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NATIONAL BUREAU OF STANDARDS - 1963
ORN P. 3324
Cont-670822--/
MASTER
. THE EXCISION OF PYRIMIDINE DIMERS IN VIVO AND IN VITRO
by R. B. Setlow and W. L. Carrier
RECEIVED BY DTIE OCT 2 1967
Biology Division
Oak Ridge National Laboratory
Oak Ridge, Tenn., U.S.A.
Oral and written presentation at
International Conference on Replication and Recombination
of Genetic Material
28 August to 1 September, 1967
Canberra, Australia
Research sponsored by the U.S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
-
.
LEGAL NOTICE
This report was prepared as an account of Government sponsored work. Neither the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Mal.es any warranty or representation, expregeed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the use
of any information, apparatus, method, or process disclosed in this report may 10t infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, or for damages resulting from the
use of any information, apparatus, method, or process disclosed tu this report,
As used in the above, "person acting on behalf of the Commission" includes any em-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
such employee or contractor of the Commission, or employee of such contractor prepares,
dioseminates, or provides access to, any information pursuant to his employment or contract
with the Commission, or his employment with such contractor.
DISTRIBUTION OF THIS QOCUMENI LS UNLIMITED
24
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Send proof to: Dr. R. B. Setlow
Biology Division
Oak Ridge National Laboratory
P. O. Box Y
Oak Ridge, Tennessee 37830, U.S.A.
.
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Introduction
The notion that microorganisms could recover from the effects of
radiation arose because the survival or the induction of mutants :
from ultraviolet radiation could be modified by many post-irradiation
treatments. (Reviews by Rupert and Harm, 1966; Kimball, 1966) The
discovery of radiation-sensitive mutants of bacteria and the finding
that these mutants differed from the wild type by only a few mutational ·
steps and that many such mutants were unable to do host-cell reactivation
(reviews by Howard-Flanders and Boyce, 1966; Rorschetai, 1967) particularized
the concept of recovery from a passive notion to an active process--the
process which we speak about as "repair of DNA." (R. B. Setlow, 1967a).
If we are to investigate the molecular mechanisms involved in repair of
DNA, we must have a convenient model system to investigate. We must be able
to observe what is repaired.
A number of experimental investigations on the effects of ultraviolet
radiation on DNA -- in particular the relation between enzymic monomerization
and biological photoreactivation--indicate that pyrimidine dimers in DNA
affect biological systems. (Review by J. K. Setlow, 1966) Such photochemical
products distort the structure of DNA and alter its interaction with
enzymes such as polymerases and nucleases. Thus, since dimers seem to
affect biological activity and since damage to DNA is repaired, it is
a reasoneble supposition that the repair systems operate on dimers. (of
course, dimers are not the only photochemical damage suffered by DNA
(Smith, 1967) but the action of repair systems on any other types of photo-
products has not been documented).
The pyrimidine dimers formed by irradiation of DNA are stable to acid.
techniques. Moreover, they probably do not turn over in cells, i.e., they are
not incorporated into polynucleotides though they may be removed from them.
Thus dimers seem to be ideal models for the investigation of the various
steps in repair. Certainly they are the best studied physico-chemical lesion
in DNA. In some strains of bacteria, lisually UV-sensitive and Hcr”, the
dimers remain in DNA for long times after irradiation. In other strains of
bacteria, usually UV-resistant and Her*, the dimers are removed selectively
from the bacterial DNA as parts of oligonucleotides. The latter process,
termed "excision", is associated in vivo with others observables such as
DNA breakdown, repair replication and the rejoining of strand breaks that
result from excision. Obviously the biological effectiveness of these
processes may be thought of as dependent on their rates compared to DNA
synthesis around a lesion (R. B. Setlow, 1967b), cr past a strand break
(Hanawalt, 1966). We have been measuring the properties of three excising
systems: E. coli, (Setlow and Carrier, 1964; Boyce and Howard-Flanders,
1964), T4 phage (Setlow and Carrier, 1966) and an in vitro system derived
from M. lysodeikticus (Carrier and Setlow, 1966). The three systems have
similar, but by no means identical, properties. We have not yet been able
to use the data to elucidate the individual steps in repair or make a ....
consistent story that will cover all the three systems we describe.
Some of the steps associated with repair of E. coli DNA (excision of
damage, DNA degradation, repair replication and rejoining of strands) have
observed for damage produced by other agents such as X-rays, (McGrath and
Williams, 1966; Kaplan, 1966) nitrogen and sulfur mustards, (Kohn, et al,
1965; Lawley and Brookes, 1965; Hanawalt and Haynes ; Rapirmeister and
5
.
Davison, 1964) mitomycin-C, (Boyce and Howard-Flanders, 1964) and nitroso-
guanidine (Cerda-Olmedo and Hanawalt, 1967). The products that are made
by the action of these agents on DNA in vivo are not as easy to detect as
are dimers.
Methods
The usual experimental procedure is to label cells with radioactive
thymidine, (most of the dimers are thymine-containing ones), irradiate them
with UV doses that leave many survivors (so that the properties of surviving
rather than dying cells are measured) and then Incubate the cells in non-
radioactive medium. At several times after irradiation the DNA is fractionated
into pieces of various sizes, for example by chromatography on Sephadex or
by acid solubility, and each fraction is hydrolized and chromatographed.
and counted so as to measure the radioactivity in t’iymine and in dimers.
If thymine-containing dimers are lost from the DNA more rapidly than is
thymine, we say that excision - the specific removal of dimer-containing
:
E. coli is particularly well suited for studies of excision since the
dimer-containing oligonucleotides remain in the cells and do not leak out :
into the medium where they are difficult to detect. The loss of dimers from
Her strains is very slow, or zero, even at low UV doses. The difference in
the rates of excision between Hcr* and Her“ strains is between 10 and 100-.
fold (Fig. 1).
.
*2 The same result could be obtained by complete breakdown of the DNA and resynthesis
without the incorporation of dimer containing oligonucleotides. Such a
pathway is an extravagant way to repair DNA. It seems to take place in the
bacterial DNA of cells infected with T-even phages (see below) but in this
case, the bacteria do not survive. :
The removal of dimers from the acid insoluble fraction of DNA'may be
imagined in terms of either of the two molecular mechanisms shown in Fig.
2. Both mechanisms yield the same end result for dimers--the dimers appear
in the acid soluble phase. Ons of them (Scheme A) requires DNA synthesis
to be effective if excision is to take place. In it, excision is the last
step in repair. In Scheme B, excision is the first step in repair and
does not require DNA synthesis. The available experimental evidence does
not favor one model over the other.
Excision in E. coli
Chemical agents that inhibit host-cell reactivation (review by Rupert
and Harm, 1966), such as caffeine (Fig. 3) and acriflavine (R. B. Setlow,
so inhibit excision. Excision is not significantly inhibited in
E. coli by chloramphenicol even up to 400 micrograms per ml nor is it
inhibited by deprivation of thymine in bacteria such as 15T-(Shuster and Boyce,
1964; and Figil). Unfortunately the latter observation cannot be taken to . .
indicate that DNA synthesis is unimportant in the excision mechanism because
associated with excision there is always some breakdown of DNA. Such break-
down could provide the small count of nucleotides necessary for synthesis and
excision. However, the process of excision is drastically inhibited by the
more general metabolic inhibitors of starvation or cyanide (Fig. 3) (Setlow
and Carrier, 1964 and unpublished). DNA breakdown in irradiated B. subtilis
is also inhibited by starvation for glucose (Searshi and Strauss, 1967).
Whatever the detailed steps in the excision process are, both models
in Fig. 2 predict that there should be an increase in single strand breaks
followed by return of the DNA to its normal molecular weight during the process
of repair following UV irradiation. The measurement of the relatively small
numbers of break per bacterial chromosome (200 ergs per square millimeter
only makes about 500 dimers per strand) can be carried out by the method
developed by McGrath and Williams (1966) in which high molecular weight,
single-stranded DNA of bacteria is isolated directly on an alkaline sucrose
gradient. The measurement of strand breaks that appear and disappear
during the normal excision process in E. coli B/r indicate that about
15 single strand breaks are open at any instant during the time that
about 30 dimers per minute are being excised (Setlow, Carrier, and Williams,
1967). Thus, on the average, breaks do not stay open for a long time
during normal excision in vivo. If agents such as KCN or starvation
acted only by inhibiting the DNA synthesis, the nuclease that makes the
incision would keep working in the model of Fig. 2A. the bacterial
DNA would have many more strand breaks than are observed in the uninhibited
cells. The large number of anticipated breaks is not observed (Fig. 4).
We conclude that the incision enzyme needs an energy source, or a cofactor
such as ATP that would not be found in starved or cyanide-poisoned cells,
or that the model in Fig. 2A is not correct. In the later case, we cannot:
describe excision as the result of independent steps of incision, followed
by polymerase 'and polynucleotide ligase action. The in cising enzyme, however,
could be part of a small number of complexes of repair-enzymes than must,
finish repair in a local region before moving on to the next lesion. .
The denial of the model in Fig. 2A would not lend support to the one
in Fig. 2B. In both models the first steps in the process are inhibited
by starvation or cyanide and such inhibitors are not anticipated for an
endonuclease that is expected to recognize warped regions in the polynucleotide:
turisit
(An exonuclease activity in Molysodeikticus that requires nucleoside di- or
· triphosphates has been reported by Tsuda and Strauss (1964). ] The other agents,
caffeine and acraflavine, that inhibit excision also do not result in an
increase in strand breaks. Their model of action in the excision system
is not known.
Excision in T4 phage
Bacterophages that carry the vt allee are only one-half as sensitive
as those, such as T2, that are v- (review by Rupertand Harm, 1966). The v+
viruses have a smaller photoreactible sector and thus they act as if there
is a dark repair system associated with the v gene that can remove some of
the damage associated with dimers and Walter Harm (1961) has shown that
in a mixed infection the vt phage are able to rescue ultraviolet irradiated
v- pliage. Ellison et al. (1960) have given evidence that the survival
of vt or v- phages is unaffected by the host used to titer them and hence
we infer that the bacterial enzyme systems of repair do not operate on the
T-even phages, although by definition of host-cell--reactivation they do
work on phages Ti, T3 and 1.
The experimental observations on excision of dimers from phage DNA in
vivo are in accord with the above biological findings (Setlow and Carrier,
1966).
Infection of E. coli with vt phage results in the excision of
dimers from the vt DNA, whereas in an infection with v- phage, the dimers
remain the the phage DNA. Excision is dependent of another phage gene,
X, that also affects UV-sensitivity and is independent of the Her character
of cells as indicated by experiments using strains Bfr, B, Bs-] and several
of the Hcr- mutants of E. coli K12 (Setlow and Carrier, unpublished data).
Some of the excision data are shown in Table 1. The data indicate that the
action of the v.gene on phage DNA takes place in cells that, by themselves,
are unable to excise dimers from their own DNA or from the DNA of T4 phages.
On the other hand, in a mixed infection with nonradi nactive v+ phage
.. plus H-thymine labeled v- phage (both irradiated with UV) we observed
the excision of dimers from the v- DNA.
The biological and the biochemical findings on repair in T4 phage
led us to expecy, that the vt allele results in the production, or activation,
of an enzyme that acts early in infection to effect the excision of
dimers. (It is not known which step or steps in excision are controlled
by the v gene.) According to this expectation, an infection made in the
presence of chloramphenicol (an agent that does not inhibit excision in
E. coli) should inhibit the action of the v gene because the early protein
could not be made. However, we have only been able to inhibit the action
of the v gene, (as measured by excision) by use of massive amounts of
chloramphenicol or by the general metabolic inhibitors of cyanide and
starvation (Table 21). Since the latter two treatments also inhibit excision
in E. coli, their action tells us little about their effects on the phage
excision system, such as whether they act by inhibiting preformed enzymes
or by preventing the synthesis of new activity. The, unexpected result that
the v gene system is only inhibited by high levels of chloramphenicol mayo
be of use in the purification of the specific enzymes or activities from
phage-infected cells since most new proteins are inhibited by low levels of
chloramphenicol.
Although the bacterial enzymes do not work to effect excision in T4
phage DNA's, the phage-induced activity does act on bacterial DNA in vivo.
If UV irradiated bacteria are infected with v+ phage in the prescence of
chloramphenicol (to prevent breakdown of the bacterial DNA and reincorporation
of nucleotides but not dimers into viral DNA), excision of dimers from the
can
are
bacterial DNA can easily be observed; whereas, if the bacteria are
infected with v- pliage, no excision is observed (Setlow and Carrier,
unpublished data).
M. lysedeuk ticus in vitro
Extracts of M. lysodeikticus usually have low nuclease activity against
native DNA. They do however have detectable nuclease activity that seems :
to be specific for irradiated or otherwise damaged DNA (Strauss, 1962).
Such extracts are able to reactivate the ultraviolet-inactivated replicative-
form (RF) of the virus g.X174 (review by RÜrschetal, 1966) and to effect the
reactivation of transforming DNA (Elder and Beers, 1965). The extracts
make single strand breaks in the irradiated but not in the unirradiated of
0X174. Thus the extracts can at least do the first step in an excision
process, and although no net excision was detected in vitro, the other
steps in repair could go on in the in vivo part of the essay for the
infectivity of the RF.
We have shown that extracts of M. lysodeikticus do excise dimers
from DNA (Carrier and Setlow, 1966). Pyrimidine dimers become acid-
soluble much more rapidly than does thymine. Thus this excision cannot
be a general breakdown of the DNA. Nor can it result from an incision on
one side of a dimer followed by an exonuclease attack on the broken poly-
nucleotide chain. The extracts do not require glucose. They act as if
they were doing the first group of steps in Fig. 2 B.
re
We have been able to fractionate the extract by DEAE chromatography
into two components: A and B. Excision requires the sequential action of
A and B and does not work if component B acts (and then is heat inactivated)
before A. Neither fraction effects excision alone, but fraction A has all
the properties of an endonuclease that acts on one side of a pyrimidine
dimer (Strauss, et al., 1966). The nature of component B and its mode of
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action is not known. The nuclease activity of M. Lysodeikticus extracts
W
se
that degrades UV-irradiated DNA also consists of two components (Nakayama,
et al 1967). It is improbable that these components are the same as those
that effect excision.
Conclusion
We have summarized what little is known about the processes of excision
in vivo and in vitro. Despite the fact that excision can effect the repair
of up to 90% of the damage resulting from pyrimidine dimers, the details of
the steps in, and associated with, excision are not known; nor is the order
of the steps--if there is an unique order--understood. The excision
mechanism is not confined to damages arising only from ultraviolet
radiation.
TABLE 1
Percentage radioactivity as dimers
in the polynucleotide fraction of
SH-thymine T4 - E. coli complexes
Time after adsorption (min)
Bacterial
Strain
Phage
-3_
2
7-10___
15-20
B
0.153
0.019
-B5-2
0.153
0.013
0.136
0.081
0.056
0.050
AB1886
0.136
0.061
v+X-
B go?
0.171
0.047
0.048
V-X+
0.122
0.154
0.162
0.148
0.144
0.169
0.090
0.096
S-1
0.095
0.116
0.091
0.094
0.076
V-X-
a Bsol
0.139
0.116
0.136
0.085
0.084
PH thymine - labeled 74 phage were irradiated with ~ 100--200e5lma
of 265 mu (w 5 lethal hits) and adsorbed to E. coli at a multiplicity of 5
phage per bacterium. At various times after infection che polynucleotide
es Was
fraction of the complexes was analyzed for radioactivity in thymine dimers
and in thymine. The precision of the values at - 3 min (before adsorption)
is + 10%.
TABLE 2
The effect of some inhibitory treatments
on the excision of dimers in E. coli Bay
cells infected with irradiated, 3H-thymine
labeled T4 v* phage.
-
.-
.-.-
-
-.
Before
Adsorption
Percentage of radioactivity in
dimers at incubation times (min)
5
10 _ 15-20
Treatment
0
No inhibitor
0.106
0.01M KCN
0.095
0.077
0,01M KCN (only
after adsorption)
-
0.121
0.092
0.094
0.093
--
0.0LM KCN
(only after
adsorption)
No inhibitor
.170
.064
.070
100 ugm/ml
chloramphenicol
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No inhibitor
.212
0.149
0.206
400 ugm/m1
chloramphenicol
0.207
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REFERENCES
Boyce, R. P. and P. Howard-Flanders: (1964). Proc. Natl. Acad. Sci. U.S.
5.7., 293.
Boyce, R. P. and P. Howard-Flanders: (1964). Zeit. Vererbungsl. 95, 345.
Carrier, W. L. and R. B. Setlow: (1966). Biochim. Biophys. Acta. 129, 318.
Cerda-olmedo, E. and P. C. Hanawalt: (1967). Mutation Res. 4, 369.
Elder, R. L. and R. F. Beers, Jr.: (1965). J. Bacteriol. 89, 1255.
Ellison, S. A., R. R. Feiner and R. F. Hill: (1960). Virology 11, 294.
Hanawalt, P. C.: (1967). Photochem. Photobiol. 5, 1.
Hanawalt, P. C. and R. H. Haynes: (1965) Biochem. Biophys. Res. Commun.
19, 462.
Harm, W.: (1961). J. Cellular Comp. Physiol. 58, Suppl. 1, 69.
Howard-Flanders, P. and R. P. Boyce: (1966). Radiation Research, Suppl. 6. p.136
Kaplan, H. S.: (1966). Proc. Natl. Acad. Sci. U.S. 55, 1442.
Kimball, R. F.: (1.966). Advances in Radiation Biol. 2, 135.
Kohn, K. W., N. H. Steigbigal and C. L. Spears: (1965). Proc. Natl. Acad.
Sci. U.S. 53, 1154.
Lawley, P. D. and P. Brookes: (1965). Nature 226, 480.
McGrath, R. A. and R. W. Williams: (1966). Nature 212, 534.
Nakayama, H., S. Okubo, Sekiguchi, M. and Y. Takagi: (1967). Biochem.
Biophys. Res. Commun. 27, 217.
Papirmeister, B. and C. T:, Davison: (1964). Biochem. Biophys. Res.
Commun. 17, 68.
Rörsch, A., P. van de Putte, I. E. Mattern and H. Zwenk: (1967). In Radiation
Research, G. Silini, ed. North-Holland, Amsterdam, p. 771.
Rupert, C. S. and W. Harm: (1966). Advances in Radiation Biol. 2, 2.
Searashi, T. and B. Strauss: (1967). Mutation Res. 4, 372.
Setlow, J. K.: (1966). In Current Topics in Radiation Research, vol. II.,
M. Ebert and H. Howard, eds. North-Holland, Amsterdam, p. 195.
Setlow, R. B.: (1964). J. Cellular Comp. Physiol. 64 Suppl. 1, 51.
Set.low, R. B.: (1967a). In Regulation of Nucleic Acid and Protein Biosynthesis,
V.V. Koningsberger and L. Bosch, eds. Elsevier, Amsterdam, p. 51.
Setlow, R. B.: (1967b). Brookhaven Symposia in Biology 20, in press.
Setlow, R. B. and W. L. Carrier: (1964). Proc. Natl. Acad. Sci. U.S. 51, 226..
-
.
.
:
.
.
.
-
-
Setlow, R. B. and W. L. Carrier: (1966). Biophysical Society 10th Ann. Meet.,
Abstracts, p. 68.
Setlow, R. B., W. L. Carrier and R. W. Williams: (1967). Biophysical Society,
l1th Annual Meet. p. 83.
Shuster, R. C. and R. P. Boyce: (1964). Biochem. Biophys. Res. Comm. 16, 489.
Smith, K. C.: (1967). In Radiation Research, G. Silini, ed., North-Holland,
Amsterdam, p. 756.
Strauss, B. S.: (1962). Proc. Natl. Acad. Sci. U.S. 48, 1670.
Strauss, B. S., T. Searashi and M. Robbins: (1966). Proc. Natl. Acad. Sci.
U.S. 56, 932.
Tsuda, Y. and B. Strauss: (1964) Biochemistry 3, 1678.
FIGURE LEGENDS
FIGURE 1.
-
-
-
Excision in E. coli following UV-irradiation at zero time with the doses,
at 265 mu , shown on the right-ordinate. Cells in synthetic medium containing
casamino acids. Left Panel: Typical Hort .. type strains. The open and
closed circles for 15T - indicate incubation without and with added thymine.
Right panel: Typical Hcr- strains.
FIGURE 2.
F
Two schemes illustrating the possible sequences of steps in the repair
of DNA.
The schemes are an outgrowth of the discussions in a symposium on
Structural Defects in DNA and their Repair in Microorganisms, Proceedings
of a Conference on Radiation Microbiology, Haynes, R. H. Wolff, S., and
Till, J., Editors, Radiation Res. Supplies 6 (1966).
FIGURE 3.
The inhibition of dimer excision in E. coli B/r, after 265 mu irradiation,
by caffeine and by starvation (left panel: irradiated with 200 ergs/mm2) and
by KCN (right panel: irradiated with 40 ergs/mm²).
FIGURE 4.
Sedimentation of E. coli DNA in alkaline sucrose. Cells labeled with
PH-thymine were irradiated with UV and were then incubated in growth medium
for the indicated times with or without KCN. Cells labeled with -4c-thymine
were not irradiated. The lysis and sedimentation of DNA (detected as radio-
activity) was by the procedure of McGrath and Williams (1966). The positions
of M, and M, indicate the calculated distances sedimented for 2.5 X10% and
9 x 10' daltons of single stranded DNA respectively. My represents approxi-
mately 1/5 of a single strand of an E. coli chromosome.
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