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66067412 H.C. $ 2.00; MN_,50
JUN 27 28
MASTER
THE REPAIR OF MOLECULAR DAMAGE TO DNA*
R. B. Setlow
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
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Paper to be presented at
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The Third International Congress of Radiation Research
Cortina, Italy
June 26 thru July 2, 1966

RELEASED FOR ANNOUNCEMENT
IN NUCLEAR SCIENZE-ABSTRACTS
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LEGAL NOTICE
This report was prepared u wa ACC at of Government sponsored work. Neither the Vallad
Buates, por ebe Commission, nor any worson acting on behalf of the Commission:
A. Makes any warranty or repron station, expressed or implied, wild rospect to the accu-
racy, completeness, or unnfulness of the information contained in this report, or that the wo
of nas information, apparatus, morbod, or procon diacioned in this roport may not infringo
privately owned rights; or
B. Assumes any Habiules with respect to the use of, or for damages resulting from the
use of any information, apparatus, metbod, or proceso disclosed in the report.
Ar ured in the abovo, "person acting on baball of the Commission" includos rayon-
ployee or c iructor of the Commission, or employee of such contractor, to the extent that .
such employs or contractor of the Commission, or employee of such contractor prepares,
disseminatos, or provides accon lo, any information pursuant to his employmrat or contract
with the Commission, or his employmont with such contractor.
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*Research sponsored by the United States Atomic Energy Commission under
contract with the Union Carbide Corporation.
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Running head:
Send proof to: Dr. R. B. Setlow
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Biology Division
Oak Ridge National Laboratory
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Oak Ridge, Tennessee 37831
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1.
INTRODUCTION
This paper reviews briefly the physico-chemical changes produced in
DNA by irradiation and the possible steps in the repair mechanisms that
operate in vivo to nullify or correct these changes. We 2.5 sume, with good
cause, that DNA is the most important biological component as far as the
radiation responses of bacteria anů viruses are concerned and that the
response of such systers to radiation depends not only on the type and the
number of physico-chemical changes produced in their DNA but also on how
they react to these changes and how they recover from them [reviews by
Haynes (1) and J. J. Setlow (2)]. Some of the recovery processes are
capable of nullifying over 90% of the initial damage to DNA. Obviously,
if all the damage to DNA is repaired, then DNA cannot be considered to be
the sensitive target.
2. CELL DEATH
u
I shall concentrate, for reasons given below, on the effects of
radiation on macromolecular changes rather than on the effects on survival
of bacteria and viruses. The killing of cells, that is the inability to
matematik tulisation at the time in
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form a macroscopic colony, and the production of mutants is normally scored
a long time after the initial treatment and it is difficult from such
observations to infer the nature of the repair process that may have gone
on earlier [review by Kimball (3)]. A second reason not to emphasize cell
survival and its relation to repair, is that the causes of cell killing are
many and in individual cases may not be known at all. Cells may die
(Table 1) because (1) DNA synthesis is permanently inhibited or because
DNA is broken down as a result of irradiation or abortive attempts at
T-1
repairing damage. For example the attempted repair of crosslinked strands
coulà lead to a double-strand break. (2) Radiation-induced changes in DNA
may result in the subsequent synthesis of incorrect DNA. (3) Radiation
may induce phages or defective phages and their multiplication may be lethal
as in most mutants of E. coli 15. Cells may also fail to form a colony
because radiation inhibits their ability to divide as in bacteria such as
E. coli 6. The inhibition of cell division occurs at small doses and it is
conceivable that the failure of cells to divide arises from the induction
of a defective prophage (ref. 4). (4) Cells may fail to form a colony
because their chromosomes cannot separate or (5) because of effects on RNA
and proteins that alter the normal cellular events.
:
3. GENERAL NATURE OF REPAIR PROCESSES
The molecular repair processes seem to be general in that they operate
not only on damage arising from irradiation but on chemically-induced changes
in DNA -- changes that arise from treatment with nitrogen or sulphur mustards,
for example. Our initial problem is simply stated. Which physico-chemical
changes in DNA are lesions and how are they repaired? The experimental
approaches to these questions have come from two directions. The first
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concerns the identification and physical properties of ultraviolet-induced
products in model. polynucleotides and in DNA in vivo and in vitro (reviews
by Smith (5) and J. K. Setlow (2)]. A major class of photoproduct in native
DNA is represented by cyclobutane dimers between adjacent pyrimidine residues
in the same strand. Such dimers interfere with enzymi, polymerization and
degradation of DNA and they account for a very large fraction of the
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photochemical changes that are responsible for the loss of biological activity,
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of transforming DNA. Moreover, they are the only known photochemical lesion
that is destroyed (probably monomerized) by photoreactivating enzymes plus
visible light. Thus, dimers have the properties of lesions and we shall
assume that they are lesions in microbial systems. (There is however no
indication that they act as lesions in the mammalian cells.) In most
UV-irradiated systems there is more than one pyrimidine dimer made in the
DNA by a mean lethal dose. Thus we could conclude either that not all dimers
are lethal or that cells have a very efficient way of repairing or bypassing
the damage represented by the dimer.
The second experimental approach to the problem of repair has been
Marketinis vi
F-1
F-1
biological and has been given a firm foundation by the discovery by Ruth
Hill of the exquisitely sensitive mutant E. coli BS-1 (ref. 6). A comparison
of the survival curves of several E. coli strains is shown in fig. 1.
Because, as far as UV sensitivity is concerned, strain Be- differs from
B/r by over a factor of 100, we shall concentrate our attention on these
two strains and use the interpretation derived from their properties to
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describe some of the intermediate cases shown in fig. 1. It is obvious that
the sensitive strain is not only more sensitive to UV radiation but also to
X-radiation and p suicide. It has been shown that it is also more sensitive.
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to inactivation by nitrogen mustard treatment (1), although, as for X-rays,
it is more sensitive by only about a factor of 3.
The way in which cells die (table 1) and the manner in which they may
repair damage (fig. 2) are under genetic control (8). Many bacterial
strains that are very UV sensitive, such as E. coli Bez, are also unable
F-2
to reactivate UV-irradiated bacteriophages such as T1 and 1 (review in 4).
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The difference in sensitivity between viruses plated on host-cell
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reactivating, hcr*, and her strains is not the factor of ~ 100 cbserved
for UV-inhibition colony formation of the cells but on].y ~ 3-5 observed
for the other inactivating treatments. There is not a good quantitative
correlation between the ability to do host cell rear:tivation and cell
survival.
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Some bacterial strains, such as E. coli B or B-2, may be sensitive
but still are able to do host-cell reactivation. Their high sensitivity
results from their dying for special reasons. E. coli B forms filaments
and E. coli B. is probably a member of the rec" class of mutants --
mutants, that in mating strains, are deficient in genetic recombination
(9) and may show a large amount of DNA breakdown following radiation.
treatment (review in 10). Such mutants in E. coli Kl2 can do hcr.
4. PHYSICO-CHEMICAL CHANGES THAT MAY BE LESIONS: (fig. 3)
Fu3
4.1. Double-strand breaks. The decay of PC, incorporated in DNA,
can result in the production of single- or double-strand breaks. The
experimental data indicate that each decay in a double-stranded DNA phage
and bacteria does not result in inactivation [review by Stend and Fuerst
(.11)]. Therefore a single-strand break is not necessarily a lethal event
(except in single-stranded phages).
The daia are consistent with the
notion that a double-strand break is a lethal event. X-rays also produce
both single- and double-strand breaks in phage DNA and at the mean lethal
dose to T7 phage there is about one double-strand break in its DNA but many
more single-strand breaks (12). Such data do not prove that double-strand
breaks are lesions because another as yet undetected radiation product could
be produced with equal frequency and this second, hypothetical one, could
be the lethal lesion. It is usually assumed that double chain breaks are
not reparable and therefore are lethal because of the small probability of
the two free ends finding one another. In phage they can be lethal because
all the phage DNA may not be injected into the host bacterium.
Dean et al. (12a) have isolated DNA from Micrococcus radiodurans after
X-ray exposure and estimated its molecular weight by viscosity and sedimentation
measurements. The molecular weight of DNA isolated from cell.s exposed to
220 kr is less than that from unirradiated cells even though such an exposure
yields 100% survival in this very resistant orga.ism. If the irradiated
cells are incubated in growth medium after irradiation the molecular weight
of the extracted DNA returns to the value for unirradiated cells before
there has been appreciable net synthesis of DNA. The authors' take these
data as indicating that this bacterium can repair double-stranded breaks".
However, since the extraction conditions themselves yield many two-stranded
breaks, the X-ray-induced, two-strand breaks could have arisen from
radiation-induced, single-strand breaks that were converted to the two-strand
breaks by the extraction procedure. If so, single strand, rather than
two-strand were being repaired. (See Sec. 5.1.)
4.2. Single-strand breaks. Single-stranded breaks are not levhal to
T7 bacteriophage (12). However exposure of E. coli Bs-1 to a mean lethal
dose of X-rays results in approximately one single-strand break per DNA
strand (13). Such breaks are lethal in this strain probably because they
result in extensive breakdown of the bacterial DNA. (In this respect they
are similar to some of the recombinationless mutants of E. coli K12.)
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Strains which do not show such breakdown are not inactivated by
single-strand breaks even though they may be UV-sensitive and cannot do
host-cell reactivation (14). Since X-irzadiated T7 phage shows the same
survival when plateå on E. coli Bs- as on B/r, we conclude that a
single-strand break is not lethal to the phage DNA in Bar, but that a
single-strand break in the bacterial DNA is a lethal event to the bacterium.
This situation has its analogue in the high UV-sensitivity of rec strains
(presumably because they excise dimers and then breakdown their DNA) even
.
though they are able to reactivate UV-irradiated Tl phage.
The evidence identifying single-strand breaks as lesions is indirect
and oniy numerological. Single-strand breaks are reparable (see below).
Perhaps our difficulty in interpreting the effects of repair mechanisms on
X-ray damage, in these seemingly simple systems is complicated by the
existence of a large number of radiation products many of which are
intrinsically irreparable.
4.3. Pyrimidine dimers. Pyrimidine dimers are the only reasonable-
well-documented radiation-induced lesion. The phenomenon of direct
photoreactivation allows us to relate dimers with the inhibition of DNA
synthesis in vivo, the formation of filaments, the inhibition of colony
ca
formation, and with the induction of some mutations.
5. REPAIR OF LESIONS
5.1. Single-strand breaks. McGrath and Williams (13) have isolated
large single-strand pieces of DNA from E. coli and therefore have been able
to detect small numbers of single-strand breaks in the bacterial DNA. They
9
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find that equal numbers of breaks are produced by irradiating E. coli B.
or B/r. In Be there is further degradation of the DNA whereas in B/r
the single-strand breaks disappear with time after irradiation. It takes
about 60 minutes to repair 20 singl.e-strand breaks per bacterial chromosome,
during which time there has been a negligible amount of net DNA synthesis,
Strand breaks also disappear with time in M. radiodurans (see Sec. 4.1).
5.2. Pyrimidine dimers. These are single-strand lesions that do not
by themselves make chain breaks. Since pyrimidine dimers are easy to label
with radioactive thymidine, it is possible to detect them in small numbers
(50 to 500 dimers per chromosome strand) and therefore it is possible to
measure these lesions at doses for which M. rediodurans or radiation resistant
strains of E. coli show 100% survival. We can thus be certain that we are
looking at properties of the surviving cells.
The lesions -- pyrimidine dimers -- do not remain in the DNA of her*
cells (fig. 4) but appear in the acid soluble fraction of the cells as
parts of small oligonucleotides (15, 16). Excision is rapid and is completed
before DNA synthesis resumes at a normal rate. It is not observed in
sensitive cells that are her®, such as B-7: The existence of cells that
show no excision lends strength to the argument that excision is part of
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the repair process. Excision of pyrimidine dimers has been observed in all
the host-cell reactivating strains of E. coli that have been investigated,
and has not been observed in the hcr
strains. It has also been detected
in M. radiodurans (17) and B. megaterium (18) but for the latter two bacteria
there are no sensitive strains which permit one to define zero or-close-to-zero
repair. Dimers are excised from the DNA of phage T3 infecting E. coli B/r
but not from T3 infecting Bo (B. Baker and R. B. Setlow, unpublished).
And, finally, the v-gene -- a gene in T4 phage that results in T4 being
twice as UV-resistant as T2 phage and whose biological effects overlap
those of pħotoreactivation -- resiults in the excision of dimers from T4
DNA in both resistant and sensitive strains of E. coli (19). The latter
result is expected from the biological observation that the survival of
T4 phage is independent of the host used to assay the irradiated phage.
The above data indicate that the excision mechanism is common to many
microbial systems. Since the usual definition of excision is the appearance
of dimers in acid soluble material, such a definition automatically means
that the dimers must be parts of small pieces of DNA. If there were
excision in two large stable single-strand DNA pieces it would not be
measured by the techniques used at present.
6. STEPS IN EXCISION
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6.1. The order of the molecular steps -- excision, degradation,
polymerization and rejoining -- is not known (20). For example, as
suggested by Rörsch (21), excision might result from a single-strand break
on one side of a dimer followed by polymerization and rejoining with release
of the dimer as the final -- rather than the first -- step.
6.2. Rate of excision. The excision process is a rapid one. It
proceeds at an initial rate of about 15 dimers per min in E. coli (fig. 5)
F-5
and approximately 10 times this rate in M. radiodurans. E. coli shows very
high survivals after 500 dimers per strand have been excised and M. radiodurans
after approximately 5000 dimers. The massive excision that is observed during
repair must be accompanied by other repair steps. Otherwise, the bacterial
DNA's would end up with many single-strand breaks and there would be a
high probability of breaks in opposite strands being close enough to result
in a double-strand scission. Double-strend breaks could also rise during
replication through a single-stranded region (22).
6.3 Repair replication. We expect that the gaps remaining after
excision of the dimer-containing oligonucleotides would be filled in by
a polymerizing mechanisa that uses the opposite strand as a template.
Such a mechanism would not work on single-stranded DNA's since they do
not contain redundant information as do the double-stranded ones. As a
matter of fact host-cell reactivation has not been demonstrated for the
single-strand phages but it has been demonstrated for the double-stranded
replicative form of such phages.
Insofar as dimers; and hence excised regions, are randomly distributed
along the chromosome, the newly synthesized DNA, should be scattered
throughout the length of the bacterial chroniosome. This type of replication
called "repair replication" has been characterized experimental.ly by
Pettijohn and Hanawalt (23) who observed that the incorporation of radioactive
bromouracil into bacterial DNA during the repair period is into light-, rather
than hybriä-density material. Repair replication has been observed in cells
treated with nitroren mustard (24). There is unfortunately no definitive
evider.ce that the newly incorporated material actually replaces the excised
regions. It would represent some other type of aberrant synthesis such as
phage DNA (25) or replication from new chromosomal origins (26) that is
observed when normal DNA synthesis is stopped by irradiation and some of the
DNA breaks down (see Sec. 6.4).
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6.4. DNA breakdown (review in ref. 10). It is difficult quantitatively
to relate the amount of repair replication with the excision of dimers because
the excision process seems to be followed in many cases by further degradation
of DNA. Such degradation could be associated with nuclease attack in the
region of the excised dimer and obviously if it proceeded extensively it
could result in so much degradation that the cell could die. It is not clear
whether degradation plays a role in the repair process. Since DNA degradation
is measured in bacterial cultures rather than in single cells' the breakdown
could represent either (a) che degradation of the DNA of dead cells or (b) a
necessary enlargement of the excised hole or (c) the degradation of large
dimer-containing oligonucleotide to mononucleotides, plus a small dimer-containing
unit. In any event degradation seems to require a single-strand break as a
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starting place. Such'a break may be put in by excising enzyme or by some agent
such as X-rays or methylmethanesulphonate (28).
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6.5. Rejoining of strands. If the DNA is to become resistant to
exonuclease attack, the single-strand breaks that remain after excision of
dimers, degradation, and polymerization will disappear by a rejoining mechanism
that is presumably similar to that observed for the repair of X-ray-induced
breaks (see Sec. 5.1). · The technique of McGrath and Williams (13) has been
used to measure the number of strand breaks during the time that UV-damage is
being repaired by the excision mechanism (fig. 5).
• The efficiency of the UV-repair process is indicated by the observed
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small numbers of single-strand breaks that exist while dimers are being
Sie
excised. Even though in E. coli, 15 dimers per minute are being excised at
a constant rate for about 30 minutes there are only approximately 15
www.come.....
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single-strand breaks at any instant. Thus the activity of the various
steps in the repair process are indeed well coordinated -- as would be
necessary if the process is to be efficient as it is.**
There is a big difference between the repair of X-ray-induced breaks
and breaks that arise as a result of excision of dimers. In 60 min only
20 X-ray breaks per strand are fixed but 500 excision-breaks per strand
are rejoined. Of course the two types of breaks probably have different
kinds of ends and there is no reason why they should be repaired at the
.
same rates.
....
Some strains of E. coli are deficient in the ability to participate
.
in genetic recombination. The extreme rec strains are sensitive to UV,
....
X-rays and other agents and are characterized, at the molecular level, by
extensive DNA breakdown after treatment with inactivating agents. Rec
strains that cannot excise dimers do not show DNA breakdown after
UV-irradiation. Rec strains may lack the rejoining enzymes or they may
not rejoin because, as a result of extensive degradation, they never get
--
a chance to attempt rejoining.
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7. CONCLUSION
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Our knowledge concerning the temporal sequences of steps such as
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excision, degradation, repolymerization and rejoining is almost nonexistent.
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We have no good, verified ideas as to tñe control mechanisms for the
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individual steps and how any such control would affect survival and mutation
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induction. Nevertheless even though we know little about the intimate
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oper
details of these repair processes it is clear that the processes are not
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limited to radiation-induced changes in DNA and that the observed response
of microorganisms and viruses to physical and chemical insults is more
dependent on the repair processes than upon the induced lesions.
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REFERENCES
1. R. H. Haynes, in Physical Processes in Radiation Biology, Academic
Press, New York, 1964, p. 51.
2. J. K. Setlow, Current Topics in Rad. Res. Vol. II, M. Ebert and
A. Howard, eds. North-Holland Pub. Co., Amsterdam, 1966, p. .
3. R. F. Kimhall, Adv. Rad. Biol. 2, 135 (1966).
4. C. S. Rupert and W. Harm, Adv. in Rad. Biol. 2, 1 (1966).
5. K. C. Smith, Photophysiol. Vol. 2, A. C. Giese, ed. Academic Press,
New York, 1964, p. 329.
6. R. F. Hill, Biochim. Biophys. Acta 30, 639 (1958).
7. R. F. Hill and E. Simson, J. Gen. Microbiol. 24, 1 (1961).
8. H. I. Adler, Adv. Rad. Biol. 2, 167 (1966).
9. A. J. Clark and A, D. Margulies, Proc. Natl. Acad. Sci. vis. 53, 451
(1965).
10. P. Howard-Flanders and R. P. Boyce, Rad. Res., Suppl. 6 (1966),
in press.
11. G. S. Stent and C. F. Fuerst, Adv. Biol. Med. Phys. 2, 1 (1960).
12. D. Freifelder, Proc. Natl. Acad. Sci. U.S. 54, 128 (1965).
12a. C. J. Dean, P. Feldschreiber and J. T. Lett, Nature 209, 49 (1966).
13. R. A. McGrath and R. W. Williams, Nature, in press.
14. B. A. Bridges and R. J. Munson, Biochem. Biophys. Res. Comm. 22, 268
(1966).
15. R. B. Setlow and W. L. Carrier, Proc. Natl. Acad. Sci. U.S. 51, 226
(1964).
16. R. P. Boyce and P. Howard-Flanders, Proc. Natl. Acad. Sci. U.S. 51,
293 (1964).
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17. M. E. Boling and J. K. Setlow, Biochim. Biophys. Acta, in press.
18. J. E. Donnellan, Jr. and R. B. Setlow, Biophys. Soc. Abstracts, 10th
Ann. Meet., 1966, p. 112.
-
19. R. B. Setlow and W. L. Carrier, Biophys. Soc. Abstracts, loth Ann.
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t
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.
.
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-
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-
-
-
-
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Meet., 1966, p. 68.
20. R. B. Setlow, Rad. Res. Suppl. 6 (1966), in press.
21. A. Rörsch, Rad. Res. Suppl. 6 (1966), in press.
22. P. C. Hanawalt, Photochem. Photobiol. 5, 1 (1966).
23. D. Pettijohn and P. Hanawalt, J. Mol. Biol. 2, 935 (1964).
24. P. C. Hanawalt and R. H. Haynes, Biochem. Biophys. Res. Comm. 19, 462
(1965).
25. H. D. Mennigmann, J. Gen. Microbiol. 41, 151 (1965).
26. R. Hewitt and D. Billen, J. Mol. Biol. 13, 40 (1965).
27. C. R. Shaffer and R. A. McGrath, Exp. Cell Res. 39, 604 (1965).
28. B. S. Strauss and R. Wahl, Biochim. Biophys. Acta 80, 116 (1964).
R.
S
haffer and R. A. McGra
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1
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Footnotes
* Research sponsored by the United States Atomic Energy Commission under
contract with the Union Carbide Corporation.
*In the one reported experiment (ref. 27) in which single cells were
observed, cells of E. coli b/r that survived X-irradiation were
observed to lose some of the radioactive label associated with DNA.
** The excision process is obviously not 100% efficient -- all dimers are
not successfully repaired even in E. coli B/r -- as indicated by the
fact that B/r shows photoreactivation.
18.
Table 1
Bacterial cells may fail to multiply after irradiation because
1. DNA synthesis is blocked.
DNA is broken or is broken down.
2. Incorrect DNA is made.
(Enzymatic machinery affected.)
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(Cells cannot divide.)
3. Phage (or defective phage) induced.
(Enzymatic machinery affected.)
(Cells cannot divide.)
4. Chromosomes, cannot separate.
5. RNA and proteins affected.
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19
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Figure Legends
Figure 1. Survival curves for several strains of E. coli. Reproduced,
(15,939-R) with permission, from Hill and Simson (7). a) UV-irradiation,
b) X-irradiation, c) p-suicide.
Figure 2. Various ways in which a lesion in DNA, a pyrimidine dimer, may
(15,935)
affect DNA synthesis or be repaired.
Figure?. Typical structure changes that may act as lesions. The changes
(15,950)
illustrated by 2), 3) and 4) are discussed in the text.
· Figure 4. Excision of thymine-containing dimers from the acid-insoluble
(13,114) fractions of E. coli B and B/r. Cells were labeled with
Hi-thymidine, transferred to nonradioactive growth medium and
irradiated, at zero time, with 200 ergs/mm of 265 mu radiation.
At various times after irradiation the radioactivity in thymine
and diners was determined for both acid-soluble and acid-insoluble
fractions. Dimers that disappear from the acid-insoluble
fraction appear in the acid-soluble fraction [Setlow and Carrier
(15)].
Figure 5. Excision of pyrimidine dimers and the appearance of single-strand
(15,590)
breaks in the DNA of E. coli B (unpublished results of R. B.
irradiated with 200 ergs/mom of 265 mu radiation -- a dose that
makes about 500 pyrimidine dimers per strand. The numbers of
dimers excised is obtained from measurements similar to those in
Fig. 4 -- assuming all dimers are excised at the same rates.
The number of strand breaks is approximately equal to the
ratio of the molecular weight of unirradiated to irradiated
DNA multiplied by the number of breaks observed in the
extraction of unirradiated cells. The latter number is about
6 (ref. 13).
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