insight into the problem but more algebraic complexity
by considering an unsynchronized group of templates such as we are actually
dealing with. The solutions of our problem using an unsynchronized start
gives results that are within about 20% of those given by Eqs. 4 ani 5 --
a difference much smaller than the uncertainties in assumption 2.
Appendix 2
In an exponentially growing culture the amount of synthesis, or uptake
of radioactive label R 11 pool sizes remain constant, 18 given by
No v.v. R = a(at/T . 1).
(6)
where a 18 a constant dependent on specific activity, DNA per cell, and
the number of cells at time zero. If, as a result of irradiation, DNA
synthesis ceases for a time to and then resumes at the same rate as
existed before irradiation
- Biala (t=to)/ - 1) tyto
u.v. .
(7)
RO
ts to
(If the bacteria are to survive, and have the normal amount of DNA per
cell, we expect at some time to find 7 less than the normal division time.)
If we plot R versus 2*/ -, Eq. (6) gives a slope a. By adding and
subtracting a 2-to/t to Eq. 7 we obtain
w.v. R = 22-to/7 (at/) + a(zeto/* - 1).
A graph of R versus (25/1 - 1) now has a slope
u•v. slope - a2-tolt :
(8)
W
Figure Legends
Feu 1 - The incorporation of (PH)thymidine by several strains of E. coll
given small doses of ultraviolet radiation (2650 Å).
Fig. 2 - A model to explain the inhibition of DNA synthesis in ultraviolet-
Irradiated E. coli Bg- and B-2 for particular distributions of
dimers
.: Fig. 3 - DNA synthesis as a function of time in irradiated sensitive strains
of E. coli. The solid curves are theoretical curves for the indicated
average number of blocks (k) per template as worked out in Appendix 1.
Time is in units of the generation time of mirradiated cultures and
synthesis in irradiated cultures is given as percentage of mirradiated
cultures.
Fig. 4 - Incorporation of (H) thymidine into irradiated cultures of E. coli
B/r versus 2/t 1 where t is time and T 18 the generation time (34 min)
of unirradiated cultures.
Fig: 5 - Dose-effect curves for inhibition of DNA synthesis in B/r by several
wavelengths. The data are taken from Fig. 4: a) delay in synthesis ;
b) relative slope after synthesis has resumed. The dashed lines indicate
the level at which the data in Table 4 were obtained.
Fig. 6 - Inhibition and recovery from v.v. irradiation (2650 Å) by strains
B and B/r as indicated by their ability to incorporate (a)thymidine.
Hg. I - Liquid holding recovery of DNA synthesis in B/r as measured by
incorporation 02 (H)thymidine. Cell suspensions were irradiated
(200 ergs/mm², 2650 A) and held for 30 minutes in growth medium without
.
glucose or casamino acids at the temperatures indicated. At time
zero nutrients and (PA) thymidine were added to cells and all
suspensions put at 37°C.
Fig. 8 - Photoreactivation (PR) of DNA, synthesis (CH)thymidine incorporation]
in E. coli B : a) 10 min illumination of cells with photoreactivating
light immediately following exposure to u.v. (10 ergs/mom, 2650 A);
b) following u.v. irradiation, synthesis was allowed to take place for
34 min before Illuminating for 10 min with photoreactivating light.
F18. 9 - Resumption of DNA synthesis by ultraviolet irradiated E. coli B
cells in the presence of chloramphenicol (CAP) at a concentration of
5 uom/mi.
Fig. 10 - DNA, RNA and protein synthesis in ultraviolet (2650 A) irradiated
E. coli B.
as indicated by incorporation of tritiated precursors,
thymidine, uridine and leucine .
MADE IN U.S.A.
Fig. 21 - DNA, RNA and protein syathesis in ultraviolet Irradiated E. coli B
as indicated by incorporation of tritiated precursors, thymidine uridine
and leucine
Chantex ORTHS SENTIMETER 46 1510
KEUFFEL & ESSER CO.

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