Molec. gen. Genet. 155, 279-286 (I977) © by Springer-Verlag 1977
The Dependence of Postreplication Repair on uvrB in a recF Mutant of Escherichia coli K-12 Robert H. R o t h m a n 1'* and Alvin J. Clark z 1 Department of Genetics and 2 Department of Molecular Biology, University of California, Berkeley, California 94720, USA
Summary. Mutants carrying recF143 or recF144 show wild type levels of host cell reactivation of UV-irradiated 2vir and wild type rates of excision gap closure in repairing UV damage to their own D N A . The same mutants showed reduced rates of postreplication repair strand joining. When u v r A - r e c F - or uvrB - r e c F - strains are tested, postreplication repair strand joining is incomplete or does not occur at fluences above 1 J/m 2. We suggest that there may be a UvrAB and a RecF pathway of postreplication repair or that the repair functions controlled or determined by uvrA uvrB and by recF m a y be similar. An intermediate in postreplication repair m a y accumulate in the u v r - r e c F - strain.
Introduction The recF143 mutation of E.coli K-12 leads to increased sensitivity to UV radiation and to an inhibition of postreplication repair ( R o t h m a n et al., 1975). Joining of D N A strands synthesized after exposure to 7.4 J/m 2 of UV radiation proceeded to completion but at a much slower rate than in an isogenic recF ÷ strain. These observations led us to suppose that another pathway of postreplication repair operated in the recF mutant. We had expected this pathway to be controlled by the recB and recC genes on three grounds: first recB and recC mutants are sensitive to UV radiation but show normal amounts of postreplication repair (Smith and Meun, 1970); second, the recB and recFmutations act synergistically to increase UV sensitivity to that observed in a recA mutant (Rothm a n et al., 1975; K a t o et al., 1977); and third, a recA * Present Address: Department of Biology, Brookhaven National Laboratory, Upton, L.I., New York 1I973 For offprints contact. Dr. A.J. Clark
mutation completely blocks postreplication repair (Smith and Meun, 1970). When tested, however, the recB recC recF triple mutant exhibited significant levels of fragment joining considered to be evidence of completed postreplication repair ( R o t h m a n et al., 1975). Later Ganesan and Seawell (1975) found that fragment joining in a recF143 strain was blocked by the addition of a uvrB mutation. Two explanations of this result were possible. First, the uvrB defect might be blocking postreplication repair. Secondly, a defect in another gene might be responsible for blocking postreplication repair. This second possibility is plausible since the uvrB mutation used was a deletion, which was detected by the chlorate resistance, biotin requirement it produces and which removes pgl, phr and probably att2 as well as chl and bio genes. In this report, we compare postreplication repair in recF143 strains carrying a uvrB301 deletion and a uvrB5 point mutation. We also further characterize the postreplication repair observed in a recF single mutant, and assess the role of recF in excision repair.
Methods and Materials Bacterial strains are listed in Table l. Techniques of UV irradiation, determination of UV sensitivity, and alkaline sucrose density gradient centrifugation have been described (Rothman and Clark, 1977).
Results Recovery after U V Irradiation The UV sensitivities for cells grown in and plated on complex medium are shown in Figure 1. recF143 caused an increase in sensitivity to radiation, but not as great as that caused by a uvrB mutation or by
280
R.H. R o t h m a n and A.J. Clark: Postreplication Repair in a recF M u t a n t
Table 1. Bacterial strains Sex
uvr
recF
reeL
Other m u t a n t alleles °
Basic genetic background b
Derivation or reference
AB2499 JC8908 JC8911 SR58
FF FF-
B5 B5 B5 B5
+ + 143 +
+ + + +
thyA18, thyR12 thyAt8, thyRi2 (?) thvA18 thyRl2 (?) recA56 thyR12
ABl157 ABl157 AB1 t57 ABl157
P. Howard-Flanders JC8566 x AB2499 Xyl + Met+[Sm R] transconjugant UV R JC9248 x AB2499 Xyl + Met+[Sm R] transconjugant UV s K.C. Smith
JC3890
F
B301
+
+
Kato et al., 1977
JC3893
F-
B301
143
+
deletion of phr, pgl, att2 ABl157 ehIA and/or chlD, all or part of bio operons same as for JC3890 ABl157
JC8403 JC9239 JC8931 JC8471
F F FF
+ + + +
+ 143 144 +
+ + + 152
ilv-332
AB 1157 ABl157 ABlI57 AB1157
Horii and Clark, 1973 Horli and Clark, 1973 JC8403 x P1 -JC7902 Ilv + transductant UV s Horii and Clark, 1973
JC3913 JC7902
FF
A6 +
143 144
+ +
uvrA6 reeB21 ree C22 sbcBl5
ABl157 AB 1 157
Kato et al., 1977 Horii and Clark, 1973
JC1664 JC1670 JC8904
FFF-
+ + +
+ 143 143
152 + 152
ilvD145
AB2277 AB2277 AB2277
AB2277 x P1-JC8471 Met + transductant UV s AB2277 x P1 -JC9239 Ilv + transductant UV s JC1670 × P1 .JC1664 Met + transductant UV s
JC8563 JC8566
Hfr Hfr
+ +
+ +
+ 152
ilv-331 metBI PO3 metBl PO3
P4X P4X
Horii and Clark, 1973 Horii and Clark, 1973 (no strain n u m b e r given)
JC9248
Hfr
+
143
+
metB1 PO3
P4X
JC8563 × P1-JC9239 Ilv + transductant U V s
Kato et al., 1977
Gene symbols and strain designations conform to the recommendations of Demerec et al., 1966, and the standard genetic m a p of E. coli (Bachmann, Low and Taylor, 1976). The following pheuotype abbreviations are used: Xyl, xylose; Met, methionine; Sm, streptomycin; UV, ultraviolet irradiation; Ilv, isoleucine and valine; + , independence when used with amino acid abbreviations, and utilizing when used with sugar abbreviations; - , dependence or uonutilizing, respectively; S, sensitive; R, resistant. Definition of PO3 is to be found in Table 2 of B a c h m a n n (1972) b Each strain has several genetic markers derived from its ultimate ancestor, unless otherwise noted in the derivation. The descendents of ABl157 inherit argE3, his-4, Ieu-6, proA2, thr-1, thi-t, str-31, galK2, lacYt, xyl-5, mtl-1, ara-14, tsx-33, supE44. Unless otherwise noted, descendents of AB2277 inherit arg-4, ilvD145, metE46, proA2, trp-3, thi-t, str-8 or str-9, gaIK2, lacYt or lacZ4, rnalA1, mtl-1 and supE44
a recL152 mutation (Rothman and Clark, 1977). The uvrB301 deletion mutant was as sensitive as the uvrB5 point mutant but the uvrB301 recF143 double mutant was somewhat more sensitive than the uvrB5 recF143 double mutant. In the uvrB5 background the reeF143 mutant was more sensitive than the recL152 mutant (Rothman and Clark, 1977). A recF143 recL152 double mutant was as sensitive as the recL152 single mutant determined previously. Two other alleles of recF, in addition to recF143, were obtained by Horii and Clark (1973). We have transduced one of these, recF144, out of the mutagenized primary mutant background. The survival curve of the recF144 single mutant is included in Figure 1. It was essentially the same as the curve for the recF143 single mutant. The sensitivities of the strain in Figure 1 were also determined when cells grown in complex medium were plated on minimal medium. Contrary to recL152, neither recF143 nor recF144 led to significant levels of shift-down recovery (data not shown, but see Rothman and Clark, 1977).
Post-Irradiation DNA Synthesis and Degradation
.
The effects of recF143 on post-irradiation D N A .synthesis and degradation were measured (Fig. 2). Log phase cultures of the reeF143 mutant and a wild type control were grown in the presence of [3H]-thymidine, washed and irradiated with 7.5 J/m 2 of UV light. Half of the culture was grown in the continued presence of [~H]-thymidine to measure post-irradiation synthesis, and half was grown in non-radioactive thymidine to measure post-irradiation degradation..Irradiation had little effect on incorporation of l a b e l in the wild-type control (Rothman and Clark, 1977) .... but drastically reduced incorporation in the recF!43 mutant (Fig. 2). The cessation of incorporation in. the unirradiated controls is due to the passage of cells from log phase to stationary phase since t h e cells were not diluted at any time during tl~e course of the experiment. D N A degradation was not enchanced by irradiation in the wild type strain (Rothman and Clark, 1977), and was slightly enchanced in the recF mutant (Fig. 2). This behavior contrasts
R.H. R o t h m a n and A.J. Clark: Postreplication Repair in a r e c F M u t a n t
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Fig. I. Survival after UV irradiation. Cells grown to log-phase in Luria broth were washed, resuspended in 56/2 buffer, and irradiated. Following irradiation the cells were diluted and plated on Luria broth agar. Symbols: o uvr + rec+ ; [] u v r B + r e c F 1 4 3 ; o u v r B + r e c F 1 4 4 + ; • r e e F 1 4 3 r e c L 1 5 2 ; z~ u v r B 5 recF+ ; • u v r B 3 0 1 recF + ; v uvrB5 reeF143; • uvrB301 recF143
l• s
0
40
_
,,
0.8 Relafive
0.6 Distance
0.4
Q2
0
Sedimenfed
Fig. 3. Effect of UV fluence on the sedimentation rate of newly synthesized D N A in a r e c F 1 4 3 mutant. Cells grown to log-phase in KB medium were washed and resuspended in M-9 buffer. Immediately following irradiation the cells were allowed to incubate in KB medium containing 100 gCi/ml [3H]-thymidine for 10 rain. The cells were then harvested by filtration, washed, and converted to spheroplasts and layered on the gradient. Symbols: • 0 J/m 2 ( 1 0 0 % = 1 4 8 1 0 c p m ) ; © 2 . 5 J / m ~ (100% 9190cpm); [] 10J/m 2 (100%=2580cpm); ± 20J/m 2 (100%=1520cpm); v 30J/m 2 (100% = 1340 cpm). The peak fractions for [l~C]-labeled T4 and 2 D N A are indicated on the abcissa
1Oil
E
0.. 10 4
-
10 3
_ I
I I 2 .5 4 Hours Fig. 2. Post-irradiation D N A synthesis and degradation by a r e c F 1 4 3 mutant. Cells were grown in KB medium containing 10 pCi/ml [3H]-thymidine and 250 ~tg/ml deoxyadenosine. Label incorporation during this period is symbolized by open circles (o) At the time indicated by the arrow, the culture was washed and resuspended in M-9 buffer and divided in half. One half was left unirradiated and the other exposed to 7.5 J/m 2 of UV radiation. One half of each of the unirradiated and irradiated p o m o n s were allowed to grow in radioactively labeled KB medium (o and 0, respectively). The other halves were allowed to grow in non-radioactive m e d i u m ([] and I , respectively). At the time indicated, 0.1 ml aliquots were withdrawn and spotted on a filter paper disc. These were dried, washed in T C A and counted for radioactivity
O
I
strikingly with the behavior of the recL152 mutant in which there is no inhibition of synthesis and no degradation as a result of irradiation (Rothman and Clark, 1977). In light of this UV-produced inhibition of net D N A synthesis in the recFmutant, we were concerned that this strain might not incorporate enough label in a 10 rain post-irradiation labeling period to detect postreplication repair. In Figure 3, however, it can be seen that label taken up after irradiation can be resolved by alkaline sucrose density gradient centrifugation into two peaks and that the mode of the low molecular weight peak is inversely dependent upon the fluence. Label incorporated by unlabeled cells after irradiation remained acid insoluble for periods up to two hours.
Postreplication Repair Joining of DNA fragments synthesized after irradiation with 10 J/m 2 was measured (Fig. 4). Label incor-
282
R.H. Rothman and A.J. Clark: Postreplication Repair in a recF Mutant Table 2. Balance sheet of label incorporated during postreplication repair experiments (Figs. 4, 5)
4000
300
Experiment 2000
200
Label remaining acid insol,
100
cpm
0
3,580 3,600 3,480 3,810 3,870
100 101 97 106 108
98 91 93 104 103
Fig. 5
0 min 30 min 50 rain 70 min 90 rain
16,600 14,700 16,100 10,300 12,700
100 88 97 62 76
92 100 101 97 71
300 I:: In
2oo
200
U
I00
+,+
0
,
F
C
400
~ 2°°
~
0
'
0.9
Relative
400
L /X T z
0.5
I~'
0.1
0.9
This also equals the amount of label layered on the gradient
20o
0,5
0.1
Percent
0 min 30 min 50 min 70 min 90 min
400 oin
% cpm recovered from gradient
Fig. 4
E
E
Chase
0
DistanceSedimented
Fig. 4. Joining of DNA fragments synthesized by the recF143 mutant after exposure to 10 J/m a UV radiation. Cells were irradiated and labeled as in Figure 3. Following the l0 min pulse label, the cells were washed of radioactive medium, resuspended in 3 X the original volume of nonradioactive KB medium and allowed to incubate for 0 (B), 30 (C), 50 (C), 70 (E), and 90 (E) min before being converted to spheroplasts. Four 60 pl aliquots of spheroplasts were drawn after each chase. One of these was layered on a sucrose gradient and the remaining three were counted independently for acid insoluble radioactivity. These counts were averaged and equalled both the amount of label added to the gradient and the fraction of input label remaining acid insoluble at the end of the chase periods (see Table 2). Panel A is an unirradiated control labeled for 10 min
porated by the r e c F I 4 3 mutant during the 10 rain post-irradiation period was resolved into two peaks. The molecular weights at the modes were 2.8 x 10a and 1.4 x 107 and the peaks contained 19% and 81% of the labeled material respectively. Complete joining of all the fragments required 90 min, as compared to 25 and 70 rain for the rec + and r e c L 1 5 2 strains irradiated with the higher fluence of 15 J/m 2 (Rothman and Clark, 1977). All label made acid insoluble during the 10 min post-irradiation pulse remained insoluble during the experiments (Table 2). Strand joining by the r e c F mutant was characterized mainly by the appearance of increasing amounts of labeled material under the high molecular weight peak with a corresponding depletion of label from the low molecular weight peak. There was, however, a slight increase in the mode molecular weight of the slowly 5000
1600
4000 1200 3000 E 0_ o
2000
1000
I
0 1
0,8
0.6
I
0.4
Rel~3%ive Dis#ance Sedimenfed
0.2
0
Fig. 5. Joining of DNA fragments synthesized by the recF143 uvrB5 mutant after exposure to 1 Jim z UV radiation. The protocol was the same as that in Figure 4 and the economy of acid insoluble radioactivity in this experiment is also in Table 2. After being labeled, cells were allowed to grow in non-radioactive medium for 0 (u), 50 (A), and 90 (v) min. The unirradiated control (o) was normalized to be presented on the same scale as the irradiated samples. The counts/rain for the unirradiated control are found on the right hand ordinate
R.H. R o t h m a n and A.J, Clark: Postreplication Repair in a recF M u t a n t
sedimenting material with increasing lengths of time after irradiation. The recF144 mutant behaved identically to the recF143 mutant (data not shown). The uvrB5 recF143 strain was tested (Fig. 5) and yielded results similar to those for the uvrB301 recF143 strain described by Ganesan and Seawell (1975). Following irradiation with 1 J/m z, the newly synthesized DNA was found mainly in a low molecular weight peak with a mode molecular weight of 2.5 × 10 7. By the end of 30 min the mode had shifted to 4.3 × 107, and by the end of 50 rain it had reached a mode of 5.5 x 107. The two-fold increase in mode molecular weight was accompanied by no more than 10% loss of acid insoluble radioactivity (Table 2), and by an absolute increase in the amount of rapidly sedimenting radioactive material. We therefore, feel that the increase in the mode molecular weight which we have observed in this strain reflects the accumulation of an intermediate in postreplication repair. Little further change in the mode molecular weight of the more slowly sedimenting material was observed during the remaining 40 rain of incubation in non-radioactive medium. There was, however, continued de-
283
3000 2000 I000 j 0
I
I
I
I
I
B. rec LI52 oE e_ 2000 1000 0 800
•_ _ ~
6O0 4O0 2O0 0
0.8
O.6
Rela#ive Disfance
O.4
O.2
0
Sedimenfed
Fig. 7. Formation of incision breaks during the first five min after irradiation. Cells were grown to log-phase in KB m e d i u m containing 10 pCi/ml [3H]-thymidine, washed and resuspended in 5 mls of M-9 buffer. The cells were irradiated with 10 (n), 20 (zx), and 30 (v) J/m 2 and allowed to incubate in non-radioactive medium for five rain before conversion to spheroplasts and layering on the gradients. An unirradiated control (o) is incIuded for each strain. The panels are identified in the figure
10 °
ld'
Id 2
/
m L
recFI43
gradation of DNA and loss of counts from all regions of the gradient. Identical results were obtained with a uvrA6 recF143 strain (data not shown).
L
m c
I()3
>
recFI44 /
>
UVFB5 uvr'B,301
Excision Repair
Td ~ -
-
uvcB5FecFI43
~6 5 dVFB5FecA56 uvr'B301 recFI43 16 6 I
0
I
I
I
I
10 20 UV FIuence(d/m 2)
I
30
Fig. 6. Host-cell reactivation of UV-irradiated 2vir. A stock culture of 2vir was diluted to 1 x 107 pfu/ml in 0.01 M MfSO 4 and irradiated. Following dilution, 0.1 ml aliquots of phage suspension was added to 0.2 ml aliquots of host bacteria and allowed to stand for 20 min to permit adsorption. The phage-host complex was spread on L C T G plates with soft agar. Symbols: o recF+ uvrB + ; [] recF143 uvrB+ ; o recF144 uvrB+ ; A recF+ uvrB5; • r e e f + uvrB301; ~7 recF143 uvrB5; • reeF143 uvrB301; • r e e f 5 6 uvrB5
The possibility of a role for recF in excision repair was tested by examining host-cell reactivation ability (HCR) and the formation and closure of incision breaks (Fig. 6). The recF143 and recF144 strains promoted the recovery of irradiated 2vir as well as did the wild type control. Surprisingly, however, the recF143 uvrB5 double mutant showed less 2vir recovery than did the uvrB5 single mutant. 2vir survival in the double mutant was as low as that observed in a uvrB5 recA56 double mutant. The uvrB30l strains behaved like the corresponding uvrB5 strains. The efficiency of incision break closure was measured by determining the fluence required to saturate the excision repair system with dimers and lead to maximal accumulation of breaks. Since excision is
284
R.H. Rothman and A.J, Clark: Postreplication Repair in a recF Mutant
rapid, most breaks will be closed within five minutes after irradiation with low fluences in excision proficient cells. Thus, more than 30 J/m 2 were required to accumulate breaks in the rec ÷ control (Fig. 7A), whereas only 10 J/m 2 led to a similar accumulation in the recL152 mutant (Fig. 7B). The recF mutant accumulated breaks with a fluence dependency similar t o that of the recF ÷ control (Fig. 7C). By these criteria, we conclude that recF does not play an important role in excision repair.
Discussion
A mutation recF143 led to greatly reduced levels of post-UV-irradiation D N A synthesis as compared to the wild type. The small amount of synthesis which did occur resulted in strands of reduced molecular weight as did the synthesis in the wild type. Furthermore, the size of the strands synthesized varied inversely as the fluence applied which is also characteristic of the wild type (Rupp and Howard-Flanders, 1968). It is unclear, therefore, whether a small subpopulation of recF143 cells is undergoing normal amounts of post-UV-irradiation D N A synthesis or whether the whole population is undergoing reduced amounts of synthesis. Regardless, the time for the conversion of these small molecular weight strands into larger molecular weight fragments was longer in the recF143 strain than in the wild type (see Fig, 6; Rothman and Clark, 1977). This seemed to be the main difference between the repair in the wild type and recF mutant. The same effect was seen with a recF144 mutant (data not shown). Sedliakova et al. (1975) have reported uvrB mutations to have an effect on postreplication repair. In confirmation we found that the slow joining of postUV-irradiation labeled D N A strands in the recF mutant was blocked by deletion or point mutations of uvrB and point mutations in uvrA. Complete blockage occurred after irradiation with 2 J/m 2 (T. Kato, personal communication) or 4 J/m 2 (data not shown) while at 0.5 J/m 2, no blockage was observed with a u v r A - recF- strain (T. Kato, personal communication). After irradiation with 1 J/m 2 (Fig. 5), incomplete blockage was observed. There was an increase in the relative amount of the more rapidly sedimenting D N A from 25% to 45% of the counts on the gradient. There was also a two-fold increase in the mode molecular weight of the more slowly sedimenting DNA. Both of these changes occurred within 50 min, after which no more repair appeared to occur and degradation, especially of the higher molecular weight strands, took place. The dependence of postreplication repair on uvrA
and uvrB in a recF mutant leads us to two possible conclusions: (1) there are at least two pathways of postreplication repair (a UvrAB and a RecF pathway); (2) uvrA and uvrB control or determine gene products which can substitute for the mutant recF product. The first possibility seems attractive in that at least three different types of postreplication repair have been proposed: (1) recombinational (Rupp and Howard-Flanders, 1968); (2) mutagenic (Witkin, 1975; Radman, 1975), and (3) excisional (Sedliakova et al., 1975; Bridges, 1977). Knowing that the recF mutation can block recombination in certain genetic backgrounds (Horii and Clark, 1973), and that uvrA and uvrB mutations block excision of pyrimidine dimers (Howard-Flanders et al., 1966), it is obvious to suggest that they block recombinational and excisional postreplication repair, respectively. That would leave mutagenic postreplication repair to explain the residual repair seen in the recF uvr double mutant. In support of this idea, UV-mutagenesis has been found to occur in recF uvrA and recF uvrB strains (Kato et al., 1977). The second possible conclusion is also attractive, however, because it allows us to explain a particular feature of our data on the u v r B - r e c F - strain. This feature is an approximately two-fold increase in the mode molecular weight of the slowly sedimenting labeled DNA. Such a shift in the mode molecular weight is also a feature of postreplication repair in the wild type (Fig. 6; Rothman and Clark, 1977) and recF single mutant (Fig. 4, this paper), but in these cases, the enlarged slowly sedimenting labeled material shifts to more rapidly sedimenting material and postreplication strand joining is completed. The second shift is rapid and slow in the wild type and recF mutant, respectively, and does not take place at all in the double mutant. This has led us to think that the two-fold change in mode molecular weight of the more slowly sedimenting material signals the formation of a particular intermediate in postreplication repair. The possible nature of this intermediate can be visualized by drawing the conventional model for recombinational postreplication repair (e. g. Ganesan, 1974) with three or more pyrimidine dimers on one parental strand (Fig. 8). If strand displacement, D-loop formation (synapsis) D-loop cleavage and ligation occur, then an intermediate is formed whose average length is two times the average pyrimidine dimer spacing on a single strand (Fig. 8). The subsequent elongation of this intermediate to complete postreplication repair strand joining would then be partially blocked in a recF mutant and totally blocked in the u v r B - r e c F - strain after exposure to fluences greater than 1 J/m 2. If there is only one route of such elongation, then the uvrA uvrB function might
R.H. R o t h m a n and A.J. Clark: Postreplication Repair in a recF M u t a n t
3'5 ' - - - i m i l u
J1
J/
II
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I
II
Synopsis
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I J-L
11
degradation Ligation
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II
II
7_-t --t -t -7111 / ...... pathetical
lP°lymerizati°n
intermediates~ Stranddisplacement ......
~
~
~
~
......
UTIII
111117 -~-mm|
mmi---
uvrA-recF
-
Fig. 8. An intermediate in postreplication repair m a y accumulate because of a hypothetical recF-uvrA- b l o c k . . A n oversimplified diagram showing a portion of the E.coli chromosome° which has replicated in the presence of 3H Thymidine. Three pyrimidine dimers (II) are in one parental strand; none are shown in the other. Parental unlabeled strands are shown by thin lines and radioactively labeled strands are shown by thick lines. Hypothethical steps in postreplication repair are n a m e d by each arrow and are shown to proceed in synchrony. The labeled strand between the first and second dimers from the left increases about two-fold in molecular weight as a result of D-loop degradation and hgation. In alkaline sucrose the strands will be separated from each other and the shift in molecular weight would be detected. The mutational block is shown at the b o t t o m of the diagram as the failure of the next step to occur. The next step is either bridge strand cleavage and ligation or further polymerization and strand assimilation. Either would result in further increase in molecular weight of the labeled strand
partially replace the recF function. It seems unlikely that the r e c k uvrA and uvrB genes are completely isofunctional because the uvrA recF + and uvrB - r e c F + strains show no excision of pyrimidine dimers (Howard-Flanders et al., 1966).
285
In the course of determining a role for recF in excision repair, we found that recF143 enchanced 2vir sensitivity in a u v r B - but not a uvrB + strain. One explanation for this, consistent with the above hypothesis, is that there is a residual amount of hostmediated 2 recovery in a uvrB mutant which is dependent upon the ability to perform postreplication repair. This component would be invisible in the uvrB + r e c F - strain because of the partially overlapping functions of uvrB and r e c F in postreplication repair. In support of the hypothesis that there is a small postreplication repair component of host cell reactivation we call attention to the failure of the recL152 mutation to reduce survival of UV-irradiated lambda in a uvrB5 background. This is correlated with the greater ability of the uvrB5 recL152 strain to perform postreplication repair (Rothman and Clark, 1977) than the uvrB5 recF143 strain. It is even possible that this postreplication repair component of host cell reactivation is induced upon infection by UV-irradiated phage. This would be the equivalent of the indirect induction of phage lambda by infection with UV-irradiated Pl phage (Rosner et al., 1968), except in this case UV-irradiated lambda would provoke indirect induction rather than be provoked by it. We have noted that addition of recA56 to a uvrB5 strain results in the same small decrease in survival of UV-irradiated lambda, recA mutants show greatly reduced levels of postreplication repair (Smith and Meun, 1970). A connection was thus made between the ability to perform postreplication repair, on one hand, and the ability to support UV-induction of a host of recA dependent properties known as " S O S " functions (Witkin, 1976), on the other. This led us to examine whether or not survival of UVirradiated lambda increased in uvr + r e c F - and uvrrecF hosts after they are exposed to UV radiation. This phenomenon, called W-reactivation (Radman, 1974), is one of the SOS functions (Witkin, 1976) and our results will be described elsewhere (Clark, Rothman and Margossian, in preparation). We, therefore, postpone further discussion of the existence of separate RecF and UvrAB pathways of postreplication repair or overlapping recF and uvrA uvrB functions. Gellert et al. (1976a) have recently isolated a new enzyme activity, DNA gyrase, which potentially has some relevance to the nature of the recF gene product. DNA gyrase determines the novobiocin and coumermycin sensitive step in DNA replication. Both DNA gyrase activity and recF are highly cotransducible with dnaA (Gellert et al., 1976b; L. Margossian and A.J. Clark, unpublished observations), reeF143, however, does not change the cell's sensitivity to either novobiocin or coumermycin (A.J. Clark, unpublished
286
R.H. Rothman and A.J. Clark: Postreplicatl0n Repair in a recF Mutant
results), nor does it lead to a reduction in gyrase activity (M. Gellert, personal communication). Acknowledgements. Robert H. Rothman was supported by U.S.
Public Health Training Grant No. GM 367-16. This research was supported by U.S. Public Health Service Research Grant No. AI 05371 from the National Institute of Allergy and Infectious Diseases.
References Bachmann, B.J. : Pedigrees of some mutant strains of Eseherichia coli K12. Bact. Rev. 36, 525 557 (1972) Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K-12. Bact. Rev. 40, 116-167 (1976) Bridges, B.A. : Recent advances in basic mutation research. Address to 6th annual meeting of the European Environmental Mutagen Society at Gernrode, October, 1976 (1977) Demerec, M., Adelberg, E.A., Clark, A.J., Hertman, P.E. : A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61-76 (1966) Ganesan, A.K. : Persistence of pyrimidine dimers during postreplication repair in ultraviolet-light irradiated Eseherichia coli K-12. J. molec. Biol. 87, 103-119 (1974) Ganesan, A.K., Seawell, P.C. : The effect of lexA and reeF mutations on postreplication repair and DNA synthesis in Eseheriehta eoli K-12. Molec. gen. Genet. 141, 189-205 (1975) Gellert, M., Mizuuchi, K., O'Dea, H., Nash, H.: DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. nat. Acad. Sci. (Wash.) 73, 3872-3876 (1976a) Gellert, M., O'Dea, M.H., Itoh, T., Tomizawa, 1.-I. : Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. nat. Acad. Sci. (Wash.) 73, 4474-4478 (1976b) Horii, Z.-I., Clark, A.J-: Genetic analysis of the Reef pathway to genetic recombination in Escherichia coli K-12: isolation and characterization of mutants. J. molec. Biol. 80, 327-344 (1973) Howard-Flanders, P., Boyce, R.P., Theriot, L.: Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53, 1119-1136 (1966)
Kato, T., Rothman, R.H., Clark, A.J.: Analysis of the role of recombination and repair in mutagenesis of Escherichia coli by UV irradiation. Genetics (in press) (1977) Radman, M. : SOS repair hypothesis: phenomenology of an inducible repair which is accompanied by mutagenesis. In: Molecular mechanisms for repair of DNA (P.C. Hanawalt and R.B. Setlow, eds,), pp. 355-367. New York: Plenum Press 1975 Rosner, J.L., Kass, L.R., Yarmolinsky, M.B.: Parallel behavior of F and P1 in causing indirect induction of lysogenic bacteria. Cold Spr. Harb. Syrup. quant. Biol. 33, 785-789 (1968) Rothman, R.H., Clark, A.J, : Defective excision and postreplication repair of UV-damaged DNA in a recL mutant strain of E.coli K-12. (in press Molec. gen. Genet.) (1977) Rothman, R.H., Kato, T., Clark, A J . : The beginning of an investigation of the role of reeF in the pathways of metabolism of UV-irradiated DNA, in Eseherichia coli. In: Molecular mechanisms for repair of DNA (P.C. Flanawalt and R.B. Setlow, eds.), pp. 291-293. New York: Plenum Press 1975 Rupp, W.D., Howard-Flanders, P.: Discontinuities in the DNA synthesized in an excision-defective strain of Escheriehia coli following ultraviolet irradiation. J. molec. Biol. 31, 291-304 (1968) Sedliakova~ M., Brozmanova, J., Slezarikova, B., Masek, F., Fandlova, E. : Function of the uvr marker in dark repair of DNA molecules. Neoplasma 22, 361-384 (1975) Shizuya, H., Dykhuizen, D-: Conditional lethality of deIetions which include uvrB in strains of Eseheriehia coli lacking deoxyribonucleic acicl polymerase I. J. Baet. 112, 676-681 (1972) Smith, K.C., Meun, D.H.C. : Repair of radiation induced damage in Eseheriehia eoli I. Effect of rec mutations on postreplication repair of damage due to ultraviolet radiation. J. molec. Biol. 51,459-472 (1970) Witkin, E.M. : Elevated mutability of polA and uvrA polA derivatives of Escherichia coli B/r at sublethal doses of ultraviolet light: evidence for an inducible error-prone repair system ("SOS repair") and its anomalous expression in these strains. Genetics 79 (suppl.), 199-213 (1975)
Communicated by E. Witkin Received June 13, 1977
The Dependence of Postreplication Repair on uvrB in a recF Mutant of Escherichia coli K-12 Robert H. R o t h m a n 1'* and Alvin J. Clark z 1 Department of Genetics and 2 Department of Molecular Biology, University of California, Berkeley, California 94720, USA
Summary. Mutants carrying recF143 or recF144 show wild type levels of host cell reactivation of UV-irradiated 2vir and wild type rates of excision gap closure in repairing UV damage to their own D N A . The same mutants showed reduced rates of postreplication repair strand joining. When u v r A - r e c F - or uvrB - r e c F - strains are tested, postreplication repair strand joining is incomplete or does not occur at fluences above 1 J/m 2. We suggest that there may be a UvrAB and a RecF pathway of postreplication repair or that the repair functions controlled or determined by uvrA uvrB and by recF m a y be similar. An intermediate in postreplication repair m a y accumulate in the u v r - r e c F - strain.
Introduction The recF143 mutation of E.coli K-12 leads to increased sensitivity to UV radiation and to an inhibition of postreplication repair ( R o t h m a n et al., 1975). Joining of D N A strands synthesized after exposure to 7.4 J/m 2 of UV radiation proceeded to completion but at a much slower rate than in an isogenic recF ÷ strain. These observations led us to suppose that another pathway of postreplication repair operated in the recF mutant. We had expected this pathway to be controlled by the recB and recC genes on three grounds: first recB and recC mutants are sensitive to UV radiation but show normal amounts of postreplication repair (Smith and Meun, 1970); second, the recB and recFmutations act synergistically to increase UV sensitivity to that observed in a recA mutant (Rothm a n et al., 1975; K a t o et al., 1977); and third, a recA * Present Address: Department of Biology, Brookhaven National Laboratory, Upton, L.I., New York 1I973 For offprints contact. Dr. A.J. Clark
mutation completely blocks postreplication repair (Smith and Meun, 1970). When tested, however, the recB recC recF triple mutant exhibited significant levels of fragment joining considered to be evidence of completed postreplication repair ( R o t h m a n et al., 1975). Later Ganesan and Seawell (1975) found that fragment joining in a recF143 strain was blocked by the addition of a uvrB mutation. Two explanations of this result were possible. First, the uvrB defect might be blocking postreplication repair. Secondly, a defect in another gene might be responsible for blocking postreplication repair. This second possibility is plausible since the uvrB mutation used was a deletion, which was detected by the chlorate resistance, biotin requirement it produces and which removes pgl, phr and probably att2 as well as chl and bio genes. In this report, we compare postreplication repair in recF143 strains carrying a uvrB301 deletion and a uvrB5 point mutation. We also further characterize the postreplication repair observed in a recF single mutant, and assess the role of recF in excision repair.
Methods and Materials Bacterial strains are listed in Table l. Techniques of UV irradiation, determination of UV sensitivity, and alkaline sucrose density gradient centrifugation have been described (Rothman and Clark, 1977).
Results Recovery after U V Irradiation The UV sensitivities for cells grown in and plated on complex medium are shown in Figure 1. recF143 caused an increase in sensitivity to radiation, but not as great as that caused by a uvrB mutation or by
280
R.H. R o t h m a n and A.J. Clark: Postreplication Repair in a recF M u t a n t
Table 1. Bacterial strains Sex
uvr
recF
reeL
Other m u t a n t alleles °
Basic genetic background b
Derivation or reference
AB2499 JC8908 JC8911 SR58
FF FF-
B5 B5 B5 B5
+ + 143 +
+ + + +
thyA18, thyR12 thyAt8, thyRi2 (?) thvA18 thyRl2 (?) recA56 thyR12
ABl157 ABl157 AB1 t57 ABl157
P. Howard-Flanders JC8566 x AB2499 Xyl + Met+[Sm R] transconjugant UV R JC9248 x AB2499 Xyl + Met+[Sm R] transconjugant UV s K.C. Smith
JC3890
F
B301
+
+
Kato et al., 1977
JC3893
F-
B301
143
+
deletion of phr, pgl, att2 ABl157 ehIA and/or chlD, all or part of bio operons same as for JC3890 ABl157
JC8403 JC9239 JC8931 JC8471
F F FF
+ + + +
+ 143 144 +
+ + + 152
ilv-332
AB 1157 ABl157 ABlI57 AB1157
Horii and Clark, 1973 Horli and Clark, 1973 JC8403 x P1 -JC7902 Ilv + transductant UV s Horii and Clark, 1973
JC3913 JC7902
FF
A6 +
143 144
+ +
uvrA6 reeB21 ree C22 sbcBl5
ABl157 AB 1 157
Kato et al., 1977 Horii and Clark, 1973
JC1664 JC1670 JC8904
FFF-
+ + +
+ 143 143
152 + 152
ilvD145
AB2277 AB2277 AB2277
AB2277 x P1-JC8471 Met + transductant UV s AB2277 x P1 -JC9239 Ilv + transductant UV s JC1670 × P1 .JC1664 Met + transductant UV s
JC8563 JC8566
Hfr Hfr
+ +
+ +
+ 152
ilv-331 metBI PO3 metBl PO3
P4X P4X
Horii and Clark, 1973 Horii and Clark, 1973 (no strain n u m b e r given)
JC9248
Hfr
+
143
+
metB1 PO3
P4X
JC8563 × P1-JC9239 Ilv + transductant U V s
Kato et al., 1977
Gene symbols and strain designations conform to the recommendations of Demerec et al., 1966, and the standard genetic m a p of E. coli (Bachmann, Low and Taylor, 1976). The following pheuotype abbreviations are used: Xyl, xylose; Met, methionine; Sm, streptomycin; UV, ultraviolet irradiation; Ilv, isoleucine and valine; + , independence when used with amino acid abbreviations, and utilizing when used with sugar abbreviations; - , dependence or uonutilizing, respectively; S, sensitive; R, resistant. Definition of PO3 is to be found in Table 2 of B a c h m a n n (1972) b Each strain has several genetic markers derived from its ultimate ancestor, unless otherwise noted in the derivation. The descendents of ABl157 inherit argE3, his-4, Ieu-6, proA2, thr-1, thi-t, str-31, galK2, lacYt, xyl-5, mtl-1, ara-14, tsx-33, supE44. Unless otherwise noted, descendents of AB2277 inherit arg-4, ilvD145, metE46, proA2, trp-3, thi-t, str-8 or str-9, gaIK2, lacYt or lacZ4, rnalA1, mtl-1 and supE44
a recL152 mutation (Rothman and Clark, 1977). The uvrB301 deletion mutant was as sensitive as the uvrB5 point mutant but the uvrB301 recF143 double mutant was somewhat more sensitive than the uvrB5 recF143 double mutant. In the uvrB5 background the reeF143 mutant was more sensitive than the recL152 mutant (Rothman and Clark, 1977). A recF143 recL152 double mutant was as sensitive as the recL152 single mutant determined previously. Two other alleles of recF, in addition to recF143, were obtained by Horii and Clark (1973). We have transduced one of these, recF144, out of the mutagenized primary mutant background. The survival curve of the recF144 single mutant is included in Figure 1. It was essentially the same as the curve for the recF143 single mutant. The sensitivities of the strain in Figure 1 were also determined when cells grown in complex medium were plated on minimal medium. Contrary to recL152, neither recF143 nor recF144 led to significant levels of shift-down recovery (data not shown, but see Rothman and Clark, 1977).
Post-Irradiation DNA Synthesis and Degradation
.
The effects of recF143 on post-irradiation D N A .synthesis and degradation were measured (Fig. 2). Log phase cultures of the reeF143 mutant and a wild type control were grown in the presence of [3H]-thymidine, washed and irradiated with 7.5 J/m 2 of UV light. Half of the culture was grown in the continued presence of [~H]-thymidine to measure post-irradiation synthesis, and half was grown in non-radioactive thymidine to measure post-irradiation degradation..Irradiation had little effect on incorporation of l a b e l in the wild-type control (Rothman and Clark, 1977) .... but drastically reduced incorporation in the recF!43 mutant (Fig. 2). The cessation of incorporation in. the unirradiated controls is due to the passage of cells from log phase to stationary phase since t h e cells were not diluted at any time during tl~e course of the experiment. D N A degradation was not enchanced by irradiation in the wild type strain (Rothman and Clark, 1977), and was slightly enchanced in the recF mutant (Fig. 2). This behavior contrasts
R.H. R o t h m a n and A.J. Clark: Postreplication Repair in a r e c F M u t a n t
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Fig. I. Survival after UV irradiation. Cells grown to log-phase in Luria broth were washed, resuspended in 56/2 buffer, and irradiated. Following irradiation the cells were diluted and plated on Luria broth agar. Symbols: o uvr + rec+ ; [] u v r B + r e c F 1 4 3 ; o u v r B + r e c F 1 4 4 + ; • r e e F 1 4 3 r e c L 1 5 2 ; z~ u v r B 5 recF+ ; • u v r B 3 0 1 recF + ; v uvrB5 reeF143; • uvrB301 recF143
l• s
0
40
_
,,
0.8 Relafive
0.6 Distance
0.4
Q2
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Sedimenfed
Fig. 3. Effect of UV fluence on the sedimentation rate of newly synthesized D N A in a r e c F 1 4 3 mutant. Cells grown to log-phase in KB medium were washed and resuspended in M-9 buffer. Immediately following irradiation the cells were allowed to incubate in KB medium containing 100 gCi/ml [3H]-thymidine for 10 rain. The cells were then harvested by filtration, washed, and converted to spheroplasts and layered on the gradient. Symbols: • 0 J/m 2 ( 1 0 0 % = 1 4 8 1 0 c p m ) ; © 2 . 5 J / m ~ (100% 9190cpm); [] 10J/m 2 (100%=2580cpm); ± 20J/m 2 (100%=1520cpm); v 30J/m 2 (100% = 1340 cpm). The peak fractions for [l~C]-labeled T4 and 2 D N A are indicated on the abcissa
1Oil
E
0.. 10 4
-
10 3
_ I
I I 2 .5 4 Hours Fig. 2. Post-irradiation D N A synthesis and degradation by a r e c F 1 4 3 mutant. Cells were grown in KB medium containing 10 pCi/ml [3H]-thymidine and 250 ~tg/ml deoxyadenosine. Label incorporation during this period is symbolized by open circles (o) At the time indicated by the arrow, the culture was washed and resuspended in M-9 buffer and divided in half. One half was left unirradiated and the other exposed to 7.5 J/m 2 of UV radiation. One half of each of the unirradiated and irradiated p o m o n s were allowed to grow in radioactively labeled KB medium (o and 0, respectively). The other halves were allowed to grow in non-radioactive m e d i u m ([] and I , respectively). At the time indicated, 0.1 ml aliquots were withdrawn and spotted on a filter paper disc. These were dried, washed in T C A and counted for radioactivity
O
I
strikingly with the behavior of the recL152 mutant in which there is no inhibition of synthesis and no degradation as a result of irradiation (Rothman and Clark, 1977). In light of this UV-produced inhibition of net D N A synthesis in the recFmutant, we were concerned that this strain might not incorporate enough label in a 10 rain post-irradiation labeling period to detect postreplication repair. In Figure 3, however, it can be seen that label taken up after irradiation can be resolved by alkaline sucrose density gradient centrifugation into two peaks and that the mode of the low molecular weight peak is inversely dependent upon the fluence. Label incorporated by unlabeled cells after irradiation remained acid insoluble for periods up to two hours.
Postreplication Repair Joining of DNA fragments synthesized after irradiation with 10 J/m 2 was measured (Fig. 4). Label incor-
282
R.H. Rothman and A.J. Clark: Postreplication Repair in a recF Mutant Table 2. Balance sheet of label incorporated during postreplication repair experiments (Figs. 4, 5)
4000
300
Experiment 2000
200
Label remaining acid insol,
100
cpm
0
3,580 3,600 3,480 3,810 3,870
100 101 97 106 108
98 91 93 104 103
Fig. 5
0 min 30 min 50 rain 70 min 90 rain
16,600 14,700 16,100 10,300 12,700
100 88 97 62 76
92 100 101 97 71
300 I:: In
2oo
200
U
I00
+,+
0
,
F
C
400
~ 2°°
~
0
'
0.9
Relative
400
L /X T z
0.5
I~'
0.1
0.9
This also equals the amount of label layered on the gradient
20o
0,5
0.1
Percent
0 min 30 min 50 min 70 min 90 min
400 oin
% cpm recovered from gradient
Fig. 4
E
E
Chase
0
DistanceSedimented
Fig. 4. Joining of DNA fragments synthesized by the recF143 mutant after exposure to 10 J/m a UV radiation. Cells were irradiated and labeled as in Figure 3. Following the l0 min pulse label, the cells were washed of radioactive medium, resuspended in 3 X the original volume of nonradioactive KB medium and allowed to incubate for 0 (B), 30 (C), 50 (C), 70 (E), and 90 (E) min before being converted to spheroplasts. Four 60 pl aliquots of spheroplasts were drawn after each chase. One of these was layered on a sucrose gradient and the remaining three were counted independently for acid insoluble radioactivity. These counts were averaged and equalled both the amount of label added to the gradient and the fraction of input label remaining acid insoluble at the end of the chase periods (see Table 2). Panel A is an unirradiated control labeled for 10 min
porated by the r e c F I 4 3 mutant during the 10 rain post-irradiation period was resolved into two peaks. The molecular weights at the modes were 2.8 x 10a and 1.4 x 107 and the peaks contained 19% and 81% of the labeled material respectively. Complete joining of all the fragments required 90 min, as compared to 25 and 70 rain for the rec + and r e c L 1 5 2 strains irradiated with the higher fluence of 15 J/m 2 (Rothman and Clark, 1977). All label made acid insoluble during the 10 min post-irradiation pulse remained insoluble during the experiments (Table 2). Strand joining by the r e c F mutant was characterized mainly by the appearance of increasing amounts of labeled material under the high molecular weight peak with a corresponding depletion of label from the low molecular weight peak. There was, however, a slight increase in the mode molecular weight of the slowly 5000
1600
4000 1200 3000 E 0_ o
2000
1000
I
0 1
0,8
0.6
I
0.4
Rel~3%ive Dis#ance Sedimenfed
0.2
0
Fig. 5. Joining of DNA fragments synthesized by the recF143 uvrB5 mutant after exposure to 1 Jim z UV radiation. The protocol was the same as that in Figure 4 and the economy of acid insoluble radioactivity in this experiment is also in Table 2. After being labeled, cells were allowed to grow in non-radioactive medium for 0 (u), 50 (A), and 90 (v) min. The unirradiated control (o) was normalized to be presented on the same scale as the irradiated samples. The counts/rain for the unirradiated control are found on the right hand ordinate
R.H. R o t h m a n and A.J, Clark: Postreplication Repair in a recF M u t a n t
sedimenting material with increasing lengths of time after irradiation. The recF144 mutant behaved identically to the recF143 mutant (data not shown). The uvrB5 recF143 strain was tested (Fig. 5) and yielded results similar to those for the uvrB301 recF143 strain described by Ganesan and Seawell (1975). Following irradiation with 1 J/m z, the newly synthesized DNA was found mainly in a low molecular weight peak with a mode molecular weight of 2.5 × 10 7. By the end of 30 min the mode had shifted to 4.3 × 107, and by the end of 50 rain it had reached a mode of 5.5 x 107. The two-fold increase in mode molecular weight was accompanied by no more than 10% loss of acid insoluble radioactivity (Table 2), and by an absolute increase in the amount of rapidly sedimenting radioactive material. We therefore, feel that the increase in the mode molecular weight which we have observed in this strain reflects the accumulation of an intermediate in postreplication repair. Little further change in the mode molecular weight of the more slowly sedimenting material was observed during the remaining 40 rain of incubation in non-radioactive medium. There was, however, continued de-
283
3000 2000 I000 j 0
I
I
I
I
I
B. rec LI52 oE e_ 2000 1000 0 800
•_ _ ~
6O0 4O0 2O0 0
0.8
O.6
Rela#ive Disfance
O.4
O.2
0
Sedimenfed
Fig. 7. Formation of incision breaks during the first five min after irradiation. Cells were grown to log-phase in KB m e d i u m containing 10 pCi/ml [3H]-thymidine, washed and resuspended in 5 mls of M-9 buffer. The cells were irradiated with 10 (n), 20 (zx), and 30 (v) J/m 2 and allowed to incubate in non-radioactive medium for five rain before conversion to spheroplasts and layering on the gradients. An unirradiated control (o) is incIuded for each strain. The panels are identified in the figure
10 °
ld'
Id 2
/
m L
recFI43
gradation of DNA and loss of counts from all regions of the gradient. Identical results were obtained with a uvrA6 recF143 strain (data not shown).
L
m c
I()3
>
recFI44 /
>
UVFB5 uvr'B,301
Excision Repair
Td ~ -
-
uvcB5FecFI43
~6 5 dVFB5FecA56 uvr'B301 recFI43 16 6 I
0
I
I
I
I
10 20 UV FIuence(d/m 2)
I
30
Fig. 6. Host-cell reactivation of UV-irradiated 2vir. A stock culture of 2vir was diluted to 1 x 107 pfu/ml in 0.01 M MfSO 4 and irradiated. Following dilution, 0.1 ml aliquots of phage suspension was added to 0.2 ml aliquots of host bacteria and allowed to stand for 20 min to permit adsorption. The phage-host complex was spread on L C T G plates with soft agar. Symbols: o recF+ uvrB + ; [] recF143 uvrB+ ; o recF144 uvrB+ ; A recF+ uvrB5; • r e e f + uvrB301; ~7 recF143 uvrB5; • reeF143 uvrB301; • r e e f 5 6 uvrB5
The possibility of a role for recF in excision repair was tested by examining host-cell reactivation ability (HCR) and the formation and closure of incision breaks (Fig. 6). The recF143 and recF144 strains promoted the recovery of irradiated 2vir as well as did the wild type control. Surprisingly, however, the recF143 uvrB5 double mutant showed less 2vir recovery than did the uvrB5 single mutant. 2vir survival in the double mutant was as low as that observed in a uvrB5 recA56 double mutant. The uvrB30l strains behaved like the corresponding uvrB5 strains. The efficiency of incision break closure was measured by determining the fluence required to saturate the excision repair system with dimers and lead to maximal accumulation of breaks. Since excision is
284
R.H. Rothman and A.J, Clark: Postreplication Repair in a recF Mutant
rapid, most breaks will be closed within five minutes after irradiation with low fluences in excision proficient cells. Thus, more than 30 J/m 2 were required to accumulate breaks in the rec ÷ control (Fig. 7A), whereas only 10 J/m 2 led to a similar accumulation in the recL152 mutant (Fig. 7B). The recF mutant accumulated breaks with a fluence dependency similar t o that of the recF ÷ control (Fig. 7C). By these criteria, we conclude that recF does not play an important role in excision repair.
Discussion
A mutation recF143 led to greatly reduced levels of post-UV-irradiation D N A synthesis as compared to the wild type. The small amount of synthesis which did occur resulted in strands of reduced molecular weight as did the synthesis in the wild type. Furthermore, the size of the strands synthesized varied inversely as the fluence applied which is also characteristic of the wild type (Rupp and Howard-Flanders, 1968). It is unclear, therefore, whether a small subpopulation of recF143 cells is undergoing normal amounts of post-UV-irradiation D N A synthesis or whether the whole population is undergoing reduced amounts of synthesis. Regardless, the time for the conversion of these small molecular weight strands into larger molecular weight fragments was longer in the recF143 strain than in the wild type (see Fig, 6; Rothman and Clark, 1977). This seemed to be the main difference between the repair in the wild type and recF mutant. The same effect was seen with a recF144 mutant (data not shown). Sedliakova et al. (1975) have reported uvrB mutations to have an effect on postreplication repair. In confirmation we found that the slow joining of postUV-irradiation labeled D N A strands in the recF mutant was blocked by deletion or point mutations of uvrB and point mutations in uvrA. Complete blockage occurred after irradiation with 2 J/m 2 (T. Kato, personal communication) or 4 J/m 2 (data not shown) while at 0.5 J/m 2, no blockage was observed with a u v r A - recF- strain (T. Kato, personal communication). After irradiation with 1 J/m 2 (Fig. 5), incomplete blockage was observed. There was an increase in the relative amount of the more rapidly sedimenting D N A from 25% to 45% of the counts on the gradient. There was also a two-fold increase in the mode molecular weight of the more slowly sedimenting DNA. Both of these changes occurred within 50 min, after which no more repair appeared to occur and degradation, especially of the higher molecular weight strands, took place. The dependence of postreplication repair on uvrA
and uvrB in a recF mutant leads us to two possible conclusions: (1) there are at least two pathways of postreplication repair (a UvrAB and a RecF pathway); (2) uvrA and uvrB control or determine gene products which can substitute for the mutant recF product. The first possibility seems attractive in that at least three different types of postreplication repair have been proposed: (1) recombinational (Rupp and Howard-Flanders, 1968); (2) mutagenic (Witkin, 1975; Radman, 1975), and (3) excisional (Sedliakova et al., 1975; Bridges, 1977). Knowing that the recF mutation can block recombination in certain genetic backgrounds (Horii and Clark, 1973), and that uvrA and uvrB mutations block excision of pyrimidine dimers (Howard-Flanders et al., 1966), it is obvious to suggest that they block recombinational and excisional postreplication repair, respectively. That would leave mutagenic postreplication repair to explain the residual repair seen in the recF uvr double mutant. In support of this idea, UV-mutagenesis has been found to occur in recF uvrA and recF uvrB strains (Kato et al., 1977). The second possible conclusion is also attractive, however, because it allows us to explain a particular feature of our data on the u v r B - r e c F - strain. This feature is an approximately two-fold increase in the mode molecular weight of the slowly sedimenting labeled DNA. Such a shift in the mode molecular weight is also a feature of postreplication repair in the wild type (Fig. 6; Rothman and Clark, 1977) and recF single mutant (Fig. 4, this paper), but in these cases, the enlarged slowly sedimenting labeled material shifts to more rapidly sedimenting material and postreplication strand joining is completed. The second shift is rapid and slow in the wild type and recF mutant, respectively, and does not take place at all in the double mutant. This has led us to think that the two-fold change in mode molecular weight of the more slowly sedimenting material signals the formation of a particular intermediate in postreplication repair. The possible nature of this intermediate can be visualized by drawing the conventional model for recombinational postreplication repair (e. g. Ganesan, 1974) with three or more pyrimidine dimers on one parental strand (Fig. 8). If strand displacement, D-loop formation (synapsis) D-loop cleavage and ligation occur, then an intermediate is formed whose average length is two times the average pyrimidine dimer spacing on a single strand (Fig. 8). The subsequent elongation of this intermediate to complete postreplication repair strand joining would then be partially blocked in a recF mutant and totally blocked in the u v r B - r e c F - strain after exposure to fluences greater than 1 J/m 2. If there is only one route of such elongation, then the uvrA uvrB function might
R.H. R o t h m a n and A.J. Clark: Postreplication Repair in a recF M u t a n t
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uvrA-recF
-
Fig. 8. An intermediate in postreplication repair m a y accumulate because of a hypothetical recF-uvrA- b l o c k . . A n oversimplified diagram showing a portion of the E.coli chromosome° which has replicated in the presence of 3H Thymidine. Three pyrimidine dimers (II) are in one parental strand; none are shown in the other. Parental unlabeled strands are shown by thin lines and radioactively labeled strands are shown by thick lines. Hypothethical steps in postreplication repair are n a m e d by each arrow and are shown to proceed in synchrony. The labeled strand between the first and second dimers from the left increases about two-fold in molecular weight as a result of D-loop degradation and hgation. In alkaline sucrose the strands will be separated from each other and the shift in molecular weight would be detected. The mutational block is shown at the b o t t o m of the diagram as the failure of the next step to occur. The next step is either bridge strand cleavage and ligation or further polymerization and strand assimilation. Either would result in further increase in molecular weight of the labeled strand
partially replace the recF function. It seems unlikely that the r e c k uvrA and uvrB genes are completely isofunctional because the uvrA recF + and uvrB - r e c F + strains show no excision of pyrimidine dimers (Howard-Flanders et al., 1966).
285
In the course of determining a role for recF in excision repair, we found that recF143 enchanced 2vir sensitivity in a u v r B - but not a uvrB + strain. One explanation for this, consistent with the above hypothesis, is that there is a residual amount of hostmediated 2 recovery in a uvrB mutant which is dependent upon the ability to perform postreplication repair. This component would be invisible in the uvrB + r e c F - strain because of the partially overlapping functions of uvrB and r e c F in postreplication repair. In support of the hypothesis that there is a small postreplication repair component of host cell reactivation we call attention to the failure of the recL152 mutation to reduce survival of UV-irradiated lambda in a uvrB5 background. This is correlated with the greater ability of the uvrB5 recL152 strain to perform postreplication repair (Rothman and Clark, 1977) than the uvrB5 recF143 strain. It is even possible that this postreplication repair component of host cell reactivation is induced upon infection by UV-irradiated phage. This would be the equivalent of the indirect induction of phage lambda by infection with UV-irradiated Pl phage (Rosner et al., 1968), except in this case UV-irradiated lambda would provoke indirect induction rather than be provoked by it. We have noted that addition of recA56 to a uvrB5 strain results in the same small decrease in survival of UV-irradiated lambda, recA mutants show greatly reduced levels of postreplication repair (Smith and Meun, 1970). A connection was thus made between the ability to perform postreplication repair, on one hand, and the ability to support UV-induction of a host of recA dependent properties known as " S O S " functions (Witkin, 1976), on the other. This led us to examine whether or not survival of UVirradiated lambda increased in uvr + r e c F - and uvrrecF hosts after they are exposed to UV radiation. This phenomenon, called W-reactivation (Radman, 1974), is one of the SOS functions (Witkin, 1976) and our results will be described elsewhere (Clark, Rothman and Margossian, in preparation). We, therefore, postpone further discussion of the existence of separate RecF and UvrAB pathways of postreplication repair or overlapping recF and uvrA uvrB functions. Gellert et al. (1976a) have recently isolated a new enzyme activity, DNA gyrase, which potentially has some relevance to the nature of the recF gene product. DNA gyrase determines the novobiocin and coumermycin sensitive step in DNA replication. Both DNA gyrase activity and recF are highly cotransducible with dnaA (Gellert et al., 1976b; L. Margossian and A.J. Clark, unpublished observations), reeF143, however, does not change the cell's sensitivity to either novobiocin or coumermycin (A.J. Clark, unpublished
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R.H. Rothman and A.J. Clark: Postreplicatl0n Repair in a recF Mutant
results), nor does it lead to a reduction in gyrase activity (M. Gellert, personal communication). Acknowledgements. Robert H. Rothman was supported by U.S.
Public Health Training Grant No. GM 367-16. This research was supported by U.S. Public Health Service Research Grant No. AI 05371 from the National Institute of Allergy and Infectious Diseases.
References Bachmann, B.J. : Pedigrees of some mutant strains of Eseherichia coli K12. Bact. Rev. 36, 525 557 (1972) Bachmann, B.J., Low, K.B., Taylor, A.L.: Recalibrated linkage map of Escherichia coli K-12. Bact. Rev. 40, 116-167 (1976) Bridges, B.A. : Recent advances in basic mutation research. Address to 6th annual meeting of the European Environmental Mutagen Society at Gernrode, October, 1976 (1977) Demerec, M., Adelberg, E.A., Clark, A.J., Hertman, P.E. : A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61-76 (1966) Ganesan, A.K. : Persistence of pyrimidine dimers during postreplication repair in ultraviolet-light irradiated Eseherichia coli K-12. J. molec. Biol. 87, 103-119 (1974) Ganesan, A.K., Seawell, P.C. : The effect of lexA and reeF mutations on postreplication repair and DNA synthesis in Eseheriehta eoli K-12. Molec. gen. Genet. 141, 189-205 (1975) Gellert, M., Mizuuchi, K., O'Dea, H., Nash, H.: DNA gyrase: an enzyme that introduces superhelical turns into DNA. Proc. nat. Acad. Sci. (Wash.) 73, 3872-3876 (1976a) Gellert, M., O'Dea, M.H., Itoh, T., Tomizawa, 1.-I. : Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc. nat. Acad. Sci. (Wash.) 73, 4474-4478 (1976b) Horii, Z.-I., Clark, A.J-: Genetic analysis of the Reef pathway to genetic recombination in Escherichia coli K-12: isolation and characterization of mutants. J. molec. Biol. 80, 327-344 (1973) Howard-Flanders, P., Boyce, R.P., Theriot, L.: Three loci in Escherichia coli K-12 that control the excision of pyrimidine dimers and certain other mutagen products from DNA. Genetics 53, 1119-1136 (1966)
Kato, T., Rothman, R.H., Clark, A.J.: Analysis of the role of recombination and repair in mutagenesis of Escherichia coli by UV irradiation. Genetics (in press) (1977) Radman, M. : SOS repair hypothesis: phenomenology of an inducible repair which is accompanied by mutagenesis. In: Molecular mechanisms for repair of DNA (P.C. Hanawalt and R.B. Setlow, eds,), pp. 355-367. New York: Plenum Press 1975 Rosner, J.L., Kass, L.R., Yarmolinsky, M.B.: Parallel behavior of F and P1 in causing indirect induction of lysogenic bacteria. Cold Spr. Harb. Syrup. quant. Biol. 33, 785-789 (1968) Rothman, R.H., Clark, A.J, : Defective excision and postreplication repair of UV-damaged DNA in a recL mutant strain of E.coli K-12. (in press Molec. gen. Genet.) (1977) Rothman, R.H., Kato, T., Clark, A J . : The beginning of an investigation of the role of reeF in the pathways of metabolism of UV-irradiated DNA, in Eseherichia coli. In: Molecular mechanisms for repair of DNA (P.C. Flanawalt and R.B. Setlow, eds.), pp. 291-293. New York: Plenum Press 1975 Rupp, W.D., Howard-Flanders, P.: Discontinuities in the DNA synthesized in an excision-defective strain of Escheriehia coli following ultraviolet irradiation. J. molec. Biol. 31, 291-304 (1968) Sedliakova~ M., Brozmanova, J., Slezarikova, B., Masek, F., Fandlova, E. : Function of the uvr marker in dark repair of DNA molecules. Neoplasma 22, 361-384 (1975) Shizuya, H., Dykhuizen, D-: Conditional lethality of deIetions which include uvrB in strains of Eseheriehia coli lacking deoxyribonucleic acicl polymerase I. J. Baet. 112, 676-681 (1972) Smith, K.C., Meun, D.H.C. : Repair of radiation induced damage in Eseheriehia eoli I. Effect of rec mutations on postreplication repair of damage due to ultraviolet radiation. J. molec. Biol. 51,459-472 (1970) Witkin, E.M. : Elevated mutability of polA and uvrA polA derivatives of Escherichia coli B/r at sublethal doses of ultraviolet light: evidence for an inducible error-prone repair system ("SOS repair") and its anomalous expression in these strains. Genetics 79 (suppl.), 199-213 (1975)
Communicated by E. Witkin Received June 13, 1977
