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Increased resistance of DNA replication to ultraviolet light damage in amino acid-starved bacteria
BIOCHIMICAET BIOPHYSICAACTA
57
BBA 97679
I N C R E A S E D RESISTANCE OF DNA R E P L I C A T I O N TO U L T R A V I O L E T L I G H T DAMAGE IN AMINO ACID-STARVED B A C T E R I A
C. O. DOUDNEY Research Laboratories, Department o[ Genetics, Albert Einstein Medical Center, Philadelphia, Pa. .r9z4z IU.S.A.)
(Received August i7th, 197o)
SUMMARY
Incubation of a log-phase culture of a ultraviolet light-resistant, tryptophanrequiring strain of Escherichia coli (B/r WP2) for 4 ° min without t r y p t o p h a n allows completion of the DNA replication cycle. When this occurs before ultraviolet light exposure an increase in ultraviolet light resistance of the DNA replication system of 2-fold (in terms of target number) over the log phase culture is produced. The projection to the ordinate of the relative rate values for inhibition of DNA synthesis after a series of ultraviolet light doses gave a target number of 4 for the log phase culture and 8 for the amino acid-starved culture. A comparable increase in resistance to the lethal effect of ultraviolet light was also seen with the same preirradiation treatment. The data suggest the possibility that the same mechanism is responsible for both the decreased ultraviolet light lethality and decreased inactivation of DNA replication. Both are presumably produced b y completion of the DNA replication cycle during amino acid starvation, since lack of thymine with a thyminerequiring substrain prevents the development of resistance.
INTRODUCTION DOUDNEY AND YOUNG1 demonstrated that below a "critical" ultraviolet light dose little non-recoverable damage to DNA synthesis is induced in Escherichia coli strain B/r WP2 but that a prolonged dose-dependent lag is induced during which the DNA synthetic system recovers and synthesis finally resumes at the rate seen with unirradiated bacteria. Above the critical dose little further lag in resumption of synthesis is induced. Non-recoverable damage is induced however so that the rate of synthesis observed is decreased. Utilizing the density gradient technique of MESELSON AND STAI-IL~ for measurement of the distribution of DNA sub-units with DNA replication, DOUDNEY3 showed that the reduced rate of synthesis of DNA caused b y ultraviolet light exposure was due to inactivation of part of the DNA rather than production of a slowed rate of synthesis of all the DNA. Recently SMITH AND O'LEARY a and SMITH5 have demonstrated a comparable pattern of inactivation of DNA replication b y ultraviolet light utilizing a modified technique for measurement of DNA replication involving the incorporation of labeled thymine in thymine-requiring strains of E. coll. Biochim. Biophys. Acta, 228 (i971) 57-66
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C . O . DOUDNEY
A comparison of the relative degree of non-recoverable inactivation of DNA replication in E. coli strain B/r WP2 with the lethal effect of ultraviolet light at various doses has been made *. The findings suggested the possibility that the increased inactivation rate for lethality seen with exposure above the critical dose is based on inactivation of DNA replication. These studies were subsequently extended to compare the degree of restitution of DNA replication produced by photoreversing light after various ultraviolet light doses with the increased survival also produced by the light s. In this study we have compared another method of increasing resistance of E. coli to ultraviolet light exposure with the change produced in resistance of the DNA synthetic system to ultraviolet light damage. A number of investigations have shown that, when certain strains of E. coli are permitted to complete their normal cycle of DNA replication in the absence of protein synthesis, considerably increased resistance to ultraviolet light inactivation is observed (consult a review by HANAWALT7 for a discussion of this effect). We have demonstrated an increase in radiation-resistance of the DNA replication system produced by amino acid starvation in E. coli strain B/r WP2. This increase is comparable to the increased survival produced b y the same preirradiation treatment of this strain.
MATERIALS AND METHODS
Organism E. coli strain B/r WP2 (tryptophan-requiring, hcr +) was used. This strain was kindly supplied to us by Dr. Evelyn Witkin some years ago. Culture growth procedures A small inoculum of bacteria was taken from a fresh, refrigerated nutrient agar slant and added to minimal medium supplemented with 4 °/zg/ml of L-tryptophan. The culture was grown in a large Erlenmeyer flask on a vigorously-rotating shaker at 37 ° until an absorbance of 0.25 was reached (measured at 660 m/~ with a Coleman model 6D spectrophotometer). The cells were then chilled in an ice bath in preparation for ultraviolet light exposure. Ultraviolet light exposure The chilled log-phase culture was spun down at io ooo rev./min in a Servall refrigerated centrifuge, resuspended in ice cold minimal medium (containing no tryptophan) to volume, spun down once more and finally resuspended to an absorbance of o.125 in the same cold minimal medium. Preirradiation amino acid starvation was accomplished by rapidly warming selected samples in a hot water bath to 37 ° and incubating for 40 min on the rotary shaker before again chilling in the ice bath. One hundred ml samples were then exposed in large, flat 25 cm × 16 cm pyrex dishes with magnetic stirring to the light from a General Electric 15 W germicidal lamp which emits most of its light around 2537 A. The cells were placed at a distance from the lamp which gave a dose rate below 2800/X, of about 12 ergs/mm ~ per sec as measured by a Hanovia model AV-97I ultraviolet light meter. The irradiated samples were collected in flasks held in an ice bath. Biochim. Biophys. Acta, 228 (1971) 57-66
ULTRAVIOLET LIGHT RESISTANCE OF DNA REPLICATION
59
Assay o/survival Small samples were taken from each flask and appropriately diluted in minimal medium, o.i ml of the final dilutant was then pipetted on the surface of Difco nutrient agar plates and spead evenly with a glass spreader. All platings were in triplicate. Incubation was for three days at 37 ° . Survival was scored at dilutions which result in IOO to 300 colonies per plate.
Postirradiation incubation L-Tryptophan was added to all flasks to a concentration of 4o #g/ml. The flasks were then rapidly warmed to 37 ° in a hot water bath and placed on a rotary shaker at 37 °. Ten ml samples were then taken at timed intervals and held in an ice bath for subsequent determination of DNA content.
Determination o/DNA The method of extraction of DNA was that of OGUR AND ROSEN8. The cells in the IO ml samples were spun out of suspension at IO ooo rev./min for 20 rain in a refrigerated centrifuge. The resulting pellet was then extracted for cold-acid-soluble material b y resuspension in ice cold 0. 5 M HC10, for 30 min. The pellet containing the nucleic acid obtained by centrifugation at IO ooo rev./min for 30 min after the above treatment was then resuspended in HC10, and heated for 50 rain at 7 °0 to hydrolyse the DNA. Analysis for DNA was b y the BtmTON 9 diphenylamine reaction with overnight development of color in the dark at room temperature.Color was measured at 61o m/~ by the Zeiss spectrophotometer with correction for the absorption at 650 m#. Salmon sperm DNA was used as the standard.
Accuracy o/the method We have adopted the above-described method for DNA determination in the past in numerous studies as being appropriate for determination of DNA in non-thymine requiring bacteria where relatively large changes in amount are measured. Recently SMITH AND O'LEAR'x"t have plesented evidence which suggests that pitfalls exist in the methods of measuring DNA synthesis kinetics b y the commonly adopted method of incorporation of labeled thymine and proposed new protocols for the determination of DNA replication b y thymine incorporation. Using the new techniques, they confirmed some of our earlier findings utilizing the chemical method described above*, 5. We have recently compared DNA replication measured by the Burton diphenylamine reaction as described above and by a modification of the SMITH AND O'LEARY technique. This involved incorporation of labeled thymine in a thymine-requiring strain of B/r in which the determination of relative amount of DNA increase was based on the amount of thymine incorporation after ultraviolet light exposure as compared to that incorporated with preirradiation growth from a very small inoculum. In these studies (to be published elsewhere) the relative DNA increase as determined b y the Burton method and b y the thymine incorporation method agreed closely. We therefore can conclude that the chemical determination of DNA utilized in these studies is accurate enough for our purposes in these experiments. All chemical data represent the averaged results of six identical experiments. The survival data represent three identical experiments. All operations were carried Biochim. Biophys. Acta, 228 (i97 I) 57-66
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c . o . DOUDNEY
out under yellow room light from General Electric "gold" fluorescent bulbs to prevent photoreactivation from the room light source.
RESULTS
E//ect o/ D N A replication cycle completion on sensitivity to ultraviolet light damage When an unirradiated subculture of the log phase culture of strain WP2 was warmed to 37 ° after holding in an ice bath during the period necessary ior ultraviolet light exposure (i.e. about 15 rain), DNA synthesis started immediately (Fig. I). When the subculture had been previously starved for t r y p t o p h a n for 4 ° min and was similarly warmed up, a delay of 3o-4o min in initiation of DNA replication was observed. This lag in resumption of DNA replication is typical of bacteria which have completed their DNA replication cycle in the absence of protein s y , thesis, as has been demonstrated by the studies of HANAWALT et al3 °. That DNA replication actually ceased in the absence of t r y p t o p h a n in 4 ° rain is demonstrated in Table I.
TABLE I T H E F O R M A T I O N OF D N A TOPHAN
BY
E. coli
STRAIN B#" we2
IN T H E P R E S E N C E A N D A B S E N C E O F T R Y P -
Unity = 3.3/*g of DNA per ml of culture. Incubation time (rain):
+ Tryptophan --Tryptophan
zo
2o
3°
4°
5°
6o
1.16 1.14
1.28 1.22
1.42 1.31
1.6o 1.43
1.87 1.41
2.4 1.39
When the log phase culture was exposed to an ultraviolet light dose of 3o sec a marked lag in DNA replication was induced (about 60 min) and the rate of synthesis when replicatior~ resumed was greatly reduced (Fig. I). However, when the amino acid-starved culture was exposed to an identical ultraviolet light dose, the amino acid starvation-caused lag in DNA replication of 30 min was increased by only about I0 min (for a total lag of 40 min). Little or no inactivation of DNA, as measured by rate of replication, was observed. We can thus conclude that the DNA replication system is considerably more resistant to ultraviolet light when exposed at the end of the replication cycle than when replication is actually occurring. That completion of the DNA replication cycle in the absence of t r y p t o p h a n was responsible for the increased resistance of the DNA synthetic system rather than some other effect of amino acid starvation was suggested by studies with a thyminerequiring strain in which thymine was ommitted from the medium during amino acid starvation. In this case the expected increase in ultraviolet light resistance of the DNA synthetic system did not develop. Kinetics o/inactivation o / D N A replication The relative efficiency of increasing ultraviolet light doses in inactivation of DNA replication in log phase and amino acid starved-bacteria was compared.With Biochirn. Biophys. Acta, 228 (1971) 57-66
ULTRAVIOLET LIGHT RESISTANCE OF DNA REPLICATION
2.5
C,+Trp
7"C,- T r l ~ y
u,,j/
2.5 UV,
<
61
< z
2.0
(3 2.c
O
o
~ 1.5
~ 1.5
1.0
~ 10
•
4'0 r:;o go ~bo 1~o 44o 460
26 4b 6b ~o 16o ~o 740 ;60
Postirradiation incubation (min)
Postirradiation incubation (rnin)
Fig. I. The effect of preirradiation t r y p t o p h a n s t a r v a t i o n on the ultraviolet light sensitivity of postirradiation D N A replication with E. coli strain B/r WP2. Before ultraviolet light exposure one subculture was incubated for 4 ° min w i t h o u t t r y p t o p h a n ( - - T r p ) at 37 ° while a n o t h e r was held in an ice b a t h in log phase ( + T r p ) . One half of each subculture was exposed to ultraviolet light (UV) (3o'sec) while the other half was held in the dark (C). All four subcultures were t h e n w a r m e d rapidly to 37 ° and samples taken for analysis of D N A c o n t e n t every Io min for I6o min. The D N A increase is expressed relative to t h a t a m o u n t present at the time of ultraviolet light exposure (unity = 3.3/*g/ml for the log phase culture and 4.6 p g / m l for the a m i n o acid-starved culture). Fig. 2. The effect of increasing ultraviolet light doses on rate of D N A replication with a log p h a s e culture of E. coli strain B/r \VP2. The e x p e r i m e n t was conducted as described in Fig. I. Various subcultures were exposed to the indicated doses of ultraviolet light and samples taken with postirradiation incubation. U n i t y = 3-3/zg of D N A per ml of culture.
10
2.5 < z o 2.0 E
UV: 3
o//
'~.
!,,,),,,,,,,
o 1.0
g
~ 1.0
4b io ~o ~6o ~o ,~o ~o Postirradiation incubation (rnin)
i
I
i
i
•
I
i
T
40 60 80 UV-exposure (see)
Fig. 3- The effect of increasing ultraviolet light doses on rate of D N A replication of a t r y p t o p h a n s t a r v e d culture of E. coli strain B/r W P 2 . The e x p e r i m e n t was conducted as described in Fig. I. Various subcultures were exposed to the indicated doses of ultraviolet light and samples t a k e n with postirradiation incubation. U n i t y = 4.6/~g of D N A per ml of culture. Fig. 4. The relative rate of D N A replication w i t h ultraviolet light exposed bacteria as c o m p a r e d to the unirradiated controls. The slope of the rate curves for D N A replication after the v a r i o u s ultraviolet light doses (Figs. 2 and 3) was determined graphically and expressed relative to t h e slope of the unirradiated controls (Fig. I ), I n each case the relatively c o n s t a n t rate of D N A replication represented b y s t r a i g h t lines between 60 and i2o min of incubation was utilized to e s t i m a t e slope for the irradiated samples and c o m p a r e d to the initial slope represented b y a s t r a i g h t line plot of the unirradiated controls. The r e s u l t a n t curves of the semi-logarithmic plot of such d a t a were projected to the ordinate to estimate t a r g e t n u m b e r s ( - - - ) .
Biochim. Biophys. Acta, 228 (1971) 57-66
62
C.O. DOUDNEY
the log phase culture only a slight a m o u n t of inactivation was seen after an ultraviolet light dose of 20 sec (Fig. 2). W i t h increasing doses, increasing inactivation was seen so t h a t after 50 sec of exposure little syuthesis of D N A could be measuredin the 16o min postirradiation test period (Fig. 2). These findings agree essentially with those reported b y DOUDNEY AND YOUNG1. W i t h the amino acid-starved culture, a dose of 30 sec produced only very slight non-recoverable inactivation and 80 sec of ultraviolet light was required for almost complete suppression of D N A replication (Fig.3). The rates of D N A replication relative to the unirradiated controls were calculated for the various ultraviolet light doses (Fig. 4). It is evident t h a t the D N A synthetic system with the amino acid-starved bacteria is about twice as resistant to ultraviolet light inactivation as with the log phase bacteria. As can be seen from Fig. 4, the inactivation kinetics for the log phase culture suggest t h a t four targets must be hit for nonrecoverable inactivation of D N A replication while the inactivation kinetics for the amino acid-starved culture suggest eight tragets must be hit for such inactivation.
Comparison o~ D N A synthesis inactivation with lethal eHect It has been established t h a t amino acid starvation produces increased ultraviolet light resistance of certain strains of E. coliL In E. coli strain B/r WP2, a comparison has been made between inactivation of D N A replication and lethality which suggests t h a t the increased rate of lethality with dose represented b y the second part of the inactivation curve above the critical dose m a y be related to inactivation of D N A replication 1. In this study, the lethal effect of ultraviolet light has been compared, both for log phase bacteria and amino acid-starved bacteria, with the effect on D N A replication (Fig. 5). It is apparent t h a t the increase in ultraviolet light resistance with amino acid starvation is of comparable magnitude to the increase in resistance of the D N A replication system produced b y the same t r e a t m e n t (Fig. 4)1.0
916' .>_ t/~16 3
20
40
60
80
UV-exposure (sec)
Fig. 5- Relative survival after exposure to various ultraviolet light doses of log phase and tryptophan-starved cultures of E. coli strain B/r WP2. The bacteria were handled as described in Fig. I and survival determined by immediate plating before postirradiation incubation. The unirradiated log phase (+ Trp) cultures showed an average of 5.8" io 8 colony-forming organisms per ml before exposure and the tryptophan-starved (--Tip) cultures showed an average of 6. 4. IO8 colony-forming organisms per ml.
Biochim. Biophys. Acta, 228 (1971) 57~66
ULTRAVIOLET LIGHT RESISTANCE OF D N A
REPLICATION
63
Furthermore, the increases in rate of inactivation or "break" in the survival curves comes at about 20 sec of ultraviolet light for the log phase culture and 5 ° sec of ultraviolet light for the amino acid-starved culture, which is about the same dose level in both cases which produce measurable inactivation of DNA replication. The results suggest the possibility of a direct relationship between inactivation of DNA replication and the increased rate of lethal inactivation over that seen below the dose causing an effect on DNA replication under these two conditions.
DISCUSSION
It is sometimes difficult to know how to interpret multitarget data of the sort which appear in the case of the inactivation of DNA replication. With log phase cultures, in which at least one replication point presumably exists somewhere on the chromosomes of most bacteria, the target number for inactivation of DNA replication projects to 4 (Fig. 4). This implies that four separate damaging events must occur in order for the possibility of some restoration event to be eliminated. Since inactivating damage to the distal end of the chromosome past the replication point would presumably involve only two hits at closely linked sites on the complimentary strands 11, these data raise the possibility that damage which inactivates DNA replication is to the proximal end of the chromosome which is already replicated. This would not be too surprising since the work of BILLENTMsuggests that at dose levels of the magnitude showing inactivation of DNA replication the site of DNA replication active at the time of exposure is inactivated b y ultraviolet light and resumption of DNA replication is from a new site probably the fixed origin (see ref. 13 for a discussion of this point). Thus it could be presumed that the functional intactness of any one of 4 sites at or near the origin of the chromosome (presumably located on the 4 strands of DNA) is adequate to allow either recombination-repair or another restorative process involving excision 14 to take place which allows the establishment of new fixed origins of replication and the initiation of DNA replication. Somewhat more surprising is the apparent increase in target multiplicity from 4 to 8 in the case of the amino acid-starved culture. If the site involved in inactivation of DNA replication is actually at the chromosomal origin as we have suggested, then it is not clear why completion of the chromosome would increase the target number to 8 since it is presumed that the block in DNA replication resulting from inhibition of protein synthesis prevents DNA replication from the fixed origin. One would have to assume in order to explain these data on this basis that the block in DNA replication produced by the absence of the amino acid is not actually at the site of initiation of the new DNA replication cycle, but that at least a small portion of the chromosomes (containing presumably the new fixed origins and the ultraviolet light target sites) replicates even in the absence of a required amino acid. This would increase the number of DNA strands at the chromosomal origin from four to eight and thus could account for the ultraviolet light inactivation data obtained. We hesitate to interpret the inactivation of DNA replication b y ultraviolet light upon the hypothesis of " a t t e m p t e d DNA replication" as suggested for cell inactivation 15 and the increase in resistance of the DNA replication system with amino acid starvation to the removal of such possibility by completion of the chromoBiochim. Biophys. Acta, 228 (1971) 57-66
64
C.O. DOUDNEY
some 15. This is in view of the work of BILLEN1~ which suggests that with dose levels sufficient for non-recoverable DNA synthesis inactivation most semiconservative DNA replication which occurs is from the chromosomal origin a n y w a y and that the old site of DNA replication is inactivated. Furthermore, if the induction of a requirement for RNA and protein formation in reestablishment of the cell's capacity for DNA replication after ultraviolet light damage is equated to the inactivation of the old site of DNA replication and consequent necessity for new fixed origin formation for recovery of DNA synthesis (see ref. 13 for a discussion of this point) then it is evident that the dose ranges shown here to inactivate the capacity for DNA replication irreversibly are sufficient also to inactivate all or almost all active sites of semiconservative DNA replication on the chromosomes. Since with both log phase bacteria and amino acid-starved bacteria, new origin formation requiring a prolonged period of time before recovery of semi-conservative DNA replication would be necessary after ultraviolet light doses sufficient to induce non-recoverable damage to DNA replication, it would appear unreasonable that " a t t e m p t e d DNA replication" at an active site of DNA replication on the chromosome could play a role in nonrecoverable inactivation of DNA replication and it suggests that this inactivation must occur at the chromosomal origin as we have proposed. It is clear that considerable reservation must be made in an interpretation of these ultraviolet light data based on target inactivation. What is established, however, is that completion of the chromosome and the occurrance of those events involved with amino acid starvation somehow functions to increase the radiation resistance of the DNA synthetic system b y about 2-fold. It is worthy of note that the slope of the curve for inactivation of DNA replication is slightly less with the culture starved for the amino acid. This might imply a slight increase in resistance of the target area itself. However, this apparent increase in target resistance could be due to an unknown secondary factor, which developed during the amino acid starvation period. It is axiomatic in modern biology that when a cell has undergone non-lepairable damage to its DNA synthetic system it will die. The corollary m a y be expressed that should a cell sustain no other lethal damage at the dose which results in such irreversible damage to DNA synthesis then death results from inactivation of DNA replication. However, the hypothesis that inactivation of DNA replication is directly responsible for cell death in radiation-resistant bacteria has not been a popular one with radiobiologists, probably because (aside from the fact that most studies of DNA inactivation kinetics have proven unreliable in reflecting the actual pattern of DNA replication 4) most strains studied show considerable lethality at doses which have little effect on DNA replication. A case in point is E. coli strain TAU, a strain which has been widely used in studies of the sort described here 7. The lower energy level required for a comparable degree of cell inactivation compared to strain B/r and the "one target" nature of the survival curve 15 suggest another more sensitive lethal mechanism is responsible for most inactivation in this organism rather than inactivation of DNA replication. Our present results suggest the possibility that one cause of death of strain B/r above the critical dose is direct inactivation of DNA replication. It must be remembered that most E. coli cells in log phase growth possess two replicating chromosomes. Since this is the case, a departure of the cell inactivation kinetics from the Biochim. Biophys. Acta, 228 (197 I) 57-66
ULTRAVIOLET LIGHT RESISTANCE OF D N A REPLICATION
65
DNA replication kinetics might be expected. Presumably inactivation of DNA replication requires only the inactivation of the individual chromosome. Cell death would presumably require inactivation of both chromosomes and would become quantitatively apparent only after enough damage to DNA has occurred that the probability of inactivation of both chromosomes is significantly high. It is evident from these results that the "break" or increase in rate of cell inactivation with dose of the survival curves comes after about 20 sec of ultraviolet light for the log phase culture and after about 50 sec of ultraviolet light for the amino acid-starved culture. These are doses after which some DNA synthesis has been inactivated as indicated by the decrease in rates of DNA replication observed. Thus it appears plausible that direct inactivation of DNA replication could account (at least, in part) for the increased rates of cell inactivation seen above these dose levels. Damage to the chromosome after which considerable DNA synthesis can still take place 4,5,1e might also be ot a lethal nature if the chromosome is no longer viable after such damage, accounting for part of the cell inactivation observed. In fact, with the lower doses below the dose showing the increased rate of inactivation it could well be that this type of damage accounts in large part for cell death since over half the bacteria die before measurable damage to DNA replication is observed. This kind of damage could in fact account for the slight decrease in rate of DNA synthesis seen in some cases after 120 min of incubation in these experiments, in line with the suggestions of BILLEN AND BRUNSx6. While this type of damage complicates our understanding of the basic nature of cell inactivation, it is evident that existance of the two types of damage could explain the biphasic nature of the B/r inactivation curve. Thus below the dose showing the increased rate ot lethality damage which does not inactivate DNA replication (at least, immediately) could be at least partly responsible for cell inactivation. At the critical dose measurable inactivation of DNA replication begins and with increased doses producing the increased rate of lethality the total lethality m a y be due to the additive effect of both types of damage to DNA. I t is apparent that above the dose level where inactivation of DNA replication begins the relative rates of cell inactivation exceed somewhat the rates of inactivation of DNA replication both with the log phase and amino acidstarved bacteria, supporting the plausibility of the concept that the rate of cell inactivation above the critical dose depends on the addition of that portion of the cells which die from damage to DNA replication to the portion already dying from causes which do not reflect direct damage to DNA replication. A possible type of lethal damage of this second type not inactivating DNA replication is damage to the complementary DNA strands at closely linked sites. NISHIOKA AND DOUDNEY 11 have suggested that this type of damage m a y be non-repairable when induced in the yet-unduplicated distal portion of the bacterial chromosome past the DNA replication fork but that such damage could be restored through recombination-repair in the proximal already-duplicated portion of tile chromosome. Since this type of damage would require only two targets (on each DNA strand of the duplex) it would be expected to occur at lower dose levels than damage inactivating DNA replication involving four targets. Damage to both strands of the chromosome in the duplex state in a localized area would be lethal since there would be no possibility of recombination-repair but when two duplexes exist recombination would be possible thereby increasing cell survival considerably 11. Thus some of the increase Biochim. Biophys. Acta, 228 (1971) 57-66
66
C.O. DOUDNEY
in resistance to lethality produced by completion of the DNA replication cycle in the absence of protein synthesis could be related to the bringing into the possibility of the recombination-repair of all the bacterial chromosome rather than only just part of it n. Since initiation of DNA replication after ultraviolet light damage is from the the chromosomal origin 1., there is no apparent reason why lethal damage of this sort to the distal of the chromosome would have an immediate effect on DNA replication. The fact that the rate of cell inactivation is decreased by amino acid starvation even at low dose levels below those producing inactivation of DNA replication supports this concept. Of course it should be kept in mind that any attempt to relate cell inactivation to immediate inactivation of DNA replication may be misleading since the possibility exists that unobserved recovery of the supposedly inactivated DNA could occur considerably later. Utilizing the density gradient analysis technique of MESELSON AND STAHL2, we have followed the fate of 15N-containing DNA in bacteria grown after ultraviolet light exposure in 14N-containing medium (Doudney, unpublished results). These studies establish that such DNA which does not replicate within 16o rain after exposure does not replicate in an 8 h period. These studies suggest but do not establish that the supposedly non-recoverable inactivation of DNA studied here is permanent in nature. ACKNOWLEDGEMENTS
The author acknowledges the valuable technical assistance of Miss Diana Ballard. He thanks Dr. David Ezekiel for valuable discussions and suggestions. This investigation was supported in part by U.S. Atomic Energy Commission contract AT (3o-1)-3893. REFERENCES I C. O. DOUDNEY AND C. S. YOUNG, Genetics, 47 (1962) 112.52 IV[. MESELSON AND F. W. STAHL, Proc. Natl. Acad. Sci. U.S., 44 (1958) 671. 3 C. O. DOUDNEY, Cellular Radiation Biology, Williams and Wilkins, Baltimore, Md., 1965, p. 12o-138. 4 K. C. SMITH AND 1V[. E. O'LEARY, Biochim. Biophys. Acta, 169 (1968) 43 o. 5 K. C. SMITH, Mutation Res., 8 (1969) 481. 6 C. O. DOUDNEY, Mutation Res., 3 (1966) 280. 7 P. C. H&NAWALT, in A. C. GIESE, Pkotophysiology, Vol. 4, Academic Press, New York, 1968, p. 2o3-251. 8 M. OGUR AND G. ROSEN, Arch. Biochem., 25 (195 o) 262. 9 K. BURTON, Biochem. J., 62 (1965) 315. IO P. C. HA.NAWALT, O. MAALOE, D. J. CUMMINGSAND M. SCHAECTER, J. Mol. Biol., 3 (1961) 156. i i H. NISHIOKA AND C. O. DOUDNEY, Mutation Res., 9 (197 o) 349. 12 D. BILLEN J. Bacteriol., 97 (1969) 1169. 13 C. O. DOUDNE¥, Current Topics in Microbiology and Immunology, 46 (I968) 116. 14 D. BILLEN AND L. H. BRUNS, J. Bacteriol., in the press. 15 P. C. HANAWALT, Photochem. Photobiol., 5 (1966) I. 16 D. BILLEN AND L. H. BRUNS, Biophys. J., in the press.
Biochim. Biophys. Acta, 228 (1971) 57-66
57
BBA 97679
I N C R E A S E D RESISTANCE OF DNA R E P L I C A T I O N TO U L T R A V I O L E T L I G H T DAMAGE IN AMINO ACID-STARVED B A C T E R I A
C. O. DOUDNEY Research Laboratories, Department o[ Genetics, Albert Einstein Medical Center, Philadelphia, Pa. .r9z4z IU.S.A.)
(Received August i7th, 197o)
SUMMARY
Incubation of a log-phase culture of a ultraviolet light-resistant, tryptophanrequiring strain of Escherichia coli (B/r WP2) for 4 ° min without t r y p t o p h a n allows completion of the DNA replication cycle. When this occurs before ultraviolet light exposure an increase in ultraviolet light resistance of the DNA replication system of 2-fold (in terms of target number) over the log phase culture is produced. The projection to the ordinate of the relative rate values for inhibition of DNA synthesis after a series of ultraviolet light doses gave a target number of 4 for the log phase culture and 8 for the amino acid-starved culture. A comparable increase in resistance to the lethal effect of ultraviolet light was also seen with the same preirradiation treatment. The data suggest the possibility that the same mechanism is responsible for both the decreased ultraviolet light lethality and decreased inactivation of DNA replication. Both are presumably produced b y completion of the DNA replication cycle during amino acid starvation, since lack of thymine with a thyminerequiring substrain prevents the development of resistance.
INTRODUCTION DOUDNEY AND YOUNG1 demonstrated that below a "critical" ultraviolet light dose little non-recoverable damage to DNA synthesis is induced in Escherichia coli strain B/r WP2 but that a prolonged dose-dependent lag is induced during which the DNA synthetic system recovers and synthesis finally resumes at the rate seen with unirradiated bacteria. Above the critical dose little further lag in resumption of synthesis is induced. Non-recoverable damage is induced however so that the rate of synthesis observed is decreased. Utilizing the density gradient technique of MESELSON AND STAI-IL~ for measurement of the distribution of DNA sub-units with DNA replication, DOUDNEY3 showed that the reduced rate of synthesis of DNA caused b y ultraviolet light exposure was due to inactivation of part of the DNA rather than production of a slowed rate of synthesis of all the DNA. Recently SMITH AND O'LEARY a and SMITH5 have demonstrated a comparable pattern of inactivation of DNA replication b y ultraviolet light utilizing a modified technique for measurement of DNA replication involving the incorporation of labeled thymine in thymine-requiring strains of E. coll. Biochim. Biophys. Acta, 228 (i971) 57-66
58
C . O . DOUDNEY
A comparison of the relative degree of non-recoverable inactivation of DNA replication in E. coli strain B/r WP2 with the lethal effect of ultraviolet light at various doses has been made *. The findings suggested the possibility that the increased inactivation rate for lethality seen with exposure above the critical dose is based on inactivation of DNA replication. These studies were subsequently extended to compare the degree of restitution of DNA replication produced by photoreversing light after various ultraviolet light doses with the increased survival also produced by the light s. In this study we have compared another method of increasing resistance of E. coli to ultraviolet light exposure with the change produced in resistance of the DNA synthetic system to ultraviolet light damage. A number of investigations have shown that, when certain strains of E. coli are permitted to complete their normal cycle of DNA replication in the absence of protein synthesis, considerably increased resistance to ultraviolet light inactivation is observed (consult a review by HANAWALT7 for a discussion of this effect). We have demonstrated an increase in radiation-resistance of the DNA replication system produced by amino acid starvation in E. coli strain B/r WP2. This increase is comparable to the increased survival produced b y the same preirradiation treatment of this strain.
MATERIALS AND METHODS
Organism E. coli strain B/r WP2 (tryptophan-requiring, hcr +) was used. This strain was kindly supplied to us by Dr. Evelyn Witkin some years ago. Culture growth procedures A small inoculum of bacteria was taken from a fresh, refrigerated nutrient agar slant and added to minimal medium supplemented with 4 °/zg/ml of L-tryptophan. The culture was grown in a large Erlenmeyer flask on a vigorously-rotating shaker at 37 ° until an absorbance of 0.25 was reached (measured at 660 m/~ with a Coleman model 6D spectrophotometer). The cells were then chilled in an ice bath in preparation for ultraviolet light exposure. Ultraviolet light exposure The chilled log-phase culture was spun down at io ooo rev./min in a Servall refrigerated centrifuge, resuspended in ice cold minimal medium (containing no tryptophan) to volume, spun down once more and finally resuspended to an absorbance of o.125 in the same cold minimal medium. Preirradiation amino acid starvation was accomplished by rapidly warming selected samples in a hot water bath to 37 ° and incubating for 40 min on the rotary shaker before again chilling in the ice bath. One hundred ml samples were then exposed in large, flat 25 cm × 16 cm pyrex dishes with magnetic stirring to the light from a General Electric 15 W germicidal lamp which emits most of its light around 2537 A. The cells were placed at a distance from the lamp which gave a dose rate below 2800/X, of about 12 ergs/mm ~ per sec as measured by a Hanovia model AV-97I ultraviolet light meter. The irradiated samples were collected in flasks held in an ice bath. Biochim. Biophys. Acta, 228 (1971) 57-66
ULTRAVIOLET LIGHT RESISTANCE OF DNA REPLICATION
59
Assay o/survival Small samples were taken from each flask and appropriately diluted in minimal medium, o.i ml of the final dilutant was then pipetted on the surface of Difco nutrient agar plates and spead evenly with a glass spreader. All platings were in triplicate. Incubation was for three days at 37 ° . Survival was scored at dilutions which result in IOO to 300 colonies per plate.
Postirradiation incubation L-Tryptophan was added to all flasks to a concentration of 4o #g/ml. The flasks were then rapidly warmed to 37 ° in a hot water bath and placed on a rotary shaker at 37 °. Ten ml samples were then taken at timed intervals and held in an ice bath for subsequent determination of DNA content.
Determination o/DNA The method of extraction of DNA was that of OGUR AND ROSEN8. The cells in the IO ml samples were spun out of suspension at IO ooo rev./min for 20 rain in a refrigerated centrifuge. The resulting pellet was then extracted for cold-acid-soluble material b y resuspension in ice cold 0. 5 M HC10, for 30 min. The pellet containing the nucleic acid obtained by centrifugation at IO ooo rev./min for 30 min after the above treatment was then resuspended in HC10, and heated for 50 rain at 7 °0 to hydrolyse the DNA. Analysis for DNA was b y the BtmTON 9 diphenylamine reaction with overnight development of color in the dark at room temperature.Color was measured at 61o m/~ by the Zeiss spectrophotometer with correction for the absorption at 650 m#. Salmon sperm DNA was used as the standard.
Accuracy o/the method We have adopted the above-described method for DNA determination in the past in numerous studies as being appropriate for determination of DNA in non-thymine requiring bacteria where relatively large changes in amount are measured. Recently SMITH AND O'LEAR'x"t have plesented evidence which suggests that pitfalls exist in the methods of measuring DNA synthesis kinetics b y the commonly adopted method of incorporation of labeled thymine and proposed new protocols for the determination of DNA replication b y thymine incorporation. Using the new techniques, they confirmed some of our earlier findings utilizing the chemical method described above*, 5. We have recently compared DNA replication measured by the Burton diphenylamine reaction as described above and by a modification of the SMITH AND O'LEARY technique. This involved incorporation of labeled thymine in a thymine-requiring strain of B/r in which the determination of relative amount of DNA increase was based on the amount of thymine incorporation after ultraviolet light exposure as compared to that incorporated with preirradiation growth from a very small inoculum. In these studies (to be published elsewhere) the relative DNA increase as determined b y the Burton method and b y the thymine incorporation method agreed closely. We therefore can conclude that the chemical determination of DNA utilized in these studies is accurate enough for our purposes in these experiments. All chemical data represent the averaged results of six identical experiments. The survival data represent three identical experiments. All operations were carried Biochim. Biophys. Acta, 228 (i97 I) 57-66
60
c . o . DOUDNEY
out under yellow room light from General Electric "gold" fluorescent bulbs to prevent photoreactivation from the room light source.
RESULTS
E//ect o/ D N A replication cycle completion on sensitivity to ultraviolet light damage When an unirradiated subculture of the log phase culture of strain WP2 was warmed to 37 ° after holding in an ice bath during the period necessary ior ultraviolet light exposure (i.e. about 15 rain), DNA synthesis started immediately (Fig. I). When the subculture had been previously starved for t r y p t o p h a n for 4 ° min and was similarly warmed up, a delay of 3o-4o min in initiation of DNA replication was observed. This lag in resumption of DNA replication is typical of bacteria which have completed their DNA replication cycle in the absence of protein s y , thesis, as has been demonstrated by the studies of HANAWALT et al3 °. That DNA replication actually ceased in the absence of t r y p t o p h a n in 4 ° rain is demonstrated in Table I.
TABLE I T H E F O R M A T I O N OF D N A TOPHAN
BY
E. coli
STRAIN B#" we2
IN T H E P R E S E N C E A N D A B S E N C E O F T R Y P -
Unity = 3.3/*g of DNA per ml of culture. Incubation time (rain):
+ Tryptophan --Tryptophan
zo
2o
3°
4°
5°
6o
1.16 1.14
1.28 1.22
1.42 1.31
1.6o 1.43
1.87 1.41
2.4 1.39
When the log phase culture was exposed to an ultraviolet light dose of 3o sec a marked lag in DNA replication was induced (about 60 min) and the rate of synthesis when replicatior~ resumed was greatly reduced (Fig. I). However, when the amino acid-starved culture was exposed to an identical ultraviolet light dose, the amino acid starvation-caused lag in DNA replication of 30 min was increased by only about I0 min (for a total lag of 40 min). Little or no inactivation of DNA, as measured by rate of replication, was observed. We can thus conclude that the DNA replication system is considerably more resistant to ultraviolet light when exposed at the end of the replication cycle than when replication is actually occurring. That completion of the DNA replication cycle in the absence of t r y p t o p h a n was responsible for the increased resistance of the DNA synthetic system rather than some other effect of amino acid starvation was suggested by studies with a thyminerequiring strain in which thymine was ommitted from the medium during amino acid starvation. In this case the expected increase in ultraviolet light resistance of the DNA synthetic system did not develop. Kinetics o/inactivation o / D N A replication The relative efficiency of increasing ultraviolet light doses in inactivation of DNA replication in log phase and amino acid starved-bacteria was compared.With Biochirn. Biophys. Acta, 228 (1971) 57-66
ULTRAVIOLET LIGHT RESISTANCE OF DNA REPLICATION
2.5
C,+Trp
7"C,- T r l ~ y
u,,j/
2.5 UV,
<
61
< z
2.0
(3 2.c
O
o
~ 1.5
~ 1.5
1.0
~ 10
•
4'0 r:;o go ~bo 1~o 44o 460
26 4b 6b ~o 16o ~o 740 ;60
Postirradiation incubation (min)
Postirradiation incubation (rnin)
Fig. I. The effect of preirradiation t r y p t o p h a n s t a r v a t i o n on the ultraviolet light sensitivity of postirradiation D N A replication with E. coli strain B/r WP2. Before ultraviolet light exposure one subculture was incubated for 4 ° min w i t h o u t t r y p t o p h a n ( - - T r p ) at 37 ° while a n o t h e r was held in an ice b a t h in log phase ( + T r p ) . One half of each subculture was exposed to ultraviolet light (UV) (3o'sec) while the other half was held in the dark (C). All four subcultures were t h e n w a r m e d rapidly to 37 ° and samples taken for analysis of D N A c o n t e n t every Io min for I6o min. The D N A increase is expressed relative to t h a t a m o u n t present at the time of ultraviolet light exposure (unity = 3.3/*g/ml for the log phase culture and 4.6 p g / m l for the a m i n o acid-starved culture). Fig. 2. The effect of increasing ultraviolet light doses on rate of D N A replication with a log p h a s e culture of E. coli strain B/r \VP2. The e x p e r i m e n t was conducted as described in Fig. I. Various subcultures were exposed to the indicated doses of ultraviolet light and samples taken with postirradiation incubation. U n i t y = 3-3/zg of D N A per ml of culture.
10
2.5 < z o 2.0 E
UV: 3
o//
'~.
!,,,),,,,,,,
o 1.0
g
~ 1.0
4b io ~o ~6o ~o ,~o ~o Postirradiation incubation (rnin)
i
I
i
i
•
I
i
T
40 60 80 UV-exposure (see)
Fig. 3- The effect of increasing ultraviolet light doses on rate of D N A replication of a t r y p t o p h a n s t a r v e d culture of E. coli strain B/r W P 2 . The e x p e r i m e n t was conducted as described in Fig. I. Various subcultures were exposed to the indicated doses of ultraviolet light and samples t a k e n with postirradiation incubation. U n i t y = 4.6/~g of D N A per ml of culture. Fig. 4. The relative rate of D N A replication w i t h ultraviolet light exposed bacteria as c o m p a r e d to the unirradiated controls. The slope of the rate curves for D N A replication after the v a r i o u s ultraviolet light doses (Figs. 2 and 3) was determined graphically and expressed relative to t h e slope of the unirradiated controls (Fig. I ), I n each case the relatively c o n s t a n t rate of D N A replication represented b y s t r a i g h t lines between 60 and i2o min of incubation was utilized to e s t i m a t e slope for the irradiated samples and c o m p a r e d to the initial slope represented b y a s t r a i g h t line plot of the unirradiated controls. The r e s u l t a n t curves of the semi-logarithmic plot of such d a t a were projected to the ordinate to estimate t a r g e t n u m b e r s ( - - - ) .
Biochim. Biophys. Acta, 228 (1971) 57-66
62
C.O. DOUDNEY
the log phase culture only a slight a m o u n t of inactivation was seen after an ultraviolet light dose of 20 sec (Fig. 2). W i t h increasing doses, increasing inactivation was seen so t h a t after 50 sec of exposure little syuthesis of D N A could be measuredin the 16o min postirradiation test period (Fig. 2). These findings agree essentially with those reported b y DOUDNEY AND YOUNG1. W i t h the amino acid-starved culture, a dose of 30 sec produced only very slight non-recoverable inactivation and 80 sec of ultraviolet light was required for almost complete suppression of D N A replication (Fig.3). The rates of D N A replication relative to the unirradiated controls were calculated for the various ultraviolet light doses (Fig. 4). It is evident t h a t the D N A synthetic system with the amino acid-starved bacteria is about twice as resistant to ultraviolet light inactivation as with the log phase bacteria. As can be seen from Fig. 4, the inactivation kinetics for the log phase culture suggest t h a t four targets must be hit for nonrecoverable inactivation of D N A replication while the inactivation kinetics for the amino acid-starved culture suggest eight tragets must be hit for such inactivation.
Comparison o~ D N A synthesis inactivation with lethal eHect It has been established t h a t amino acid starvation produces increased ultraviolet light resistance of certain strains of E. coliL In E. coli strain B/r WP2, a comparison has been made between inactivation of D N A replication and lethality which suggests t h a t the increased rate of lethality with dose represented b y the second part of the inactivation curve above the critical dose m a y be related to inactivation of D N A replication 1. In this study, the lethal effect of ultraviolet light has been compared, both for log phase bacteria and amino acid-starved bacteria, with the effect on D N A replication (Fig. 5). It is apparent t h a t the increase in ultraviolet light resistance with amino acid starvation is of comparable magnitude to the increase in resistance of the D N A replication system produced b y the same t r e a t m e n t (Fig. 4)1.0
916' .>_ t/~16 3
20
40
60
80
UV-exposure (sec)
Fig. 5- Relative survival after exposure to various ultraviolet light doses of log phase and tryptophan-starved cultures of E. coli strain B/r WP2. The bacteria were handled as described in Fig. I and survival determined by immediate plating before postirradiation incubation. The unirradiated log phase (+ Trp) cultures showed an average of 5.8" io 8 colony-forming organisms per ml before exposure and the tryptophan-starved (--Tip) cultures showed an average of 6. 4. IO8 colony-forming organisms per ml.
Biochim. Biophys. Acta, 228 (1971) 57~66
ULTRAVIOLET LIGHT RESISTANCE OF D N A
REPLICATION
63
Furthermore, the increases in rate of inactivation or "break" in the survival curves comes at about 20 sec of ultraviolet light for the log phase culture and 5 ° sec of ultraviolet light for the amino acid-starved culture, which is about the same dose level in both cases which produce measurable inactivation of DNA replication. The results suggest the possibility of a direct relationship between inactivation of DNA replication and the increased rate of lethal inactivation over that seen below the dose causing an effect on DNA replication under these two conditions.
DISCUSSION
It is sometimes difficult to know how to interpret multitarget data of the sort which appear in the case of the inactivation of DNA replication. With log phase cultures, in which at least one replication point presumably exists somewhere on the chromosomes of most bacteria, the target number for inactivation of DNA replication projects to 4 (Fig. 4). This implies that four separate damaging events must occur in order for the possibility of some restoration event to be eliminated. Since inactivating damage to the distal end of the chromosome past the replication point would presumably involve only two hits at closely linked sites on the complimentary strands 11, these data raise the possibility that damage which inactivates DNA replication is to the proximal end of the chromosome which is already replicated. This would not be too surprising since the work of BILLENTMsuggests that at dose levels of the magnitude showing inactivation of DNA replication the site of DNA replication active at the time of exposure is inactivated b y ultraviolet light and resumption of DNA replication is from a new site probably the fixed origin (see ref. 13 for a discussion of this point). Thus it could be presumed that the functional intactness of any one of 4 sites at or near the origin of the chromosome (presumably located on the 4 strands of DNA) is adequate to allow either recombination-repair or another restorative process involving excision 14 to take place which allows the establishment of new fixed origins of replication and the initiation of DNA replication. Somewhat more surprising is the apparent increase in target multiplicity from 4 to 8 in the case of the amino acid-starved culture. If the site involved in inactivation of DNA replication is actually at the chromosomal origin as we have suggested, then it is not clear why completion of the chromosome would increase the target number to 8 since it is presumed that the block in DNA replication resulting from inhibition of protein synthesis prevents DNA replication from the fixed origin. One would have to assume in order to explain these data on this basis that the block in DNA replication produced by the absence of the amino acid is not actually at the site of initiation of the new DNA replication cycle, but that at least a small portion of the chromosomes (containing presumably the new fixed origins and the ultraviolet light target sites) replicates even in the absence of a required amino acid. This would increase the number of DNA strands at the chromosomal origin from four to eight and thus could account for the ultraviolet light inactivation data obtained. We hesitate to interpret the inactivation of DNA replication b y ultraviolet light upon the hypothesis of " a t t e m p t e d DNA replication" as suggested for cell inactivation 15 and the increase in resistance of the DNA replication system with amino acid starvation to the removal of such possibility by completion of the chromoBiochim. Biophys. Acta, 228 (1971) 57-66
64
C.O. DOUDNEY
some 15. This is in view of the work of BILLEN1~ which suggests that with dose levels sufficient for non-recoverable DNA synthesis inactivation most semiconservative DNA replication which occurs is from the chromosomal origin a n y w a y and that the old site of DNA replication is inactivated. Furthermore, if the induction of a requirement for RNA and protein formation in reestablishment of the cell's capacity for DNA replication after ultraviolet light damage is equated to the inactivation of the old site of DNA replication and consequent necessity for new fixed origin formation for recovery of DNA synthesis (see ref. 13 for a discussion of this point) then it is evident that the dose ranges shown here to inactivate the capacity for DNA replication irreversibly are sufficient also to inactivate all or almost all active sites of semiconservative DNA replication on the chromosomes. Since with both log phase bacteria and amino acid-starved bacteria, new origin formation requiring a prolonged period of time before recovery of semi-conservative DNA replication would be necessary after ultraviolet light doses sufficient to induce non-recoverable damage to DNA replication, it would appear unreasonable that " a t t e m p t e d DNA replication" at an active site of DNA replication on the chromosome could play a role in nonrecoverable inactivation of DNA replication and it suggests that this inactivation must occur at the chromosomal origin as we have proposed. It is clear that considerable reservation must be made in an interpretation of these ultraviolet light data based on target inactivation. What is established, however, is that completion of the chromosome and the occurrance of those events involved with amino acid starvation somehow functions to increase the radiation resistance of the DNA synthetic system b y about 2-fold. It is worthy of note that the slope of the curve for inactivation of DNA replication is slightly less with the culture starved for the amino acid. This might imply a slight increase in resistance of the target area itself. However, this apparent increase in target resistance could be due to an unknown secondary factor, which developed during the amino acid starvation period. It is axiomatic in modern biology that when a cell has undergone non-lepairable damage to its DNA synthetic system it will die. The corollary m a y be expressed that should a cell sustain no other lethal damage at the dose which results in such irreversible damage to DNA synthesis then death results from inactivation of DNA replication. However, the hypothesis that inactivation of DNA replication is directly responsible for cell death in radiation-resistant bacteria has not been a popular one with radiobiologists, probably because (aside from the fact that most studies of DNA inactivation kinetics have proven unreliable in reflecting the actual pattern of DNA replication 4) most strains studied show considerable lethality at doses which have little effect on DNA replication. A case in point is E. coli strain TAU, a strain which has been widely used in studies of the sort described here 7. The lower energy level required for a comparable degree of cell inactivation compared to strain B/r and the "one target" nature of the survival curve 15 suggest another more sensitive lethal mechanism is responsible for most inactivation in this organism rather than inactivation of DNA replication. Our present results suggest the possibility that one cause of death of strain B/r above the critical dose is direct inactivation of DNA replication. It must be remembered that most E. coli cells in log phase growth possess two replicating chromosomes. Since this is the case, a departure of the cell inactivation kinetics from the Biochim. Biophys. Acta, 228 (197 I) 57-66
ULTRAVIOLET LIGHT RESISTANCE OF D N A REPLICATION
65
DNA replication kinetics might be expected. Presumably inactivation of DNA replication requires only the inactivation of the individual chromosome. Cell death would presumably require inactivation of both chromosomes and would become quantitatively apparent only after enough damage to DNA has occurred that the probability of inactivation of both chromosomes is significantly high. It is evident from these results that the "break" or increase in rate of cell inactivation with dose of the survival curves comes after about 20 sec of ultraviolet light for the log phase culture and after about 50 sec of ultraviolet light for the amino acid-starved culture. These are doses after which some DNA synthesis has been inactivated as indicated by the decrease in rates of DNA replication observed. Thus it appears plausible that direct inactivation of DNA replication could account (at least, in part) for the increased rates of cell inactivation seen above these dose levels. Damage to the chromosome after which considerable DNA synthesis can still take place 4,5,1e might also be ot a lethal nature if the chromosome is no longer viable after such damage, accounting for part of the cell inactivation observed. In fact, with the lower doses below the dose showing the increased rate of inactivation it could well be that this type of damage accounts in large part for cell death since over half the bacteria die before measurable damage to DNA replication is observed. This kind of damage could in fact account for the slight decrease in rate of DNA synthesis seen in some cases after 120 min of incubation in these experiments, in line with the suggestions of BILLEN AND BRUNSx6. While this type of damage complicates our understanding of the basic nature of cell inactivation, it is evident that existance of the two types of damage could explain the biphasic nature of the B/r inactivation curve. Thus below the dose showing the increased rate ot lethality damage which does not inactivate DNA replication (at least, immediately) could be at least partly responsible for cell inactivation. At the critical dose measurable inactivation of DNA replication begins and with increased doses producing the increased rate of lethality the total lethality m a y be due to the additive effect of both types of damage to DNA. I t is apparent that above the dose level where inactivation of DNA replication begins the relative rates of cell inactivation exceed somewhat the rates of inactivation of DNA replication both with the log phase and amino acidstarved bacteria, supporting the plausibility of the concept that the rate of cell inactivation above the critical dose depends on the addition of that portion of the cells which die from damage to DNA replication to the portion already dying from causes which do not reflect direct damage to DNA replication. A possible type of lethal damage of this second type not inactivating DNA replication is damage to the complementary DNA strands at closely linked sites. NISHIOKA AND DOUDNEY 11 have suggested that this type of damage m a y be non-repairable when induced in the yet-unduplicated distal portion of the bacterial chromosome past the DNA replication fork but that such damage could be restored through recombination-repair in the proximal already-duplicated portion of tile chromosome. Since this type of damage would require only two targets (on each DNA strand of the duplex) it would be expected to occur at lower dose levels than damage inactivating DNA replication involving four targets. Damage to both strands of the chromosome in the duplex state in a localized area would be lethal since there would be no possibility of recombination-repair but when two duplexes exist recombination would be possible thereby increasing cell survival considerably 11. Thus some of the increase Biochim. Biophys. Acta, 228 (1971) 57-66
66
C.O. DOUDNEY
in resistance to lethality produced by completion of the DNA replication cycle in the absence of protein synthesis could be related to the bringing into the possibility of the recombination-repair of all the bacterial chromosome rather than only just part of it n. Since initiation of DNA replication after ultraviolet light damage is from the the chromosomal origin 1., there is no apparent reason why lethal damage of this sort to the distal of the chromosome would have an immediate effect on DNA replication. The fact that the rate of cell inactivation is decreased by amino acid starvation even at low dose levels below those producing inactivation of DNA replication supports this concept. Of course it should be kept in mind that any attempt to relate cell inactivation to immediate inactivation of DNA replication may be misleading since the possibility exists that unobserved recovery of the supposedly inactivated DNA could occur considerably later. Utilizing the density gradient analysis technique of MESELSON AND STAHL2, we have followed the fate of 15N-containing DNA in bacteria grown after ultraviolet light exposure in 14N-containing medium (Doudney, unpublished results). These studies establish that such DNA which does not replicate within 16o rain after exposure does not replicate in an 8 h period. These studies suggest but do not establish that the supposedly non-recoverable inactivation of DNA studied here is permanent in nature. ACKNOWLEDGEMENTS
The author acknowledges the valuable technical assistance of Miss Diana Ballard. He thanks Dr. David Ezekiel for valuable discussions and suggestions. This investigation was supported in part by U.S. Atomic Energy Commission contract AT (3o-1)-3893. REFERENCES I C. O. DOUDNEY AND C. S. YOUNG, Genetics, 47 (1962) 112.52 IV[. MESELSON AND F. W. STAHL, Proc. Natl. Acad. Sci. U.S., 44 (1958) 671. 3 C. O. DOUDNEY, Cellular Radiation Biology, Williams and Wilkins, Baltimore, Md., 1965, p. 12o-138. 4 K. C. SMITH AND 1V[. E. O'LEARY, Biochim. Biophys. Acta, 169 (1968) 43 o. 5 K. C. SMITH, Mutation Res., 8 (1969) 481. 6 C. O. DOUDNEY, Mutation Res., 3 (1966) 280. 7 P. C. H&NAWALT, in A. C. GIESE, Pkotophysiology, Vol. 4, Academic Press, New York, 1968, p. 2o3-251. 8 M. OGUR AND G. ROSEN, Arch. Biochem., 25 (195 o) 262. 9 K. BURTON, Biochem. J., 62 (1965) 315. IO P. C. HA.NAWALT, O. MAALOE, D. J. CUMMINGSAND M. SCHAECTER, J. Mol. Biol., 3 (1961) 156. i i H. NISHIOKA AND C. O. DOUDNEY, Mutation Res., 9 (197 o) 349. 12 D. BILLEN J. Bacteriol., 97 (1969) 1169. 13 C. O. DOUDNE¥, Current Topics in Microbiology and Immunology, 46 (I968) 116. 14 D. BILLEN AND L. H. BRUNS, J. Bacteriol., in the press. 15 P. C. HANAWALT, Photochem. Photobiol., 5 (1966) I. 16 D. BILLEN AND L. H. BRUNS, Biophys. J., in the press.
Biochim. Biophys. Acta, 228 (1971) 57-66