Molec. gem Genet. 138, 179---192 (1975) © by Springer-¥erlag 1975
DNA Degradation in Minicells of Escherichia coli K-12 II. Effect of fecal and recB21 Mutations on DNA Degradation i n Minicells a n d D e t e c t i o n o f E x o n u c l e a s e V A c t i v i t y * George G. K h a c h a t o u r i a n s Department of Microbiology, University of Saskatchewan, Saskatoon, Canada, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee M. C. P a t e r s o n University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee, and Laboratory for Molecular Genetios, Leiden State University, Leiden, The Netherlands R o n a l d J. S h e e h y University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee B. V a n D o r p Laboratory for Molecular Genetics, Leiden State University, Leiden, ~etherlancls T. E. W o r t h y Institute of Radiation Biology, University of Tennessee, Knoxville, Tennessee Received January 16, 1975 Summary. The properties of minicell producing mutants of Escherichia coli deficient in gentle recombination were examined. Experiments were designed to test recombinant formation in conjugal crosses, survival following UV-irradiation in cells, and the state of DNA metabolism in minicells. The REC- phenotypes are unaffected by rain+l- genotypes in whole ceils. In contrast to minicells produced by rec+ parental cells, minicells from a recB21 strain have limited capacity to degrade linear, Hfr transferred DNA. The lack of a functional recA gene product, presumably involved in inhibiting the recBC nuclease action(s), permit~ unrestricted I-Ifr DNA breakdown in minicells produced by a f e c a l strain. This results in an increase in TCA soluble products and in the formation of small DNA molecules that sediment near the top of an alkaline sucrose gradient. Unlike the linear DNA, circular duplex DNA from plasmids R64-11 or 2de, segregated into the minicells, is resistant to breakdown. By using in vitro criteria, and [3~P]-labelled linear DNA from bacteriophage T 7 for substrate, we found that the ATP-dependent exonuclease of the recBC complex (exo V) is present in re~+ and recA- minicells, and is lacking in the recB21 mutant. In fact, the absence of a functional exo V in recBC- minicells results in isolation of larger than average ]tiff DNA from minicells. We suggest that recombination (REC) enzymes segregate into the polar minicells at the time of minicell biogenesis. This system should be useful for studies on DNA metabolism and functions of the recBC and recA gene products.
Introduction
A n a l y s i s of r e c o m b i n a t i o n b e t w e e n pieces of D N A in a living b a c t e r i a l cell has i n d i c a t e d t h a t a set of genes, rec A, B, a n d C, p l a y a n i m p o r t a n t role in con* Paper 1 in series, see Khachatourians et al., 1974. 13 lV~olec,gen. Genet. 138
180
G.G. Khachatourians et al.
trolling this process (Clark, 1971, 1973, and references therein). Bacteria conraining mutations in the rec genes are recombination deficient (REC-) (Clark and Margulies, 1965; Barbour, 1972; Horii and Clark, 1973). REC- bacteria also display abnormalities in cell division (Green, Greenberg and Donch, 1969; Inouye, 1971), growth and viability (Haefner, 1968; Hertman, 1969; Willets and Mount, 1969; Capaldo-Kimball and Barbour, 1971; Webb and Lorenz, 1972; Capaldo, Ramsey and Barbour, 1974), repair of damage to DNA induced by ultraviolet and ionizing irradiation (Horii and Suzuki, 1968; Morimyo, Horii, and Suzuki, 1968; Miura and Tomizawa, 1968; Kato, 1972; Shlaes, Anderson, and Barbour, 1972; Youngs and Bernstein, 1973) and production of bacteriophages lambda (Hertman and Luria, 1967; Hertman, 1969; Wackernagel and Radding, 1973), T a (Wackernagel, t972), and T~ (Wackernagel and Hermanns, 1974). Of the various classes of rec gene products (Mount, 1971 ; Tomizawa and Ogawa, 1972; Clark, 1973), the recBC enzyme complex of Escherichia coli especially exonuclease V (an ATP-dependent exonuclease, hereafter referred to as exo V), has been studied widely (Buttin and Wright, 1968; Oishi, 1969; Barbour and Clark, 1970; Goldmark and Linn, 1970, 1972; Wright, Buttin, and Hurwitz, 1971; Nobrega, Rola, Pasetta-Nobrega, and Oishi, 1972; Tomizawa and Ogawa, 1972; Mackay and Linn, 1974; Wackernagel and Hermanns, 1974). No attempts have been made to correlate function(s) of rec enzymes with their intracellular localization. Such localization, aside from being interesting in germs of intracellular compartmentalization of, for example, DNA-specific enzymes, is important in our understanding of aspects of metabolism of donor chromosomal Hfr DNA or phage DNA in a rec- bacterium. Basically, one can hypothesize that (1) rec gene products, because of their intimate role in chromosomal functions, are associated strictly with the "nuclear region", or (2) are diffused throughout the cell. We have made use of an E. coli cell division mutant which produces small polar ceils (miniceUs) that lack chromosomal DNA (Adler, Fisher, Cohen, and Hardigree, 1967). The biogenesis of these minicells shares some steps with normM cell division in E. coli (Khachatourians, Clark, Adler, and Hardigree, 1973; Zusman and Krotoski, .1974). Minicells, because of their unique properties, have been useful in Various studies in cell physiology (Dvorak, Wetzel, and Heppel, 1970) and molecular biology of plasmids (Levy, 1974; Levy, McMurry, and PaL met, 1974; Inselburg, 1974; for a recent reviewsee Frazer and Curtiss, 1975). In the present paper we present results of in vivo studies on minicells from rcc+ and rec- strains for degradation of single-stranded Hfr DNA or circular duplex DNA. In addition, we have in vitro results which establish the presence of recBC (exo V) activity in cells and minicells from wild type E. coli, and its absence in r e c B - strains. • Materials and Methods Bacterial Strains and Growth Conditions
The E. toll strains are listed in Table 1. Nomenclature followsthe proposals of Demerec, Adelberg, Clark, and ttartman (1966). Growth conditions and media employed were described previously (Paterson and Roozen, 1972; Paterson and Setlow, 1973; Khachatourians, Sheehy, and Curtiss, 1974).
DNA Degradation in rec- Minicells
181
Table 1. Bacterial strains a Strain
rec
rain
genotype
genotype
Mating/or Chromosome plasmid type genotype
g209
-~
--
F+
prototroph strs
Z526
fecal
--
F+
leu- lac- 2strrmetB -
Z876
~
--
Hfr
prototroph strs 2-
g925
+
minAB-
F-
thr- leu- lacY- gal- str r thi-
Z1009 gll18
+
minAB-
fecal
minAB-
R64-11 F'(KLF-1)
)/1120 g1169
~
minAB-
recB21
--
FF+
g1178
-[-
minAB-
F-
same as )/1120, except real +
zl197
fecal
minAB-
F-
same as zl178, except thy +
Z1200 )/1256
recB21
minAB-
F-
recA1
minAB-
2dv
same as )/1169 same as zl197
same as g925 F'-thr+ leu+/thr - leulacY- gal- str s thi-
same as Z925, except t h y A thr- ara- leu- pro- lactsx- ~- his- str- arg- thi-
a Only relevant mutations other than rec are noted for the sake of brevity.
Determination o/U V Sensitivity
To determine the response of a strain to UV-irradiation, growth phase (108 cells/ml) were centrifuged and resuspended in phosphate buffer (100 mM K~HPOa, 100 mM KH2POa; pI-I 7.0) at a concentration of 10s cells/ml. Stirred samples (1-2 mm in depth) of bacteria were irradiated with a 15-W low pressure mercury germicidal lamp. This light source predominantly emits UV rays at 254 n m at an incident UV fluence rate of 60 J/m~/min. After suitable dilutions, irradiated and non-irradiated cultures were plated on nutrient agar plates. Cell viability was estimated by the colony forming ability of various samples after 24-48 h at 37 ° C (in the dark). To prevent photoreactivation, all UV experiments were performed under yellow light (General Electric "gold" fluorescent lamps). Assay/or
Genetic R e c o m b i n a t i o n
The cross-streak method for Hfr and F - matings (Berg and Curtiss, 1967) was employed to assess recombination capability of bacterial strains. After mating, bacteria were plated on minimal agar medium ML (Cur~iss, 1965) supplemented with 0.5% glucose, 200 tzg/ml streptomycin sulfate (Squibb), 7.5lzg/ml thiamine, and 1.5% Difco agar. Recombinant thr + leu + colonies were scored after 48 h incubation at 37 ° C. Harvesting and Mating M i n w e l l s
Minicells were purified by sedimentation through 5-20% sucrose (w/v) gradients. F minicells were mated subsequently with [aH-methyl]-thymidine-labelled Hfr cells on 0.22 tz Millipore filters (HA type, 45 ram; Millipore Corp., Bedford, Ma.) as described earlier (Sheehy, Orr, and Curtiss, 1972; Khachatourians et al., 1974). Determination o/Radioactivity in Minicells
100 ~zl samples containing 10~-10Sminicells from appropriate incubation media were spotted on W h a t m a n 3 MM paper discs (W. and R. Balston Ltd., England). Filters were immediately submerged in ice cold 10% trichloroacetic acid (TCA) and washed again with 18"
182
G.G. Khachatourians et al.
5% TCA and 95% ethanol. After drying, approximately 3 ml of 5% (w/v) 2,5-bis(2-[5-tertbutylbenzoxazolyl])-thiophene in toluene was added to the vials and radioactivity was determined by counting samples in a Packard Triearb Scintillation Spectrometer (Packard Instalment Co., Downers Grove, I1.).
Velocity Centri/ugation in Alkaline Sucrose Gradients Velocity sedimentation analysis of Hfr DNA isolated from minicells was performed using the alkaline sucrose gradient technique of ~cGrath and Williams (1966). Minicells were lysed exactly as given in Khachatourians et al. (1974). Gradients were spun at 35000 tee/rain at 21 ° C for 130 rain in an SW 56 swinging rotor driven by a Beckman Model L3-50 ultracentrifuge. The radioactivity from gradient tubes was assayed by collecting drops from the bottom of the gradient tubes on paper strips (Carrier and Sctlow, 1971) and were counted as described above.
A T P. Dependent D N ase Assay The procedure for the preparation of crude protein extracts from both minicells and parental cells of various strains was similar to the initial steps in the purification of exo V described by Goldmark and Linn (1972). Briefly, cultures were grown for 8 h at 37 ° C in a synthetic nutrient medium (final concentration about 8 × l0 s cells/ml) after which minicells were separated from parental cells as described above. All subsequent operations were conducted at 0-4 ° C. The separated fractions were suspended in 10 mM Tris.HC1-200 mM MgClf 5 mM fl.mereapto-ethanol-2 mM ethylcndiaminetetraacetic acid (pH 7.5), disrupted by sonisation (four I min pulses from a Raytheon Sonic Oscillator), and then centrifuged to remove cell debris. The superuate was treated with streptemycin sulfate (0.4%, w/v), centrifuged and the superuatant fraction was precipitated with ammonium sulfate (60% saturation). The pellet was dissolved in 10 ~ Tris. HC1 (pit 7.5) at a protein concentration of 0.1 mg/ml (determined by the method of Lowry, Rosebrough, Farr and Randall, 1951). This solution was used as the crude extract. ATP-dcpendent DNase activity present in crude extracts was assayed b y measuring the TCA soluble radioactivity released from [8~p] labelled T~ DNA (specific activity 3 × 104 cpm/~g) (Van Dorp, Caulen, and Pouwels, 1974). The reaction mixture (total volume 300 ml) contained: 20 ~M Tris.HC1 (pH 9.0), 10 ~ I MgS04, 2 ~g [s~p] labelled T 7 DNA, and 0.4 ATP. A second reaction mixture without ATP served as a control to measure the background level of ATP-independent nuelease activity. The reaction was initiated by introducing, with thorough mixing, 0.1 ml of crude extract. After a 30 rain incubation at 30° C, the reaction was stopped by adding 0.2 ml bovine serum albumin (1.25 mg/ml) and 0.2 ml 6% HCIO~. Finally the acid soluble radioactivity present in both ATP-containing and ATP-lacking samples was measured in a Nuclear Chicago planchet counter.
Results
Characteristics o/Minicell.producing Strains De/icicnt in Genetic Recombination I n a d d i t i o n t o t h e criterion of genetic crosses used t o c o n s t r u c t rec- minicell-producing d o u b l e m u t a n t s (Table 1), r e c o m b i n a t i o n deficient p h e n o t y p e s were t e s t e d in t h e s e s t r a i n s b y t h r e e s u p p l e m e n t a r y criteria. (1) R e c o m b i n a n t f o r m a t i o n i n conjugal crosses (Clark a n d Margulies, 1965). T h e r e c o m b i n a t i o n deficiency in m i n i c e l l - p r o d u e i n g strains ~1197 (fecAl) a n d Z1200 (recB21) was e x a m i n e d b y c o m p a r i n g t h e i r a b i l i t y t o c a r r y o u t general genetic r e c o m b i n a t i o n in crosses w i t h a n F ' thr+ leu+ s t r a i n ( z l l l S ) or H f f thr+ leu+ s t r a i n (Z876). T h e i r a b i l i t y t o p e r f o r m functions r e q u i r e d for t h e p r o d u c t i o n of v i a b l e recomb i n a n t offspring is shown in T a b l e 2. (2) I n a b i l i t y t o r e p a i r U V or X - r a y i n d u c e d d a m a g e t o b a c t e r i a l D N A as m e a s u r e d b y colony f o r m i n g a b i l i t y (Clark a n d Margulies, 1965; t t o w a r d - F l a n d e r s a n d Boyce, 1966; H o r i i a n d Suzuki, 1968). U V s u r v i v a l curves i n Fig. 1 clearly r e v e a l t h a t recA- a n d recB- m i n i c e l l - p r o d u c i n g
DNA Degradation in rec- Minicells
183
I00;
10
to
0
0
.
10
1
20
~
30
qO
50
UV FLUENCE [Jim 2)
Fig. 1. UV survival curves of minicell-producing E. cvli strains having different Ree genotypes. Stirred samples of chilled cultures, suspended in phosphate buffer at l0 s eells]ml, were exposed to indicated fluenees of germicidal light and plated to measure colony-forming ability as described in Materials and Methods. Symbols: D, Z925 (rev+); I , Z1009 (tee+); e, zl197 (recA-); o, Z1200 (recB-); A, Z1256 (reeA-)
Table 2. Transfer of thr+ leu+ from various donors a Recipient strain
Z526 Z925 Z1009 Zl 169 Zl178 Zl197 X1200 Z1256
rec
genotype
fecal rec+ rec+ recB21 rec+ fecal recB21
reval
Min pheuotype
Recombinant formation with donors Z976 (Hfr)
zll18 (F'-KLF1)
iYIin+ Min-
-+
+ +
Min-
+
+
Min+ MinMinMinMin-
-+ ---
+ + + +
--
+
a Estimated from the presence ( + ) or absence (--) of thr + leu+ recombinant colonies in the standard cross streaks tests on selective minimal agar plates.
184
G.G. Khachatourians et al.
strains are more sensitive to UV than their tee+ parents. Although not shown here (for the sake of clarity) the rain genotype does not affect UV sensitivity (unpublished data). (3) The kinetics of UV-induced degradation of labelled chromosomal DNA indicate extensive degradation in reeA- as well as reeArain- strains and some DNA degradation in recB- and r e c B - rain- mutants (Paterson, Khachatourians, and Worthy, 1972). These results were in complete agreement with those of Howard-Flanders and Boyce (1966). We confidently conclude that all three criteria used confirm that recA- or recB- gene introduced into our minicell forming strains result in expression of the REC- phenotypes in these cells. Size oI the H l r D N A Isolated Post-conjugally 1tom Minicells We know now that single stranded Hff DI~A transferred from Hfr strain Z876 into minicells and isolated thereafter (60 min mating on filter membranes at 37 ° C) is 22-24 × 106 dalton weight average molecular weight (Sheehy et al., 1972; Khachatourians et al., 1972; Khachatourians et al., 1974). We examined the size of Hfr Z876 DNA transferred to F- recipient minicells isolated from midlog cultures of tee- strains. Mid-log cultures were chosen because such minicells have reduced amounts on non-specific exonucleolytic activity toward conjugally transferred Hfr DNA (Khachatourians et al., 1974). Immediately after mating, recipient minicells and donor Hfr cells were chilled and separated. The molecular weights of single-stranded DlqA transferred into tee +, recA- and recB- minicells were analyzed by velocity sedimentation. These results are shown in Table 3 (third column). The DNA found in reeA-, r e c B - and tee + minicells shows differences in weight average molecular weights (Mw). The largest size DNA was found in recB- minicells. We suspect that this reflects the role of exo V in the weight average molecular weight of DI~A recovered from minicells (see below).
The reeBC enzyme has several distinct properties (Oishi, 1969; Barbour and Clark, 1970; Wright, Buttin and Hurwitz, 1971; MeKay and Linn, 1974) such as an ATP-dependent exonuclease that digests both single-stranded and duplex DNA, and an endonuclease that exhibits absolute specificity for singlestranded D~qA. Circular duplex DNA is completely resistant to any action of the exo V (Goldmark and Linn, 1970, 1972; Mackay and Linn, 1974), even when the circles contain nicks. However, gaps as small as 5 nueleotides allow degradation of the circles (MacKay and Linn, 1974). Since the phenotypes associated with rec seem to have a quantitative effect on the M w of single-stranded linear DNA found in post-conjugation harvested minicells (Table 3), we next examined the degradation of Hfr DNA in such minicells. We also examined various plasmid DNA (e.g., strains Z1256, Z1009, Table 1) which are segregated into minicells and found mostly as covalently closed circular duplexes, some catanated and some linear forms (see Inselburg, 1974 and references therein). We have previously shown that minicells contain nuclease(s) activity which, under optimal physiological conditions can attack and degrade DI~A in vivo (Sheehy et al., 1972; Khachatourians and Riddle, 1973; Khachatourians et al., 1974). To determine whether DNA degradation activity is in any way altered in strains Z 1297 (recA-) or z l l 0 0 (recB-) in association with the absence of a functional exo V we con-
DNA Degradation in rec- Minicells
185
i
Q i
O
100
•
•
D
0
m
n
[] •
[]
g-
8o
l--o
GO-
0 0
I qO
I
I BO
r
INCUBATION
I ,,i 120
TIME
I
,i 160
I 200
(min)
Fig. 2. Kinetics of DNA degradation in minieells derived from E . coli strains having different Rec genotypes. [aH]-dThd labelled DNA present in minicells was either from conjugally (Hfr) transferred (linear) (o, e, A, A) or plasmids ~dv and R64-11 segregated into minieells during their production (covalently closed circular, and open linear) (D, II). After suitable periods of incubation minieell samples were withdrawn and the TCA-insoluble [att]-thymidine radioactivity remaining was counted and expressed as a percentage o~ that present in the corresponding unineubated sample. Symbols: A, %925 (rec+); [], %!009 (rec+); e, %1197 ( f e c A l ) ; o , %1200 (recB21); II, %1256 (recAll) ....
Table 3. Sedimentation analysis of Hfr DNA in minieells of E. coli Strain
rec
genotype
X925 %1197 Z1200
rec + fecal recB21
Weight average molecular weight a to
tl8o
25 x 106 23 x 106 36 x 10e
19 × 106 17 × 10e 25 x 106
Breaks per l0 s daltonb 2.5 3.1 2.4
a Weight average molecular weight determined immediately after the :conjugation (to) or 180 rain incubation (tlso). For details see Methods. b For a reference on calculation see Paterson (1975).
d u c t e d m a t i n g s b e t w e e n H f f s t r a i n X 876 donor cells a n d recipient minicells of different rec genotypes, T h e effect of post-conjugal i n c u b a t i o n on b r e a k d o w n of labelled D N A i n t o T C A soluble m a t e r i a l was measured.: T h e d a t a presented i n Fig. 2 shows the solubilization of [3H]-thymidine l a b e l l e d D N A i n minicells
186
O.G. Khachatourians et al. Mw , DALTONS. "|
2,1o
1o
El . . . . .
s
'1
I
2~,o
A
r~ IO
8
|
1o~
,0~
e'
2,,o
'lo"
lo" "
C
B
IM tw
Ixl n," >-
oo
Z;
/o
iP
~"l
/
12J
o,q
?o
ne
•
,,o
o
.
D~
F-
4o
~ ~'
,I
0
/
10
i
i
20
I
; ...... I
0
"1
I'
i
i
10 20 FRACTION NUMBER
o
"1
~o '
~o
I
Fig. 3A--C. Alkaline sucrose density gradient profiles of Hfr DNA from mated minicells. Minicells from late log cultures of Z925, zl197 and ZI200 after mating with [3H]dThdlabelled Hfr Z876 were examined for the DI~A recovered before (o) or after (o) 180 rain incubation at 37° C. The radioactivity profiles are shown for DNA from reo+ (A), fecAl (B) and revB21 (C) minicells. M w values are given in the upper abscissa
produced from recA1 and recB21 parental cells. When compared to recA- minicells recB- or rec+ minicells do not degrade their resident DNA as extensively, providing the D N A subject to attack is linear, and is not covalently closed circular duplexed or open forms. The difference in the final amounts of DNA degraded into acid soluble fractions is significantly lower in recB- minicells. This seems to be due to the absence ol the recBC coded exo V. Detection of ATP-dcpendent DNase Activity in Crude Minicell Extracts
These results on DNA degradation in minicells prompted us to assay for the presence of exo V in crude extracts prepared from minicclls and, as a control, parental cells containing functional reeB C gene products. This was achieved b y measuring the acid-soluble products released from [a~P]-labelled T 7 D N A during incubation with protein extracts of various rec strains. As can be seen in Table 4 both re~+ and recA- minicells clearly possess ATP-dependent DNase with an activity comparable to t h a t found in parental cells. Conversely, both cells and minicells of the recB- strain, Z 1200, lack this activity, as expected. I t seems likely then t h a t the DNA breakdown observed in minicells containing linear duplex or single-stranded H f r DNA (but not covalently closed circular R-factor or 2 d r DNA) stems from the action of exo V. The changes in the size of DNA molecules present in minicells at the end of a 3 h incubation have also been examined b y sedimentation through alkalinesucrose gradients. The sedimentation profiles are shown in Fig. 3. The most important feature of these results was the decrease in size of D N A recovered (Table 3) and the presence of a minor peak from f e c a l minicells found near the
DNA Degradation in rec- Minicells
187
Table 4. ATP-dependent DNase activity from cells or minicells Strain
Z925 gl009 9¢1197 g1200 Z1256
Plasmid
-R64-11 --2dr
rec genotype
(rec+) (rec+) (recA) (recB) (recA)
Percentage of [82PIT7DNA rendered acid soluble a cells
minicells
41 62 70
50 42 53
5
3
68
56
a Corrected for background level of ATP-independent nuclease activity.
top of the alkaline sucrose gradient (Fig. 3B). i~1200 (recB21) minicells lack such small molecules (Fig. 3 C). The radioactive DNA sedimenting as the minor peak in Fig. 3 B amounts to 25 % of total radioactivity recovered from the gradient. These results indicate that in r e c A - minicells, some endonuclease(s) produce a number of single strand scissions yielding a population of low molecular weight DNA molecules, possibly due to endonucleolytic activity associated with recBC enzyme. Interestingly enough, these molecules are not degraded into TCA-soluble fragments to any significant level. The kinetics of the formation of these molecules remain to be examined. An analysis of the weight- and numberaverage (Mn) molecular weights (Table 3) for DNA remaining undegraded in rec+, reeA- or rec B-minicells demonstrate an interesting correlation, i.e., rec B - minicells contain a DNA equal to that recovered in rec+ or r e c A - minicells immediately after mating (see Fig. 2; Khachatourians et al., 1974). Single stranded breaks per DNA molecule are greater in minicells from r e c A - than from r e c B - or rec+ strains (Table 3); these figures do not differ significantly to allow an assessment of recBC associated endonuclease activity. These breaks could be due to nonspecific endonucleases. However, we know that they cannot arise from the action of endonuelease I (end I) of E. cell since a comparison of end I - and end I+ minicell producing strains does not show a difference in the number of single strand breaks per DNA molecule (Khachatourians et al., 1972; Glatzer and Curtiss, 1972). Discussion We have used the chromosomal-DNA-free minicells of E . cell to examine the presence of enzymatic activity of recA and recBC coded proteins, rec coded enzymes, among other functions ascribed to them (see Introduction) determine the recombination proficiency of a bacterial cell (Clark, 1973). The activity of recB recC coded DNase complex and more importantly, that of the ATP-dependent exo V, is responsible for destruction of the biological functions of a linear double stranded DNA. For example, DNA from phage T 4 (Wackernagel, 1972), inverted 2 (Wackernagel and Radding, 1973), and T~ DNA (Wackernagel and Hermanns, 1974) are good substrates for exo V action. The present studies show that of the four DNase activities associated with recB recC nuclease complex (see Introduction) the ATP-dependent exo V is present in rec+ or f e c A l mini-
188
G.G. Khachatourianset al.
cells and is lacking in recB21 (strain g 1200) cells and consequently minicells produced by this strain. Furthermore, we show that there is no significant difference in exo V activity in crude extracts of R + and 2dv + minicells (strains g 1009 and Z 1256) and minicells from strains g925 and Z 1197 which are devoid of such plasmid DNA (i.e. R-, 2dv-). The consequence(s) of lack of recA protein, with its inhibitory action on the ATP-dependent recBC nuclease of E. coli (Hour, Van de Putte, DeJonge, Schuite and Oosterban, 1970) is consistent with our results. There is more unrestricted exonuclcase activity in strains g 1197 and Z1256 (Fig. 2 and Table 3). However, we have not measured the activities of nuclease(s) other than exo V in recBC complex or other gene coded nuclease(s). Our previous studies have shown the presence of DNase activity for Hfr transferred DNA (Sheehy et al., 1972; Khachatourians et al., 1972, 1974) and colicin E2 induced DNase activity for the breakdown of double stranded R +, 2dv, and )tb588 DNA (Khachatourians and Riddle, 1973; Khachatourians and Saunders, unpublished data). The present iu vivo data further clarifies this by showing differences in the DNA breakdown in minicells from rec+ and rec- strains. Sedimentation profiles from recA minicells containing an Hfr DNA show a bimodal distribution (Fig. 3B). This is consistently found only in recA- strain and was absent in minicells from rec+ or recB- strains (Sheehy et al., 1972; Khachatourians et al., 1972, 1974). Exo V must be involved in the formation of these small DNA molecules. Hfr transferred DNA in miniceUs is found in both single and double stranded forms (Cohen et al., 1968a, b; Khachatourians et al., 1974). The studies of MacKay and Linn (1974) indicate that the breakdown of double stranded linear DNA by exo V results in production of single stranded DNA fragments, 100-500 nuclcotides in length. These fragments would be rendered TCA soluble by single strand specific exonuclease(s), e.g. by activity of the recBC complex or other specific nuclease(s). The results presented here (Fig. 3) confirm the reported results (Goldmark and Liun, 1970, 1972; MacKay and Linn, 1974) that covalently closed circular duplex DNA is not a substrata for exo V action. Since the in vivo DNA degradation in minicells is largely an energy (ATP) dependent process (Khachatourians et al., 1972, 1974; Khachatourians and Riddle, 1973) and our in vitro data shown here indicates that an absence of ATP dependent exo V results in a 95% loss of DNase activity, we suggest that the main component of ATP-dependent DNA degradation in minicells is due to recBC enzymes and specifically to exo V. During conjugation, DNA is transferred from a male donor cell to recipient from the 5' end (Ohki and Tomizawa, 1968). The recBC enzyme complex lacks a unique polarity for DNA (MacKay and Linn, 1974) and either 3' or 5' ends of single strandedbroken linear DNA would serve a substrate for rec BC enzyme(s). Hence, there is good reason to expect that some of the single stranded Hfr DNA found in minicells will serve as substrata for recBC enzyme(s). The 3'-->5' activity of exo VII (Chase and Richardson, 1974) could also be involved here and is presently under investigation. However, we find negligible amounts of ATP-independent nuclease activity in crude extracts of minicells. The Hfr single stranded DNA found in conjugated minicells from recB21 parents was 36 × l0 s dalton (Table 3) immediately after harvesting, whereas that in rec+ minicells was 22 × to 25 × 10s dalton (Table 3, and Sheehy et al., 1972; Khachatourians et al., 1974).
DNA Degradation in rec- Minieells
189
Because 40% more radioactively labelled H f r D N A is recovered in r e c B 2 1 minicells after the completion of mating, this strain is particularly useful for isolation of larger pieces of conjugally transferred H f r DNA. The difference between D N A degradation in r e c B 2 1 versus recA1 or rec + minicells is also compatible with the earlier observations on gene expression after conjugal transfer. Joshi and Siddiqi (1968) found t h a t after transfer of the phosphatase gene from an H f r into rec+ and rec- recipient cells, enzyme synthesis came to a halt in the rec- cells. Similarly, D u b n a u and Maas (1969) examined the difference in the expression of newly t r a n s f e r r e d / a c z gene in l a c z - f e c a l or lac z - rec B 21 recipients. Measuring the rates'of induced synthesis of fl-galaetosidase after m a t i n g with H f r lac + donors, t h e y found substantially more e n z y m e synthesizing capacity in the r e c B 2 1 strain. I n both cases (recA1 or r e c B 2 1 ) , there was a progressive inactivation of genetic capacity of the zygotes to synthesize fl-galactosidase. The s t u d y of the fate of double and single stranded DNA-metabolism in minicell system is a unique system for the s t u d y DNA-nuclease interactions i n rive. The fact t h a t r e c B C (exo V) and r e c A gene products segregate, and t h a t their biological activities are found in minicells makes the system more useful for the s t u d y of recombination events in minicells. These experiments are presently in progress. Acknowledgement. This research was initiated at the Biology Division, Oak Ridge National Laboratory which is operated by Union Carbide Corporation under contract with U.S. Atomic Energy Commission. The work was supported by a Medical Research Council (MI~C) Canada Postdoctoral Fellowship (to G.G.K.), Public Health Service Predoetoral Fellowship, GM-1974 from National Institute of General Medical Sciences (to R.J.S. and M.C.P.) and Oak l~idge Associated Universities Graduate Participant Award (to T.E.W.). Further work was supported by MRC-Canada Postdoctoral Fellowship (to M.C.P. in Netherlands) and a Teaching ~nd Research Grant from University of Saskatchewan, College of Medicine (to G.G.K.). We are grateful to Drs. 1~. Curtiss, R. Fujimura, F. Hamilton, S. Niyogi and P. O'Neil for helpful discussions.
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Chase, J . W . , Richardson, C.C.: Exonuclease VII of Escher~ohia ooli. Purification and properties. J. biol. Chem. 249, 4545-4554 (1974) Clark, A. J.: The beginning of a genetic analysis of recombination proficiency. J. Cell Physiol. 70, 165-180 (Suppl. 1) (1967) Clark, A. J. : Toward a metabolic interpretation of genetic recombination of E. coli and its phages. Ann. Rev. Microbiol. 25, 437-464 (1971) Clark, A. J.: Recombination deficient mutants of E. ooli and other bacteria. Ann. Rev. Genet. 7, 67-86 (1973) Clark, A. J., Margulies, A. D. : Isolation and characterization of recombination-deficient mutants of Escheriohla coli K-12. Prec. nat. Acad. Sci. (Wash.) 58, 451-459 (1965) Cohen, A., Fisher, W. D., Curtiss, R. III, Adler, H. I.: DNA isolated from Eacheriohia coli minicclls after mating with F cells. Proc. nat. Acad. Sci. (Wash.) 61, 61-68 (1968a) Cohen, A., Fisher, W. D., Curtiss, R. I I I , Adler, H. I.: The properties of DNA transferred to minicells during conjugation. Cold Spr. Harb. Syrup. quant. Biol. 83, 635-641 (1968b) Curtiss, R. I I I : Chromosomal aberrations associated with mutations to bacteriophage resistance in Escherichia coli. J. Bact. 89, 28-40 (1965) Demerec, M., Adelberg, E. A., Clark, A. J., Hartman, P. E. : A proposal for a uniform nomenclature in bacterial genetics. Genetics 54, 61-76(1966) Dubnau, E., Maas, W . K . : Inactivation of a newly transferred gene in recombination-deficient recipients of Escherichia coll. Molec. gen. Genet. 198, 305-312 (1969) Dvorak, It. F., Wetzel, B.K., Heppel, L . A . : Biochemical and cytochemical evidence for the polar concentration of periplasmic enzymes in a minicell strain of Escher~chia coli. J. Bact. 104, 543-548 (1970) Frazer, A., Curtiss, R.C.: Production, properties and utility of bacterial minicells. Curr. Top. ~Iicrebiol. Immunol., 69, 1-84 (1975) Glatzer, L. G., Curtiss, R. C. : Evidence for the presence of F at the lead region of conjugally transferred Hfr DNA. Abstracts Ann. t~Iceting Amer. Soc. Microbiol., p. 31 (1972) Goldmark, P. J., Linn, S.: An endonuclease activity from Escherichia coli absent from certain rcc- strains. Proc. nat. Acad. Sci. (Wash.) 67, 434-441 (1970) Goldmark, P. ft., Linn, S.: Purification and properties of the rec BC DNase of Escherichia voli K-12. J. biol. Chem. 247, 1849-1860 (1972) Green, M. H. L., Greenberg, J., Donch, J. : Effect of a recA gene on cell division and capsular polysaccharide production in a lon strain of Escherichia coll. Genet. Res. (Camb.) 14, 159-162 (1969) I{aefner, K.: Spontaneous lethal sectoring, a further feature of Escherichia coli strains deficient in the function of rec and uvr genes. J. Bact. 96, 652-659 (1968) ttertman, I., Luria, S. : Transduction studies on the role of rec+ gene in the ultraviolet induction of prophage lambda, ft. molec. Biol. 28, 117-133 (1967) Hertman, J. M.: Survival, DI~A breakdown and induction of prophage in a Escherichla coti K12 rec A u v r B double mutant. Genet. Res. (Camb.) 14, 291-307 (1969) Horii, Z., Clark, A. J.: Genetic analysis of the RecF pathway to genetic recombination in Escherichia coli K12: isolation and characterization of mutants. J. molee. Biol. 80, 327-344 (1973) Horii, Z., Suzuki, K.: Degradation of the DNA of Escherichia coli K-12 Rec- (JC 1669b) after irradiation with ultraviolet light. Photochem. Photobiol. 8, 93-105 (1968) Hour, A., Van De Putte, P., De Jonge, A. J. R., Schuite, A., Oosterbaan, R. A.: Interference between the recA product and an ATP-dependent exonuclease in extracts of E. coli. Biochem. biophys. Acta (Amst.) 224, 285-287 (1970) Howard-Flanders, P., Boyce, R . P . : DNA repair and genetic recombination: Studies on mutants of Escherichia coli defective in these processes. Radiat. Res., Suppl. 6, 156-184 (1966) Inouye, M.: A pleiotrepic effect of the recA gene of Escherichia coli: Uncoupling of cell division from DNA replication, g. Bact. 106, 539-542 (1971) Inselburg, g. : Replication of Coliein E1 plasmid DNA in minicells from a unique replication initiation site. Proc. nat. Acad. Sci. (Wash.) 71, 2256-2259 (1974) Joshi, .G.P., Siddiqi, O.: Enzyme synthesis following conjugation and recombination in Eschcrichia coll. J. moles. Biol. 82, 201-210 (1968)
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Kate, T.: Excision repair characteristics of recB- res- and uvrC- strains of Escherichia coli. J. Bact. 112, 1237-1246 (1972) Khachatourians, G. G., Clark, D. J., Adler, H. L, tIardigree, A. A. : Cell growth and division in Escherichla cell: a common genetic control involved in cell division and minicell formation. J. Bact. 116, 226-229 (1973) Khachatourians, G. G., Riddle, J. : Mechanism of action of colicin E2: Plasmid DI~A breakdown in minicelts of Es~heri~hia ~oti. Abstracts Ann. Meeting Amer. See. iYIicrobioI., G221 (1973) Khachatourians, G. G., Sheehy, R. J., Curtiss, R.C.: Fate of conjugally transferred DNA in E. coli minicells. Abstracts Ann. Meeting Amer. Soc. Microbiol., p. 31 (1972) Khaehatourians, G. G., Sheehy, R. J., Curtiss, R. III.: Fate on conjugally transferred DNA in minicells of •scherichia coli K-12. Melee. gem Gent. 128, 23-42 (1974) Knshner, S. R., Nagaishi, H., Clark, A. J. : Indirect supression of recB and recC mutations by exonuelease I deficiency. Prec. nat. Aead. Sci. (Wash.) 69, 1336-1370 (1972) Levy, S. B.: R factor proteins synthesized in E. coli minicells, I. Incorporation studies with differen~ !~ factors and detection of DNA-binding proteins. J. Bac~. 129, 1451-1463 (t974) Levy, S.B., McMurry, L., Palmer, E.: R factor proteins synthesized in E. coli minicells II. Membrane-associated R factor proteins. J. Baet. 120, 1464-1471 (1974) Lowry, O. H., Rosebrough, N. J., Farr, A.L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 198, 265-275 (1951) MacKay, V., Linn, S.: The mechanism of degradation of duplex deoxyribonucleic acid by the recBC enzyme of Escherichia cell K-12. J. biol. Chem. 249, 4286-4294 (1974) MeGrath, R.A., Williams, R . W . : l~econstruction in vice of irradiated Escherichia cell deoxyribonucleic acid: the rejoining of broken pieces. :Nature (Lend.) 212, 534-535 (1966) Miura, A., Tomizawa, J.-I. : Studies on radiation sensitive mutants of E. coll. I I L Participation of the Roe system in induction of mutation by U.V. irradiation. Melee. gen. Goner. 103, 1-10 (1968) Morimyo, M., Horii, Z., Suzuki, K.: Appearance of low molecular weight DNA in a Revmutant of Escherlchia cell K12 irradiated with X-rays. J. Radiat. Res. (Japan) 9, 19-25
(1968) Mount, D. W. : Isolation and genetic analysis of a strain of Escherichia cell K-12 with an amber rec A mutation. J. Baet. 107, 388-389 (1971) Nobrega, F. G., Rola, F. H., Pasetta-Nobrega, M., Oishi, M.: Adenosine triphosphate associated with adenosine triphosphate-dependcnt deoxyribonuclease. Prec. nat. Aead. SoL (Wash.) 69, 15-19 (1972) Ohki, M., Tomizawa, J.-I.: Asymmetric transfer of DNA strands in bacterial conjugation. Cold Spr. Harb. Syrup. quant. Biol. 88, 651-658 (1968) Oishi, M. : An ATP-dependent deoxyribonuclease from Escherichia cell with a possible role in genetic recombination. Prec. nat. Acad. Sci. (Wash.) 64, 1292-1299 (1969) Paterson, M. C.: Use of a purified lesion recognizing enzyme to assay DNA repair in cultured animal cells. In: Proceedings of the Vth International Congress of Radiation Research, Seattle, Washington, U.S.A. (H. I. Adler, O.F. Nyegard and W . K . Sinclair, eds.). New York: Academic Press 1975 (in press) Paterson, M.C., Khachatourians, G. G., Worthy, T . E . : Strand-rejoining repair of y-raydamaged DNA in rec A-minicell producing strain of Escherichia coli. Abstracts Ann. Meeting Amer. See. Microbiol., G.222 (1973) Paterson, M.C., Roozen, K . J . : Photoreactivation, excision, and strand-rejoining repair in R factor-containing minicells of Escherichla coli K-12. J. Bact. 119, 71-80 (1972) Paterson, M.C., Setlow, R. B. : Endonucleotyic Activity from Micro~occus luteus that acts on y-Ray-Induced Damage in Plasmid DNA of Escherichia col{ Minicells. Prec. nat. Acad. Sol. (Wash.) 69, 2927-2931 (1972) Sheehy, R. J., Orr, C., Curtiss, R. I I L : Molecular studies on entry exclusion in Escherich{a coli minicells. J. Bact. 112, 861-869 (1972) Shlaes, D.M., Anderson, J . A . , Barbour, S.D.: Excision repair properties of isogenic recmutants of Escherichia coli K-12. J. Bact. 111, 723-730 (1972) Tomizawa, J.-I., Ogawa, H.: Structural genes of ATP-dependent deoxyribonuclease of Escherichia coll. Nature (Lend.) New Biol. 289, 14-16 (1972)
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Dr. Ronald J. Sheehy Department of Biology Morehouse College Atlanta, Ga. 30314, U.S.A. Dr. B. Van Dorp Laboratory for Molecular Genetics Leiden State University 64 Wassenaarseweg Leiden, 24 The Netherlands Dr. T. E. Worthy Division of Rheumatic and General Diseases Department of Medicine Duke University Medical Center Durham, North Carolina 27706 U.S.A.