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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS -1963
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2240
CONt-66062001
CFSTI PRICES
H.C. $ 7.00; MN 150
Repair of DNA
JUN 27 68
To be given as part of an International Symposium
- Det er en zaterations deportiva MASTER
on Regulatory Mechanisms in Nucleic Acid and Protein Biosynthesis,
to be held at
Lunteren, The Netherlands,
june 5 thru. 10, 1966
R. B. Setlow
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee

RELEASED FOR ANNOUNCEMENT *
IN NUCLEAR SCIENCE ABSTRACTS
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LEGAL NOTICE
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Research sponsored by the United States Atomic Energy Commission under
contract with the Union Carbide Corporation.
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Send proof to:
Dr. R. B. Setlow
Biology Division
Oak Ridge National Laboratory

P. O. Box Y
Oak Ridge, Tennessee 37831, U.S.A.
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INTRODUCTION
The realization that the observed effects of physical and chemical
agents on cells and viruses was a reflection not only of changes in their
macromolecular constituents but also in the ability of such systems to
recover from or repair the damages induced in them, t-* took place at
about the same time as the discovery of thymine dimers” -- an important
class of photochemical products in DNA.,' The ability to particularize
the concept of "repair of biological damage" to "repair of damage to DNANO
came subsequently from the demonstration that such dimers act as lesions
to biological systems, to and the ability to detect small numbers of such
photoproducts in DNA.
The repair systems,
seem to work on native DNA -- DNA that
apparently contains two copies of information. I shall summarize the
biological and physico-chemical evidence for the existence of repair
systems for DNA and describe what little is known about the detailed
workings of such systems. Most of our knowledge on this subject comes
from investigations of the effects of UV-radiation on viruses and cells.
Biological Evidence for Repair of DNA
The sensitivity of microorganisms to killing and induced mutations is
under genetic control and small genetic differences between two strains may
lead to differences in UV-sensitivity by more than 100-fold.-),14 It is
not reasonable to ascribe such big differences to different numbers of
UV-induced products or to different numbers of nuclear bodies in the
different strains. The simplest explanation for the difference is that a
resistant strain, for example, E. coli B/r, has a repair mechanism for
ni
ning
vltraviolet damage, whereas a sensitive one such as E. coli B, does not.
Moreover the survival of many strains of E. coli (but usually not the most
sensitive ones") depends markedly on environmental factors after irradiation
and thus on factors other thari the initial damage.+? For example, if cells
are held in liquid medium before plating on agar, higher survivals are
observed, whereas if plated on agar containing acriflavine, lower survivals
are found. Similar post-irradiation effects are observed for UV-induced
mutations. 19
The UV-sensitivity of many bacteriophages depends on the bacterial
strain used to titer them. The bacteria that give low viral sensitivity
are said to do host-cell reactivation and are described as her*. Such cells ·
are also radiation resistant, whereas the herº cells are sensitive to U.
These results indicate that the cellular repair machinery works on DNA.
However, her usually changes the viral sensitivity by about a factor of 3,
whereas the extremes in UV sensitivity of bacteria differ by much greater
actors.
Phages 12 and T4 ere very similar. They ão not show the phenomenon..
of host-cell reactivation but T2 is twice as sensitive as T4. The difference
in sensitivity is under genetic control (the v-gene in T4), and the survival
of phage T2 is enhanced by the presence of the v-gene injected into host
cells by T4 phage.15
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The generalization that bacteria that are sensitive to one physico-chemical
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agent are sensitive to others is true for the extremes of sensitivity, but
may break down for some intermediate cases. For example, UV-sensitive, her
strains of E. coli are also sensitive to X-irradiation, pe suicide, nitrogen

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mustard, and mitomycin c. stoc These findings, and the results of studies
on the effects of several agents combined,º indicate that at least parts
of the repair systems that have been identified for ultraviolet damage are
general and are not specific for pyrimidine dimers.
Dj.fficulties with the Repair Arguement
The biological data are consistent with and support the notion that
repair systems operate to remove damage from DNA. However, the arguments
given above for bacteria are weak for two reasons: (1) They are based
primarily on survival and mutation data -- data that are normally obtained
24 hours after the initial event. The correlation of such observations
with molecular changes at earlier times is difficult to make. 9
(2) It is
reasonable to suppose: that small genetic differences could result ir.
completely different responses to photoproducts, rather than to differences
s
2
in repair. For example, radiation and chemical treatments induce prophages
and such induction results in the killing of the recipient cells, even though
the phage itself may be defective. Such defective phages are known for many,
of the strains of E. coliTrets 2-29) and their presence can account for
the greater sensitivity of this strain as compared to E. coli B/r. A second
possible difference in response is shown by the fil mutants of E. coli and
the lon mutants of K12. These strains fail to divide after many physical
and chemical treatments and such a failure has been correlated with the
inability of the cells to form macrocolonies (and therefore be classed as
survivors)res 14). Although phage induction and division inhibition are
photoreactivable, they have not been correlated with known repair mechanisms.
Yet these responses may affect drastically the observed survival curves of
bacteria.

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The Identification of Reparable Damage
Pyrimidine dimers (thymine-thymine, cytosine-thymine, and cytosine-
cytosine) are formed in relatively large numbers as a result of l-irradiation
of DNA. Trei 20). Moreover they are stable to acid hydrolysis and are easy
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to isolate and to detect. Dimers have photochemically-reversible properties
that are distinct from other known products in polynucleotides, such as
chain breaks, cross links, and hydrates.V, The reversibility of dimers
has led to their identification as inhibitors of nucleasa action and of
calf thymus DNA polymerase, and as being responsible, at high doses, for
over 50% of the inactivation of the biological activity of transforming
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DNA. (rers. 10 gadt). Pyrimidine dimers have not explained all the effects of
UV radiation on biological systems or all the effects on DNA.** They are,
LE-
however, the only known photochemical product that has been identified with
the inactivation of DNA. All the pyrimidine dimers in native DNA are
.
destroyed (probably monomerized) by photoreactivating enzyme in the presence
of 400 mu radiation. Other photoproducts are not affected by such treatment.
12
17
The extension of the preceding argument (that related dimers to the
-.*
inactivation of DNA) to the identification of dimers as lesions in irradiated
ti
bacteria and viruses proceeds by the following four steps (reviewed in
refs. 10, 11). (1) of the known photoproducts in native DNA only dimers are
present in sufficient numbers (one or more per mean lethal dose) to account
for the observed inactivation. (2) Ultraviolet irradiation inhibits DNA
synthesis in bacteria. The inhibition is reversed by illuminating cells
with 400 mu radiation (1.e., the inhibition 18 photoreactivable) and the
fraction of dimers destroyed by in vivo photoreactivation equals the dose
reduction factor for the inhibition of DNA synthesis. (Photoreactivation,
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of course, also enhances the 'survival of irradiated bacteria. Observations
of survival, however, do not distinguish between direct photoreactivation --
the destruction of dimers -- or indirect photoreactivation -- an effect on
the cellular machinery that does not result in the destruction of dimers but
in more efficient repair). (3) Most of the pyrimidine dimers in DNA are
thymine-containing ones. Substitution of thymine by bromouracil decreases
the numbers of dimers and at the same time eliminates the phenomenon of
host-cell reactivation and photoreactivation. (4) The various phenomenological
reactivation effects such as liquid holding restoration, host cell reactivation
and v-gene reactivation are not additive with photoreactivation but overlap
it. Presumably they all act on similar lesions.
The UV-induced inhibition of DNA synthesis in many cells is only
temporary, whereas in very sensitive strains it may be permanent. Such
sensitive strains may be thought of as dying for a trivial reason -- they
cannot make DNA and therefore do not form a macroscopic colony. Figure 1.
shows four easily-identifiable patterns for the inhibition of DNA synthesis
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in bacteria. The sensitive strain Be-, is inhibited by as little as 10
pyrimidine dimers per chromosome (2 ergs/momlet. 29). There are at least
two patterns to the ability of E. coli to recover from UV irradiation. One,
such as exemplified by E. coli B/r, represents a rapid recovery and the other
as represented by E. coli B-z is slow and only observed at low doses.
Micrococcus radiodurans seems to have the best repair system of all bacteria."
It can recover the ability to make DNA and give rise to a colony at doses
which initially convert 1% of its thymine to dimers (see below). Note that
there is a reasonable correlation between the ability of cells to recover
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DNA synthesis and their ability to form colonies after irradiation. The
usefulness of the observations in Figure 1 is that they give a time scale
in which to measure repair phenomena even though they do not indicate what
the mechanisms of recovery or repair are. We suppose that the time for
which bacterial DNA synthesis is inhibited represents the time for cells
to recover from or repair the damage. For doses (100 ergs/com) to E. coli
B/r that produce up to approximately 1000 dimers/chronosome the time for
recovery of DNA synthesis (the hypothetical time for repair) is proportional
to dose.
We come to the conclusion that recovery or repair in bacteria takes
place before DNA synthesis may resume. In higher cells -- cells with a
well defined s period -- the conclusion has been reached that s represents
an end point for the repair of premutational damage.
The various strains of E. coli may be lumped into several broad
catagories that indicate their general biological and biochemical responses
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to radiation (Table 1). Somewhat similar catagorizations have been made
>
by Howard-Flanders and Boyce,” Rörsch et al., 44 and Mennigman. 45
NA
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Molecular Mechanisms for Recovery and Repair
We describe below several recovery mechanisms that operate on damags
represented by pyrimidine dimers. Dimers are not the only type of change
that may be repaired but they represent the lesion for which most data
exist and the only one that has been followed extensively. We divide the
recovery or repair mechanisms into three categories: (1) The danage is
reversed; (2) some of the damage may be ignored or bypassed; and (3) the
damage is removed. A highly schematic representation of these three processes
is shown in Fig. 2.
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WIR
(1) Reversal of damage. Enzymic photoreactivation was discussed
briefly above. The monomerization of dimers is more rapid for native DNA.
than for denatured DNA and does not occur if the dimers are in small
oligonucleotides. The fraction of UV-lethality that may be reversed by
photoreactivation 18 0.9 for the transforming DNA of H. influenzae and
0.8 for E. coli at low doses.
These figures indicate that a large
fraction of biological damage may be ascribed to the formation of pyrimidine
dimers. Cells that lack the photoreactivating enzyme (defined in terms of
the ability of extracts to reactivate UV-inactivated transforming DNA) are
not photoreactivable, nor are dimers destroyed in vivo by irradiation with
400 mu light. Photoreactivation seems to be a much more efficient process
than the other two discussed below.
(2) Bypass of damage. There is no good reason why a dimer must be
an absolute block to synthesis in vivo. Polymerization could take place
around such a distorted group of bases at a much slower rate than normal
and thus accourt for a lag in DNA synthesis. Studies on an in vitro DNA
polymerase system from calf thymus glands using a UV-irradiated primer have
been interpreted as indicating that slow polymerization around such blocks
is possible and that in the process of such polymerization the "wrong" bases
may be inccrporated into DNA.*The polymerase product made from irradiated
primer was deficient in ApA sequences as would be expected if there were
noncomplementary incorporation opposite TT dimers (the major photoproduct).47
The notion that a biological system is able to bypass blocks at a slow rate,
and sometimes make mistakes in the process, is an attractive one, but there
18 no direct experimental evidence for it although obviously it may be used
to relate dimers to lethality and to mutagenesis.
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The bypass process cannot be the major one responsible for the
resumption of DNA synthesis in resistant strains of bacteria. If it were,
rapid resumption of synthesis after large UV doses would begin only after
the amount of synthesis represented one complete replication. In actual
practice synthesis resumes rapidly in E. coli B/r and M. radiodurans before
anything near chromosome synthesis has been achieved. However, the ability
of cells such as E. coli Bez to survive reasonable numbers of dimers could
result from such a process.
In E. coli B, it takes about 5 dimers per strand to inhibit DNA
synthesis. This result should not be taken as direct evidence for
polymerization around dimers in vivo, because the synthesis that is measured
has not been identified with particular chromosomal regions. It could
represent synthesis arising from the reinitiation of the chromosome origin,
or synthesis of an extra chromosomal element, for example. It is worth
emphasizing that measurements of incorporation of radioactivity into
macromolecules give no indication as to whether they represent what we like
to think of as synthesis of the bacterial chromosome.
Ultraviolet irradiation of mammalian cells results in a decreased rate
of DNA synthesis but not the marked cessation of syn'hesis that is observed
for bacteria.
Even though large numbers of dimers are formed in mammalian
cells, there is no evidence that dimers act as blocks to synthesis or that
they have any effect on the cells whatsoever. Perhaps the bypass process
describes mammalian cells adequately. Excision of dimers (considered below)
has been looked for and has not been found in mammalian cells.49
(3) Removal of damage. This process represents the removal of damaged
regions from DNA. It is analyzed, in a very crude fashion, as the removal
of dimers from the TCA insoluble fraction of cells or extracted DNA. If
the dimers are parts of oligonucleotides that are greater than 5 or 6
nucleotides long they will be acid-insoluble and will not be detected as
contributing to excision. In E. coli the process of rapid excision 18
observed in ner* strains (Fig. 3a) but not, or very weakly, in her strains
(Fig. 3b). When excision 18 observed it occurs in a time less than or
equal to that for which DNA synthesis is inhibited (compare Figs. 2 and 3).
Associated with the excision of diners as parts of acid-soluble oligonucleotides
is the loss of photoreactivability of dimers and the further degradation of
the bacterial DNA (see below). At doses that produce little inactivation
the number of excised dimers per chromosome may be as high as 1000 for
E. coli and 5,000 for M. radiodurans. Repair by excision involves loss of
DNA and it requires a structure with redundant information if the DNA of
a cell is to be returned to the sequences that existed before irradiation.
We can easily imagine that there must be other steps associated with the
excision of dimers such as (a) possible further degradation of the DNA;
(b) "repair replication" in the excised regions; and (c) rejoining of the
repaired regions.
Repair by excision obviously must involve a number of steps (of which
excision is the easiest to measure) and it is easy to conceive that changes
in the relative rates of the various steps could affect drastically the
ability of a cell to survive such a process. For example, if single-strand
degradation following excision were rapid compared to replication, double-
strand breaks would arise. Such breaks presumably would be lethal."
The sequence of steps in the removal of damage and its replacement may
be thought of in the two extreme ways shown in Fig. 4. One requires DNA
synthesis for excision to take place. The other looks upon excision as
the initial step. In any actual biological system, both may be taking
place but existi.ng data do not distinguish well between them or rule out
the possibility that the other processes in Fig. 2 are taking place
simultaneously.
Repair by excision -- and its as summed associated steps -- is such
a neat, conceptual package that the experimental. observation of any one
of these steps is taken as an indication of the existence of a repair
mechanism. We consider briefly what is known about these steps.
STEPS ASSOCIATED WITH REPAIR
Excision of pyrimidine dimers' into acid soluble material. Excision
is inhibited by aeroflavin° but is not inhibited by coloramphenico129 nor
does it require the presence of exogeneous thymine in cells such as E. coli
KUU
or under conditions in which practically all the dimers are excised (Fig. 3).
Unfortunately, the latter data may not be taken as evidence that excision
is independent of DNA synthesis, because the internal pools of thymine may
be augmented by some breakdown of bacterial DNA (see below) and this pool
could suffice for the small amount of synthesis (perhaps less than 1%) that
might be necessary if synthesis were related to excision. The observed
rate of excision does depend on some metabolic conditions. Table 2 shows,
in the bacterium E. coli 15 T A U bar that the excision rate for TT and CT
dimers are about the same and that both rates are sharply. depressed 11 cells
are incubated in the absence of glucose but not if incubated without vracil
or amino acids. Some bacteria, such as E. coli Bs-3 (see Fig. 3), excise
dimers at a very slow rate -- a rate so slow that it is just about
unobservable at the high doses usually used to detect excision.
Degradation of DNA. At large UV doses there is degradation (~ 10%)
of the DNA of hcr* cells but not of her* ones. Similar degradation is
also observed in many her“ cells which have been treated with agents such
as X-rays that are thought to make single-strand breaks by themselves."
It is not obvious that this further degradation is a necessary step in the
repair process. It is just as reasonable to suppose that it represents
the degradation of the DNA of non-surviving cells -- those that do not
survive the radiation treatment because of an abortive attempt at repair.
Nevertheless, the presence of DNA breakdown is well correlated with the
production of single-strand breaks in DNA either directly or by excision
and because excision involves the production of chain breaks. DNA
breakdown is an easy way to screen for existence of excision steps.
Mutants of E. coli that are deficient in the ability to undergo
genetic recombination (rec mutants) are UV-sensitive.” Their DNA undergoes
an extensive breakdown that is initiated by single-strand breaks.
Mutants
that are rec and also cannot do excision would be expected to show DNA
breakdown after X- but not after UV-irradiation.
Repair Replication."* Radiation resistant cells that are grown in
bromouracil-containing medium during the time that DNA synthesis is almost
completely inhibited by UV irradiation incorporate some bromouracil into
DNA but not in a normal semi-conservative way. Incorporated bromouracil is
observed in cell density gradients at the normal light density and acts as
if it were randomly distributed along light strands. This so-called "repair
replication" fits either scheme in Fig. 4, but there are no good data on
--
the amounts of such synthesis, nor is there direct experimental evidence
that the newly synthesized regions are distributed randomly along the
bacterial chromosome. The conceptual difficulty in the identification of
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such regions arises from the existence of further degradation of the DNA.
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Such degradation could result in a large internal pool of thymine and, as
a result, the exogeneous bromouracil would be mixed with a large amount
of endogeneous thymine. Any normal type synthesis would be mostly thymine
with a little bromouracil and the incorporated bromouracil would be found
at the density of light DNA. Such synthesis could result from the initiation
of new chromosome origins” as a result of UV irradiation, or the induction
of lysogenic phages.23-25
Single-Strand Breaks in DNA
Both schemes in Fig. 4 indicate that single-strand breaks should appear
during repair of DNA and that they should disappear as repair reaches
completion. We have measured the number of single-strand breaks in bacterial
DNA's during the excision process by a technique developed by McGrath and
Williams.50 The technique involves lysis of cells in alkalai and zone
sedimentation in alkaline sucrose gradients and isolates single-stranded
DNA from E. coli of molecular weight 2.5 X 10° daltons -- approximately 1/6
of a chromosome strand. Therefore it permits us to measure small numbers
of chain breaks during excision. Figure 5 shows sedimentation patterns of
E. coli DNA during repair. Breeks appear and then disappear as excision is
completed. The maximum rate of excision is in the neighborhood of 15 dimers/minute
and the fact that a maximum of 15-20 single-strand breaks are observed indicates
either that the remaining steps are very rapid or that scheme II in Fig. 4
"
15
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is the proper one to consider. However, treatments that decrease the
rate of excision do not result in increases in the observed number of
single-strand breaks. Thus in the absence of glucose, the excision rate
is decreased34 and so are the number of observed single-strand breaks
(Fig. 5). In the presence of acriflavin the excision rate 18 decreased
by about a factor of 10, (ref. 50) but the number of observed chain breaks
observed is unchanged. Thus these data do not distinguish between the
two possibilities shown in Fig. 4, but they do indicate that the steps in
excision take place rapidly in a concerted fashion and are under metabolic
control.
The final rejoining recombinationless mutants”999 -- with their
big DNA breakdown once a single-strand break has been made -- may be
defective in the final rejoining step. However one could also explain
their properties in terms of an excess of nuclease or a defect in repair
replication. Note (Fig. I and Table 1), that the introduction of the rec
character into uvrA cells changes the manner in which DNA synthesis is
inhibited from that shown in Fig. Ib to that in Fig. la. If we speculate
that DNA synthesis in urA goes around dimers, as in Fig. 2, part 2,
whereas in rec" uvrA cells it does not, we identify rec" with the inability
to polymerize around dimers. We are thus led to associate rec" with a
defect in polymerization or repair replication.
CONCLUSIONS AND SUMMARY
Table 3 catalogs the biological systems and the physico-chemical
lesions that have been related to the various steps that we believe are
involved in repair by excision. The temporal sequence of the independence
of such steps is poorly known and their relations to survival not really
known at all.
The repair step that we call
excision -- the appearance of lesions
in the acid-soluble fraction of cells -- is related to the recovery of
cells from physical and chemical Insults. Many lesions are reparable but
none are known to be as effectively repaired as pyrimidine dimers.
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Different levels of dimer repair can explain the 100-fold differences in
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A
UV-sensitivities of sensitive and resistant bacteria strains. The sequence
of steps, or the cellular agents, if any, that control these steps, in
recovery processes involving excision are not known. Excision requires
an energy source but in view of the data of Table 1 it does not require
large amounts of protein, RNA, or DNA synthesis.
Cell free systems that can do at least some steps in biological
reactivation have been described, +2,04 and preparations that can effect
excision of dimers from native DNA ref. 60), degradation of one strand of
native DNAⓇS, and repair of these single strand regions have been
described but there is no guarantee that such in vitro systems are related

..
to the repair processes that go on in vivo. In any event the repair
processes in cells seem to be well coordinated and are able to eliminate
over 99% of the initial UV damage. However the repair processes are not
100% efficient. Even such a rezistant cell as E. coli B/r is photoreactivable,
a finding that implies that a dimer can be lethal. The failure of repair
may result for example from (a) mistakes during the repair process; (b) the
overlapping of single-strand breaks arising from excision (especially at
high doses); and (c) the improbable polymerization around a block with its
attendant incorporation of incorrect bases before the block has been repaired
by an excision mechanism.
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?W. Harm, J. Cell. Camp. Physiol., 58 Suppl. 1 (1961) 69.
PR. F. Kimball, J. Cell. Comp. Physiol., 58 Suppl. 1 (1961) 163.
*M. M. Elkind, H. Sutton and W. B. Moses, J. Cell. Comp. Physiol., 58
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SR. Beukers and W. Berends, Biochim. Biophys. Acta, 41 (1960) 550.
A. Wacker, Progr. Nucl. Acid. Res., 1 (1963) 369.
'K. C. Smith, in A. C. Giese, Photophysiology, Vol. 2, Academic Press,
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°R. H. Haynes, in Physical Processes in Radiation Biology, Academic
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°R. B. Setlow and W. L. Carrier, J. Mol. Biol., in press.

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(1965) 24.
r
..
29
P. Howard-Flanders, and L. Theriot, Genetics, 53 (1966) .
*UK. W. Kohn, N. H. Steigbigal and C. L. Spears, Proc. Natl. Acad. Sci. U.S.,
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608.
429. D. Lawley and P. Brookes, Nature, 206 (1965) 480.
*M. E. Boling and J. K. Setlow, Biochim. Biophys. Acta, in press.
**A. Rörsch, P. van de Putte, I. E. Mattern, C. A. van Sluis and H. Zwenk,
Proc. 3rd Int. Congr. Radiation Res. (1966) p
.
4344
H. D. Mennigiman, Zentralblatt f. Bakteriol. Parasit. Infektionskrank. u.
Hyg., 198 (1965) 282.
40F. J. Bollum and R. B. Setlow, Biochim. Biophys. Acta, 68 (1963) 599.
*'R. B. Setlow, W. L. Carrier and F. J. Bollim, Abstracts Biophys. Soc.
9th Ann. Meeting, 1965, p 111.
40J. E. Cleaver, Nature, 209 (1966) 1317.
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20.
15
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.
. S
.
.
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3°B. S. Baker and R. B. Setlow, unpublished observations.
NN
--
S
"YR. B. Setlow and W. L. Carrier, Abstracts Biophys. Soc. 10th Ann.
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-
-
.
*+
$
-
-
FOOTNOTES
* Research sponsored by the U.S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
† Filament formation is inhibited (and colony survival is markedly enhanced)
in the lon mutants of E. coli by temperatures above 37°C or by growth
on agar that contain: pantoyl lactone. Such treatments have been
reported to give a striking increase in the survival of E. coli Bs-1
(ref. 16) but most investigators have observed increases in resistance
that are less than 2-fold (refs. 17, 18). .
--
=-
** For example: E. coli is more sensitive to UV-radiation at temperatures
below freezing but fewer dimers are formed under such conditions
(ref. 27).
'Indirect evidence indicates that such breaks may not be lethal in the
very resistant microorganism M. radiodurans (ref. 51).
.
1
2
17TYng
ņable 1. Possible grounines for hante
Table 1. Possible groupings for bacterial strains in terms of biochemical and phenotypic responses to radiation
.

Pattern of
inhibition
Host-
Group*
~ 37%
dose
(erg/mm)
of DNA
Excision
of dimers
cell
| DNA breakdown
after
References
synthesis
Fig. 1
react.
Examples
UV
X-ray
K12.
..B
E. coli Exc
Rec
0.5
a
+
uvrA rec
Baul
28,33-36
Exc
1
ra lon
37
Filt
Exc
-
..
+ | urA,B,C 's-3
... ... ...WP2 hcr
| 38, 39
:
Rec
.
+
#
++
rec
Bs.11
Filt
+
+
+
+
lon
в
I
2570 .
induced
phage
+
+
+
wild
500
+
+
K12
M. radiodurans
6000
++
* Exc": do not excise dimers into acid soluble form.
Rec: deficient in genetic recombination.
Filt: form long non-septate filaments after IV.
Table 2
Dimers remaining in the acid-insoluble fraction of E. coli 15 TAU bar
labeled with 'H-thymidine and irradiated with
Time in growth
CT/T
TT/T
medium after UV
mediunt
none
0.044
0.088
30 min
none
0.024
0.035
- uracil
0.028
0.049
- uracil - amino acids
0.024
0.056
- uracil
- amino acids,
0.038
0.070
- glucose
* Medium: M9 plus uracil (10 ugm/mi), casamino acids (2.5 mg/ml), and
tryptophan (100 ugm/mi). No exogenous thymine added.
I
1.
- 3 .
FR
7
-
-
--
-
.
-
-
--
Table 3
Some of the biological systems in which the presumed steps in repair
by removal and replacement have been observed
Excision of dimers
Breakdown of DNA
Reference
Reference
E. coli her* strains*
34,35
35,36,38
E. coli her*: after UV, mitomycin C, X-rays
her": after X-rays but not UV
B. subtilis uvr*: after UV and methyl methane
sulfonate (moms)
by M. lysodeikticus extract after UV, nitrogen
B. megaterium
T3 phage in B/r
T4 v* in Bs-1
by M. lysodeikticus extract
- 67
61.,62
60
mustard and mms
*Such strains also excise
40-42
sulfur- and nitrogen-mustard
products
"Repair replication"
Reference
E. coli 15 TAU bar following UV and nitrogen mustard
54,63
SAYTLAR
i
Figure Legends
Fig. 1. The effect of UV-irradiation (265 m) on subsequent DNA synthesis
in irradiated cultures. The numbers next to the curves and the values
of Dz, represent doses in ergs/mm.
a) E. coli Bs-1 (Doubling time: 35 min). Data from refs. 28 and 30.
b) E. coli Bez (Doubling time: 35 min). Data from refs. 30 and 31.
c) E. coli B/r (Doubling time: 35 min). Data from refs.28 and 30.
a) M. radiodurans (Doubling time: 90 min). Data from ref. 32.
Fig. 2. Schematic pictures of possible ways in which pyrimidine dimers,
as exemplified by TT, affect DNA synthesis and may be affected by
cellular repair mechanisms.
Fig. 3a. Excision of dimers from the acid insoluble fraction of UV-resistant
cells at various times of incubation in growth medium after irradiation.
For E. coli 15 T ; 0; incubation with thymine; 0, incubation without
thymine. Refs. (31,34,43,50).
Fig. 36. Lack of extensive excision, even at low doses, in her“ strains
of E. coli. Ref. (31,34).
Fig. 4. Two possible schemes illustrating possible steps in repair of
damaged DNA by the mechanism given in Fig. 2, part 3. Only a small
section of a DNA molecule, containing a thymine dimer, is shown. The
steps are IA: excision; IA': degradation (a possible alternate pathway);
IB: repair replication; IC: rejoining. Another possibility is IIA:
single strand breaks; IIB repair replication; IIC: rejoining and excision.
26
-
.
-
-.
-
-
.
Fig. 5. Sedimentation of E. coli DNA in an alkaline sucrose gradient
.-
in
--
.
the SW-39 rotor. E. coli B, an excising strain, was labeled with
PH-thymidine, and then irradiated with 200 ergs/mom? of 265 mu radiation.
Cells were then grown in M9 plus casamino acids (except for one sample)
and at various times aliquots analyzed for dimers in acid-insoluble
form and by sedimentation. Sedimentation was for 90 min at 30,000 rpm.
The centrifuge tubes were punctured at the bottom and X fractions
collected and assayed for radioactivity (Ref. 31).
.
.
-
.
*
?
-
-
-
-
=
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Fit
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