Vol. 77, No. 4, 1977
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APURINIC ACID ENDONUCLEASE IMPLICATED IN DNA BREAKAGE IN ESCHERICHIA COLI SUBJECTED TO MILD HEAT
Nicholas Grecz and S. Bhatarakamol Microbial Biophysics Laboratory, Department of Biology Illinois Institute of Technology, Chicago, Illinois 60616 Received
July
1,1977
Summary: Alkaline sucrose gradient sedimentation studies reveal that mild heat of 50-60°C triggers DNA strand breakage in E. cold cells. DNA breakage is substantially reduced by enzyme inhibitors, p-ehloromercuribenzoate or HgCI 2 added before heating. This suggests that DNA breakage is due to enzyme action rather than to direct action of heat itself. Under identical conditions of heating, a mutant of E. cold deficient in apurinic acid endonuclease (BW 2001) yields substantially fewer DNA breaks than wild type cells or ligase deficient strain is7. These results are consistent with involvement of endonuclease in production of DNA single strand breaks in E. cold subjected to mild thermal stress. The important recognition from this study concerning the role of the cell's endonuclease in thermal damage is that mild heating (50-60°C) is able to trigger endonucleolytic DNA breakage which seems to be the initial event in heat injury. M~id heat (52°C) has been reported to induce DNAbreaks (1-4).
Furthermore~
viability
(4).
ill bacteria
DNA strand breakage is related to loss of cell
In contrast
to the rather extensive knowledge of DNA repair
in heat injured cells (5), the n~lecular mechanism of DNA strand breakage by mild heat has so far evaded scientific understanding. DNA scission may be attributed hydrolysis,
or depurination
to chemical hydrolysis,
(2).
The observation
Thermally
enzymatic
induced
(endonuclease)
that DNA scission depends
on either the particular strain or the physiological
state of the organisms
(1,2), cannot be readily reconciled with the idea of direct action of heat by thermal hydrolysis
or depurination.
reasoning Sedgwick and Bridges with "attack by nucleases or the physiological heating."
On the weight of this negative
(2) concluded
that DNA breakage was consistent
(exo- and/or endonucleases,
state of tlle organisms)
The recen~ avaibility
released or activated by mild
of mutants deficient in endonuclease
(6) made it possible for us to re-examine
Copyright © 1977 by Academic Press, Inc. All rights o/reproduction in any form reserved.
depending on the strain
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this problem and to obtain
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evidence implicating specifically apurinic acid endonuclease in the production of DNA single strand breaks in cells subjected to mild heat. Materials and Methods: Mutants of E. coli used were BW 2001, ts7, and B/r. E. coli BW 2001 is a polyauxotrophic mutant with only 8% of normal endonuclease activity at 37°C in its cell extracts (6). Previous studies (6,7) seemed to suggest that BW 2001 was deficient in endonuclease II, and that endonuclease II was identical with exonuclease III and 3'phosphatase. Subsequently, endonuclease II was separated from exonuclease III as well as from 3'-phosphatase (8). Moreover, on careful reexamination E. coli BW 2001 was shown tO be deficient in apurinic acid endonuclease, but to have endonuclease II, exonuclease III, and 3'phosphatase activities. E. coli ts7 is a temperature sensitive, polyauxotrophic, DNA-ligase-defective derivative of E. coli TAU-bar (9) containing i-3% DNA-joining activity of that of the wild type parent (i0,ii). E. coli B/r is a heat and radiation resistant (1,12) strain o£ high DNA repair capacity with a full complement of endonuclease and DNA ligase molecules. Cells were cultured on a shaker in L-broth at 25°C. DNA was labeled for four generations in L-broth with added 2.5 ~ Ci/ml [3H] thymidine, harvested by centrifugation, washed and resuspended in 0.01 M Tris-buffer, pH 8 to a final concentration of 2 x 108 cells/ml (13). Heating was conducted in 0.01 M Trls-buffer, pH 8 at two cardinal temperatures, 51°C, the lowest temperature in our experiments at which detectable DNA breakage occurred accompanied by progressive loss of cell viability, and 60°C, the temperature at which a plateau was reached of some 230-280 DNA breaks per chromosome and no cell viability could be recovered after heating of any of the strains (14). Heat induced single strand breaks in the DNA were assessed by alkaline sucrose gradient centrifugation and the MW of the DNA fragments was calculated as described (13) employing the method of Town et al. (15) using a value of 0.38 for ~. Cell survival was evaluated on the surface of L-agar pla~es. RESULTS AND DISCUSSION In order to test the hypothesis of Sedgwick and Bridges (2) that DNA breakage may be due to nucleolytic attack by the cell's own enzymes, we have first attempted to inhibit these enzymes by well known enzyme inhibitors, p-chloromercuribenzoate (p-CMB)j and HgCI 2.
When added at the
start of heating, 0.2mM p-CMB reduced the number of DNA strand breaks in the chromosome by 47% (Table i) thus suggesting that DNA breakage may have been indeed due to enzyme action.
At 100°C, DNA-breakage is due to
thermal hydrolysis (14) as evident from the drastic reduction at this temperature of the effect of p-CMB.
Essentially similar results were
obtained with 0.2mM HgCI 2. A genetic approach concerning the effect of heat on E. coli strains deficient in the endonuclease and ligase systems is summarized in Fig. i.
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Table i.
Temp. of heating °C
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Effect of enzyme inhibitor, p-chloromercuribenzoate on DNA breakage in E. coli B/r.
Addition a/ of p-CMB D b/
DNA-fragment ~ x l 0 -6 , daltons c!
Number of SSB per half d/ % of chromosome control
60
-
5.1
5.12
273
i00
60
+
6.5
9.65
145
53
i00
-
1.44
0.18
7778
i00
I00
+
1.50
0.20
7000
90
a/ Addition of 0.2 mM (final concentration pCMB (p-chloromercuribenzoate) at the start of heating before the desired temperature was reached to log-phase cells suspended in 0.01 M Tris-buffer, pH 8. b/ Sedimentation distance D = ZXiYi/ZYi where Yi is the fraction of radioactivity in the i-th fraction at a distance Xi from the meniscus (16) calculated from alkaline sucrose gradient (5-20%), pH 12.5 (17) centrifuged at 30,000 rpm for 90 min in a Beckman L2-65B ultracentrifuge in a Beckman SW 50.1 rotor. c/ Calculated from (18): MI/M 2 = (DI/D2) I/0"38 = (DI/D2)2"36 where M 1 = 6xlO 7 daltons, the MW of T4 phage DNA at pH 12.5 and D 1 = 13.0 for T 4 [3H]-DNA. The sedimentation distance D 2 for E. coli [3H]-DNA are shown in column 3. The exponent ~=0.38 in the above formula has been used in conformity with Town et al (15). d/ The number of single strand breaks calculated from the formula: #SSB=(Mo/Mn)-I where Mo is the MW of the E. coli chromosome, 2.8 x 109 daltons double stranded or 1.4 x 109 daltons single stranded in the alkaline sucrose gradient; Mn are the MW of the DNA fragments given in column 4.
Fig. a (unheated control) shows that extraction of [3H] DNA from the three strains for alkaline sucrose gradient sedimentation experiments yielded nearly coincident main peaks at a distance of 0.6-0.7 from the meniscus.
Apparently, the intrinsic fragility of the DNA from is7 and
B/r showed only minor differences, while strain BW 2001 exhibited slightly retarded DNA fragility which may perhaps be attributed to this mutant's deficiency in endonuclease (6, 8).
In view of these nearly identical
DNA sedimentation profiles in unheated control cells, the differences between the strains in DNA breakage when subjected to mild heat (Fig. ib,
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T4
51
p.0e
a
,~ /
,,n 4-z o t)
5
~
"o
b
J
# 0 I--
0.25
0.50
0,75
l.O
RELATIVE DISTANCE FROM MENISCUS
Figure i.
Alkaline sucrose gradient sedimentation profiles of 3H-DNA from E. coli B/r (o), BW2001 (A) and ts7 (A) heated for 60 min at (a) 25~C, (b) 51°C, and (c) 60°C. Log phase 3H-labeled cells harvested from L-broth were suspended in 0.01 M Tris-buffer, pH 8 for heat treatment. Methods for heating and alkaline sucrose gradient centrifugation are described in footnotes to Table i.
c), may be assumed to be due specifically to differences in DNA breakage and DNA rejoining in these mutants.
Thus, heating at 51°C for 60 min
resulted in characteristic DNA fragmentation (Fig. ib) in which strain ts7 suffered by far the most DNA breaks of the three strains tested. This is evident from the large shift of the ts7 [3H] DNA peak toward the meniscus.
In comparision, the endonuclease II defective mutant
BW 2001 sustained a considerably smaller number of DNA breaks.
More
important, BW 2001 showed significantly fewer breaks than the highly resistant and highly repair competent strain B/r.
The same basic
results Were obtained after heating at 60°C for 60 min (Fig. ic) except for the generally greater extent of DNA breakage at the higher temperature.
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Table 2.
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Effect of heating at 52°C on loss of viability of E. coli mutants defective in endonuclease II (BW2001) or DNA ligase (ts7) activity as compared with repair competent strain B/r.
Time of heating
(min)
........... BW2001
Cqlony count, .n/no ts7
a/ B/r
0
1.00
1.00
1.00
3
0.45
0.25
0.60
5
0.24
0.08
0.43
a/ Log phase cells were added i:i0 into pre-heated 0.01 M Tris-buffer, pH 8 so as to achieve instantaneous equilibration to 52°C. Heated ceils taken at the indicated time intervals, were spread on L-agar plates, and incubated at 25°C for 48 hrs. n/no = fraction of survivors where n= = initial number of cells~ n = number of cells surviving a given heat treatment.
Since BW 2001 showed a 30-80% lower cell recovery after heating than B/r (Table 2) in spite of the substantially
smaller number of DNA breaks,
it is clear that BW 2001 cannot have a more efficient repair system than B/r, and therefore it is logical to attribute
the reduced number of DNA
breaks in BW 2001 Co this mutant's genetic deficiency The conclusion
that DNA breakage is due to enzyme action is supported
by the effect of enzyme inhibitors, to relatively
large DNA fragments
with endonucleolytic of noticeable
in endonuclease.
and by the fact that DNA is broken
(106-107 dalton)
chromosome cleavage
(Table i).
consistent However,
absence
loss of soluble phosphate in any of the strains seems to
rule out the postulated
(2) involvement
molecular events of thermal injury.
of exonuclease in tlde initial
The simplest explanation of these
results therefore is that mild heat triggers endonuclease single strand breaks, characteristic
specifically
to produce
and that the ability to produce DNA breaks is a
genetic trait related to the molecular mechanism of heat
injury.
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ACKNOWLEDGEMENTS: This work was supported by U.S. Army Research Office Grant #DAHCO 4-75-G-0112, and U.S.-Hungary Research Cooperation Program FHR 03/147. REFERENCES i.
Bridges, B.A., Ashwood-Smlth, M.J., and Munson, R.J. (1969) J. Gen. Microbiol., 58, 115-124. 2. Sedgwick, S.G. and Bridges, B.A. (1972) J. Gen. Microbiol., 71, 191193. 3. Matsumoto, S. and Kagami-lshi, Y. (1970) J ~ . J. Genet., 45, 153-160. 4. Woodcock, E., and Grigg, G.W. (1972) Nature New Biol______t., 237 , 76-79. 5. Pauling, C., and Beck, L.A. (1975) J. Gen. Microbiol., 87, 181-184. 6. Yajko, D.M., and Weiss, B. (1975) Proc. US Nat. Acad. Sci., 72, 688-692. 7. Weiss, B. (1976) J. Biol. Chem., 251, 1896-1901. 8. Kirtikar, D.M., Cathcart, G.R., and Goldthwait, D.A. (1976) Proc. US Nat. Acad. Sci., 73, 4324-4328. 9. Pauling, C., and Hamm, L. (1968) Proc. US Nat. Acad. Sci., 60, 1495-1502. 10. Gottesman, M.M., Hicks, M.L., and Gellert, M. (1973) J. Mol. Biol., 77, 531-547. ii. Konrad, E.B., Modrich, P., and Lehman, I.R. (1973) J. Mol. Biol., 77, 519-529. 12. Witkin, E.M, (1967) Brookhaven Symp. Biol. 20, 17-55. 13. Alur, M.D., and Grecz, N. (1975) Biochem. Biophys. Res. Comm., 62, 308-312. 14. Bhatarakamol, S., and Grecz, N., J. Bact., (in the press). 15. Town, C.D., Smith, K.C., and Kaplan, H.S. (1971) J. Bact., iO5 , 127-135. 16. Kaplan, H.S. (1966) Proc. US Nat. Acad. Sci., 5_~5, 1442-1446. 17. McGrath, R.A., and Williams, R.W. (1966) Nature, 212, 534-535. 18. Studier, F.W. (1965) J. Mol. Biol., Ii, 373-390.
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