DNA content, synthesis and integrity in dividing and non-dividing cells of rec− strains of Escherichia coli K12

J. Mol. Bid.

(1975) 91, 53-66

DNA Content, Synthesis and Integrity in Dividing and Non-dividing Cells of Ret- Strains of Escherichia coli K12 FLORENCE

N. CAPALDO~

AND STEPHEN

D. BARBOUR

Department of Microbiology Case Western Reserve University School of Medicine Cleveland, Ohio 44106, U.S.A. (Received 3 September 1973, and in revised form 20 September 1974) Using a method to separate dividing from non-dividing cells of RetEscherichia co&i K12 (Capaldo t Barbour, 1973), we found: (1) Non-dividing cells of recA-, are defective in DNA synthesis.

(2) recArecA-

TecB-

recB-

recC-

and recA-

strains of

recB- recC- strains

non-dividing cells contain little or no DNA; recB- recC- and recC- non-dividing cells contain normal amoxmts of DNA.

(3) DNA synthesized in cultures of the recB- recC- and recA- recBstrains is made in the dividing cells and then segregated irreversibly, function of subsequent cell division, into the non-dividing cells.

recCas a

(4) Old DNA in recA- recB- recC- cells contains an increased number of single-strand breaks compared to newly synthesized DNA of tho same strain or to both old and new DNA from ret + cells ; no additional breaks in old DNA of the recB- recC- strain have been detected. We propose that recA+, recB+, recC+ gene products are involved in repair of DNA chain breaks that arise during cell growth. According to this view, mutation in the ret genes results in defective repair. This may lead to failure to replicate DNA, loss of viability, degradation of the DNA in the case of the recA- strain, and accumulation of DNA chain breaks in the recA - recB- recC- strain.

1. Introduction Strains of Escherichia coli K12 carrying mutations in any of the three ret genes, recA, recB and recC have a reduced ability to carry out genetic recombination and an increased sensitivity to agents which damage DNA, such as ultraviolet light, ionizing radiation and mitomycin C (Willetts $ Clark, 1969; Kapp & Smith, 1970). Moreover, a mutation in the recA gene results in increased rates of DNA degradation in unirradiated and u.v.-irradiated cultures, whereas mutations in the recB and recC genes result in reduced rates of DNA degradation and the absence of exonuclease V, an ATP-dependent deoxyribonuclease (Willetts t Clark, 1969; Oishi, 1969; Barbour & Clark, 1970; Tomizawa $ Ogawa, 1972). Finally, all three ret genes play a vital role in maintaining cell viability (Capaldo-Kimball & Barbour, 1971; Capaldo et al., 1974; Haefner, 1968; Hertman, 1969). t Present address: Department Stanford, Calif. 94306, U.S.A.

of Biochemistry, 53

School

of Medicine,

Stanford

University,

54

F. N. CAPALDO

AND

S. D.

BARBOUR

Ret- strains have significantly longer culture-doubling times than isogenic Ret+ strains in a variety of media (Capaldo-Kimball & Barbour, 1971). Viability (defined as the ratio of colony-forming cells to microscopically observable particles) ranges from 50% in a recA mutant to 18% in a recA - reck- recC- culture (Capaldo-Kimball & Barbour, 1971; Capaldo et al., 1974). Strains carrying mutations in either p&I or dam-3 (DNA adenine methylase) as well as in one of the ret genes (A, B or C) can be constructed only if one of the mutations is conditional and the strain is maintained under permissive conditions (Gross et al., 1971; Monk & Kinross, 1972 ; Smirnov et a$., 1973; Marinus dz Morris, 1974). Similarly, a strain carrying both the conditional Zig-4 mutation and a recA mutation is viable only at low temperature where the strain is phenotypically Lig + , and a strain carrying both lig-4 and recB is more viable at low than at high temperature (Gottesman et al., 1973). However, the specific biochemical role of the ret gene products in normal cell growth is thus far unknown. Cultures of Ret- strains are composed of three classes of cells: (i) viable cells, which can give rise to at least 20 generations of progeny; (ii) residually dividing cells, which can give rise to fewer than 20 generations of progeny (probably an average of fewer than six) ; and (iii) non-dividing cells, which are incapable of a single division (Capaldo et al., 1974). Viable cells are distinguished by their ability to form colonies when plated on solid medium. Non-dividing cells are distinguished by their inability to elongate when exposed to low concentrations of penicillin. Residually dividing cells elongate in the presence of penicillin, but are unable to give rise to a sutlicient number of generations of progeny to form a visible colony (Capaldo et al., 1974). One approach to understanding the role of the ret genes in growth is to study the non-dividing cells produced during the growth of Ret- strains. Since several of the functions of the ret genes involve the repair of DNA which has been damaged by irradiation and chemicals, it is plausible that the ret gene products might be required to repair DNA damage produced during normal growth processes such as transcription and replication. The non-dividing cells, produced as a result of the absence of normal ret gene products, would be likely to reveal this damage. Using the method previously described (Capaldo & Barbour, 1973) for isolating Ret- non-dividing cells, we have examined the content, synthesis, and integrity of DNA in the non-dividing as well as the dividing cells of recA-, recB- recC- and recA- recB- recC- strains. We have found that the non-dividing cells of all three strains are severely impaired in their ability to synthesize DNA. recA - non-dividing cells contain little or no DNA, whereas recB- recC- and recA- recB- recC- non-dividing cells contain normal amounts of DNA. Old DNA present in recA- recB- red- cells accumulates single-strand breaks or gaps.

2. Materials and Methods (a) Bacterial strains and medizcm used were E. coli K12. Their ret genotypes

All bacterial strains and other phenotypic properties are listed in Table 1. Nomenclature conforms to that of Demerec et aE. (1966). The genealogy of 504583, JC4684 and JO4688 is given by Capaldo-Kimball & Barbour (1971). SDB1006 was constructed by mating JC7505 (Hfr Thy+ Str’ ThrIlvSpcp recA56’ recB.21, obtained from A. J. Clark) with SDB1307 (a Thyderivative of JC4584, obtained by trimethoprim selection), selecting Thy + Thr + Ilv + recombinants and soreening these for the extreme U.V. sensitivity characteristic of recA - and recA- recB- strains. The presence in SDB1006 of the recC- allele was shown by the absence of complement&ion when SDB1006 was mated with Hfr JC5426 which is recA + recB+ red%!1 (Willets & Clark,

DNA

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Bacterial strains strain no. JC4683 JC4684 JC4688 SDB1006 SDB1307t

ret genotype

Reference

+ B21 c22 A56 A56 B21 C22 B21 C22

Berbour & Clark (1970) Cepaldo-Kimball & Barbour Barbour & Clark (1970) This paper This paper

All strains are F-,

His-,

Gal-,

Thi-,

(1971)

SW and EndA-.

t This strain is Thy-.

1969). SDB1307 is a Thyderivative of JO4584 obtained (Stacey & Simson, 1965). EM9 glycerol medium (Capaldo & Barbour, 1973) made acids was used for all experiments. (b) Isolation

of non-dividing

by

trimethoprim

selection

with

vitamin-free

Casamino

cella

The following procedures were used to obtain the results shown in Figs 1, 2 and 3. Methods for growth of the strains, penicillin treatment and velocity sedimentation have been desoribed (Capaldo & Barbour, 1973). All wash buffers were modified to contain 1 mg unlabeled thymidine/ml. Since uniform labeling of Reo- cultures with [14C]leucine results in the same ratio of counts per particle for both dividing and non-dividing cells, we have in these experiments grown our cells in the presence of 1 &i [U-14C]leucine/ml (SchwarzMann, 310 mCi/mmol for at least five generations and used the 14C radioactivity in each fraction as a measure of the number of cells present. Cells of all strains, not penicillintreated, sediment in a sharp band in the upper half of the gradient, as illustrated in Fig 3 of Capaldo & Barbour (1973). After penicillin treatment, all the cells in a WC+ culture grow into long filaments, and, sa a result, sediment in a broad band in the lower half of the gradient. However, sedimentation of penicillin-treated recB- WCC- and recArecB- recC- cultures reveals two distinct, though overlapping bands of cells. The band in the lower half of the gradient, analogous to that found with penicillin-treated ret+ cells, is comprised of cells which, prior to penicillin treatment, were dividing. As a result of penicillin treatment, these cells elongate, as do ret + cells. The band in the upper half of the gradient, analogous to that found with cells not treated with penicillin, is composed of cells which, prior to penicillin treatment, were not dividing. Approximately 40% of the cells in each of these Retcultures are non-dividing (recB- red-, 40.7f8.1; recArecB- WCC-, 46.0f9.2) and appear in this upper band (Capaldo & Barbour, 1973; Capaldo, unpublished data). However, in a recA- culture, the percentage of non-dividing cells is only 17.0f4.1 (Capaldo & Barbour, 1973; Capaldo, unpublished data). As a result, the non-dividing cells do not sediment in a distinct band apart from the dividing cells, but rather form a shoulder on the slower moving side of the dividing cell band (fractions 16 to 20). Approximately two-thirds of the total non-dividing cells are found in the shoulder of [rV]leucine radioactivity, the remainder overlap with the main band of radioactivity (Capaldo & Barbour, 1973). In all experiments using this isolation procedure, all radioactive labeling (both with [14C]leucine and with [3H]thymidine). and when appropriate, chasing with unlabeled thymidine, was carried out prior to penicillin treatment. Cultures were treated with penicillin in the presence of excess unlabeled leucine and unlabeled thymidine. Only the sedimentation profiles of penicillin-treated cultures will be shown. The gradients were analyzed by puncturing the bottom of the tube with a needle and collecting I2-drop fraotions (approx. 0.2 ml each, 23 fractions per gradient) onto Whatman

56

F. N. CAPALDO

AND

S. D.

BARBOUR

3MM filter discs. After collecting all fractions, the centrifuge tube was rinsed with 0.2 ml of 20% sucrose which was then spotted on a filter. After air drying the filters, each filter was saturated with 0.2 ml of 10% trichloroacetic acid. The filt,ers were again air dried, then washed three times with 5% trichloroacetic acid, twice with 95% ethanol and once with acetone, dried under a heat lamp and counted as described below. (c) Alkaline

sucrose gradients

The single-strand molecular weight distribution of the DNA was determined by alkaline sucrose sedimentation (Town et al., 1970). Six-drop fractions were collected onto Whatman 3MM filter discs, and the filters were washed as described above and counted as described below. The molecular weight of the DNA was calculated using the equations developed by Abelson & Thomas (1966) and Studier (1965). (d) Radioactive

labeling

of DNA

DNA Eyrynthesis in dividing and non-dividing cells was determined in the following mamer. Cultures were uniformly labeled with [14C]leucine, and then were pulse-labeled with [methyL3H]thymidine (New England Nuclear, 20 Ci/mmol) at 50 &i/ml for onetenth of a generation. Deoxyadenosine was present at 250 rg/ml and unlabeled thymidine at O-5 pg/ml. DNA content in dividing and non-dividing cells was determined by uniformly labeling cultures with [3H]thymidine. Cultures were grown overnight in 5 ml medium containing 1 ,.&i [14C]leucine/ml, 250 pg deoxyadenosine/ml (sufficient to allow incorporation of the [3H]thymidine during the overnight growth), 5 &i [3H]thymidine/ml and 0.5 pg unlabeled thymidine/ml (labeling medium). In the morning the cultures were diluted to an optical density at 650 nm of 0.4 absorbance units, using fresh labeling medium, and allowed to undergo one doubling. Each culture was diluted 1 : 1 with labeling medium and allowed to double. This dilution was repeated two more times. Each culture was then subjected to the procedure for isolating the non-dividing cells. DNA segregation was followed by uniformly labeling cultures with 1 &i [14C]leucine/ml during overnight growth. In the morning the cultures were diluted to an O.D. at 650 nm of O-3 absorbance units using fresh medium containing [r4C]leucine and allowed to undergo one doubling. The cultures were then diluted to an O.D. at 650 nm of 0.33 absorbance unit,s using fresh medium containing [14C]leucine. [3H]thymidine was added to a final concentration of 50 &X/ml, deoxyadenosine to 250 pg/ml and unlabeled thymidine to 0.5 pg/ml. The cultures were allowed to undergo one doubling and then were diluted 25-fold into fresh medium containing [r4C]leucine and 1 mg unlabeled thymidine/ml (a lOOO-fold excess). The cultures were incubated for three or six generations (diluted once during this time), Each culture was then subjected to the procedure for isolating the non-dividing cells. For single-label alkaline gradients, newly synthesized DNA was specifically labeled by following the same labeling protocol as described for the DNA synthesis experiment, except that the uniform labeiing with [r4C]leucine was omitted and the pulse length was increased to one-half of a generation. Old DNA was specifically labeled by following the same labeling protocol as described for the DNA segregation experiment, except that the uniform labeling with [14C]leucine was omitted and the culture was chased for six generations. Under these conditions, 60 to 70% of the radioactive label is found in the nondividing cells. For double-label alkaline gradients, Ret + cells were labeled with 20 &i [methyZ-14C]thymidine/ml (New England Nuclear, 51.5 mCi/mmol) for two doublings. Deoxyadenosine was present at 250 pg/ml final concentration. Retcells were labeled with 20 &i [“HIthymidinelml for one doubling. Deoxyadenosine was present at 250 rg/ml and unlabeled thymidine at 0.5 pg/ml final concentration. (e) Scintillation

counting

Dry filters were added to 10 ml scintillation fluid (4 g of 2,5diphenyloxazole and 0.1 g of l,&bis-2-(5-phenyloxazolyl)-benzene per liter of toluene) and assayed for radioactivity in a Packard tricarb liquid scintillation spectrometer. In single-label experiments 3H

DNA radioactivity counted with

IN

REC-

was counted with 30% e ffi ciency. 12% and i4C with 40% efficiency. (f) Biochemid

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double-label

67 experiments

3H was

assays on unfraCtiO?Uhd cultures

Exponentially growing cells (approx. 2 x lOa particles/ml) were harvested at 6000 revs/mm for 10 min in the GSA rotor of the Sorvall RCBB centrifuge. The cells were washed twice with MS salts (Capaldo-Kimball & Barbour, 1971) and resuspended in M9 salts to a concentration of 3 X lOlo particles/ml. The amount of DNA/ml of the cell suspension was assayed using the modified Dische diphenylamine technique (Burton, 1956). Deoxyadenosine (California Biochemical Research) was used as a standard. The amount of protein/ml of the cell suspension was assayed according to the method of Lowry et al. (1951). Bovine serum albumin WM used as a standard. The total number of cells/ml of the cell suspension was determined by using a LevyHausser counting chamber at a magnification of 400 x .

3. Results (a) DNA synthesis Figure

1 shows

pulse-labeled

Rottom

with

the results of an experiment in which Ret + and Ret - cultures were [3H]thymidine for one-tenth of a generation. In each panel, the

Fraction no

FIG. 1. DNA

Bottom

synthesis

Fraction no

in dividing

and non-dividing cells. SBD1006. -O-O-, [14C]leucine

(a) JC4583; (b) JC4684; (0) JC4688; (d) radioactivity; --A-A-, [“H]thymidine radioactivity. Cultures were uniformly labeled with [“Cjleuoine and then were pulse-labeled with [3H]thymidine for one-tenth of a generation. Each culture w&8 treated with penicillin and was sedimented through a neutral sucrose gradient to separate dividing and non-dividing cells. The gradients were collected and each fraction was analyzed for 3H and ‘% radioactivity.

F. N. CAPALDO

68

AND

S. D. BARBOUR

profile of incorporated [14C]leucine indicates the regions of the gradient in which cells have banded. In the ret+ culture (a) there is a single broad band of dividing cells, and, coincident with this, a band of incorporated [3H]thymidine. In the recB- redand recA- recB- recC- cultures (b) and (d) two bands of cells, dividing and nondividing, can be seen. However, only the dividing cell band contains a substantial amount of incorporated [3H]thymidine. Similarly, in the recA- culture (c), there is very little incorporated [3H]thymidine found in the shoulder of non-dividing cells. These results indicate that the non-dividing Ret- cells present in a liquid culture before penicillin treatment are severly impaired in their ability to incorporate r3H]thymidine into DNA. We have obtained identical results with SDB1307, a Thyderivative of JC4584 (data not shown). Therefore, we consider it unlikely that the non-dividing cells are able to synthesize DNA (using endogenously synthesized thymidine), but are unable to incorporate the exogenously supplied radioactive thymidine. Rather, they appear to be impaired in DNA synthesis itself. Two possible reasons for this impairment in DNA synthesis are either, that non-dividing cells do not contain DNA or, that non-dividing cell DNA is in some way damaged. The following experiments test these possibilities. (b) DNA content Using biochemical assays, we have determined the amount of DNA and protein per particle in unfractionated Ret + and Ret- cultures. These results are summarized in Table 2. Whereas the recB- recC- and recA- recB- recC- strains contain the same TABLE

2

Biochemical assays on unfractionated cultures

Strain

mg protein

pg DNA/

mg protein/ 1O’O partiales

/% DNA/ lOlo partiales

Average number of chromosomes per particle?

ret + recB- red’recA recA - ret B - red -

22.8 23.8 16.8 22.6

3.11 3.63 3.41 3.69

70.9 86.4 67.3 83.4

2.13 2.69 l-12 2.60

t Baaed on a molecular

weight

of 2 x 10°.

amount of DNA per milligram protein as the ret+ strain and somewhat more (20%) when calculated on a per particle basis, the recA - cells contain only 75% as much DNA per milligram protein as the ret + strain and 80% as much when calculated on a per particle basis. Hout & Schuite (1973) have reported 15% less DNA per gram wet weight in recA- cells compared with ret+ cells. To determine whether the lower amount of DNA in the recA- culture results from a specific absence of DNA in the non-dividing cells, we uniformly labeled Ret+ and Ret- cultures with [3H]thymidine and isolated the non-dividing cells. Figure 2 shows the results of this experiment. In the ret+ culture (a) there is a single broad band of dividing cells and, coincident with this, a band of incorporated [3H]thymidine. In the recB- recC- and recA- recB- recC- cultures ((b) and (d)) two bands of cells,

DNA 3.0

IN

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REC 12

I.3

6

” 16IO ri0 148g :E 12-

“0 25 x .g 2.0 ” 0 Yi .c 15 ‘: -; IO JI 05

5 4

n

n

7

69 recf3- recc-

(b)

5 “0 4 .G x < ‘” 3 <

ZIO-

6 -2 4

2

6 5 ,’ Pf , :?,

6-

2

3 342 I 2-

Bottom

Fraction no

I

I

nl

Bottom

I

Fraction r-o.

FIQ. 2. DNA content of dividing and non-dividing cells. (a) JC4683; (b) JC4684; (a) JC4588; (d) SDB1006. -O-O-, [“C]leucine radioactivity; -A--A-, [3H]thymidine radioaotivity. Cultures were uniformly labeled with [“C]leucine and with [3H]thymidine. Each culture was treated with penicillin and was sedimented through a neutral auorose gradient to separate dividing and non-dividing cells. The gradients were collected and each fraotion was analyzed for SH and l*C radioactivity.

dividing and non-dividing, can be seen and both bands contain incorporated [3H]thymidine. In both cases there is a slightly higher ratio of 3H/14C radioactivity in the non-dividing cell band. In contrast, in the recA- culture (c), the ratio of 3H/14C radioactivity in the region of the gradient which is enriched for non-dividing cells (fractions 17 to 20) is only 40% of the ratio of 3H/14C radioactivity in the main band of cells (fractions 5 to 15). These results indicate that the non-dividing recA- cells contain little or no DNA, whereas the non-dividing recB- recC- and recA- recBrecC- cells contain normal or slightly higher than normal amounts of DNA. If the recA- non-dividing cells (17% of the culture) completely lacked DNA but the dividing cells had the normal (ret+) amount, one would expect the unfractionated recA culture to contain 83% as much DNA as the ret+ culture. The observed values (75 to 85%) agree closely with the predicted value. (c) DNA segregation Since recB- red- and recA- recB- recC- non-dividing cells contain DNA but are defective in the synthesis of DNA, one would expect that the DNA found in these cells was originally synthesized in dividing cells, segregated into residually dividing cells, and finally trapped in non-dividing cells. Figure 3 shows the results of an ex-

60

F. N. CAPALDO

AND

S. D. BARBOUR

6----r-~---r---(b)

Fraction no.

FIG. 3. Segregation of DNA into non-dividing cells. (a) JC4683; (b) JC4684; (c) SDB1006. -O-O--, [‘*C]leucine radioactivity; -A-A-, [3H]thymidine radioactivity. Cultures which had been uniformly labeled with [‘*C]leucine were puke-labeled with [3H]thymidine for one generation, and then were grown in the presence of exoess unlabeled thymidine for three generations. Each culture was treated with penicillin and was sedimented through a neutral sucrose gradient to separate dividing and non-dividing cells. The gradients were collected and eaoh fraction was analyzed for 3H and “C radioactivity.

periment in which Ret+ and Ret- cuhures were p&e-labeled with [3H]thymidine, then chased with unlabeled thymidine for three generations. In the ret+ culture (a) there is a single broad band of dividing cells, and, coincident with this, a band of incorporated r3H]thymidine. In the recB- recC- and recA- recB- recC- cultures ((b) and (c)) two bands of cells, dividing and non-dividing, can be seen, and in both cultures more than half of the incorporated [3H]thymidine is found in the nondividing cell band. After a six-generation chase, 60 to 70% of the incorporated [3H]thymidine has moved from the dividing cell band into the non-dividing cell band (data not shown). These results indicate that the DNA present in recB- red?- and recArecB- recC- non-dividing cells was originally synthesized in dividing cells. In a recAculture (data not shown) there is no movement of incorporated [3HJthymidine into the non-dividing cell shoulder after a three or a six-generation chase, consistent with an absence of DNA in these cells. (d) DNA damage The products of all three ret genes are required for the repair of X-ray-induced single-strand breaks in the DNA. Single-strand breaks may occur during DNA

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replication and perhaps also during transcription, and it has been suggested that the inviability of Ret - strains is a result of improper or inadequate repair of these breaks (Capaldo-Kimball & Barbour, 1971; Capaldo & Barbour, 1973; Monk et al., 1973). We have specifically looked for an accumulation of single-strand breaks in newly synthesized and in old DNA using alkaline sucrose gradients. The results are shown in Figure 4.

recB-

recA-

recC-

recB-

recC-

recB-

Bottom

Fraction no

Bottom

ret C-

Froctlon no.

FIG. 4. Alkaline sucrose gradients of newly synthesized and old DNA. (a), (b) and (c) Newly synthesized Reo- DNA is labeled with [sH]thymidine (-A-A-) and Ret+ (JC4683) DNA, run as a marker in each gradient, is labeled with [14C]thymidine (--O--O--). (a) JC4684 (100% = 1271 irC cts/min, 4608 3H cts/min;) (b) JC4688 (100% = 1262 l&C ots/min, 3448 sH &s/mm); (c) SDB1006 (100% = 1193 “C! cts/min, 3220 3H cts/min). The data shown in (d) are taken from four representative single label ([3H]thymidine) gradients. For each strain, triplicate gradients containing either newly synthesized or old DNA were prepared and centrifuged at the same time using the six-place SW60.1 rotor. Each experiment was done twice. In every gradient of recA- recB- re.cC- old DNA we observed the abnormal DNA distribution illustmted in (d). (0, 0) JC4683; (A, A) SDB1006. (A) Newly synthesized DNA; ( A ) old DNA. A single curve has been drawn to fit the two ret + sets of data and the recA - recBrecC- newly synthesized DNA date. 100% = 27,993 cts/min (0); 1306 cts/min (a); 9186 ots/min (A); 1277 ots/min (A).

In Figure 4 (a), (b) and (c), [3H]DNA from each of the Ret- strains labeled for one generation (predominantly dividing cell DNA) is compared with l*C-labeled ret+ marker DNA run in the same gradient. In all cases, the single-strand molecular weight distribution of newly synthesized Ret- DNA is indistinguishable from that of Ret+ DNA. Figure 4 (d) compares the single-strand molecular weight distribution of old DNA from the ret+ and from the recA- recB- red- strains with newly synthesized DNA

02

F. N. CAPALDO

AND

S. D. BARBOUR

from the same strains. When ret+ cells are labeled with [3H]thymidine for one generation and chased with unlabeled thymidine for six generations there is no observable change in the single-strand molecular weight distribution of the DNA compared with DNA from unchased cells. However, when a recA- recB- recC- culture is pulsed and chased in the same manner there is a significant decrease in the size of the DNA (d). We observed no increase in the amount of smaller molecular weight DNA when either the recA- or the recB- recC- cultures were chased for six generations. These results indicate that there is an accumulation of single-strand breaks or gaps in the old DNA of recA- recB- recC- cells.

4. Discussion The products of the three ret genes, A, B and C are required for normal growth and viability of E. wli K12 (Capaldo-Kimball & Barbour, 1971). During normal growth of Ret- mutants an event may occur in a cell which restricts the number of subsequent generations of progeny to which that cell will be able to give rise (Haefner, 1968; Capaldo et al., 1974). The nature of this event is not known, nor is it known whether this event reflects a single lethal event or the accumulation of sublethal damage to a critical level. In the studies reported in this paper terminal non-viable cells were used specifically, i.e. those which can no longer divide. These cells may exhibit most clearly the damage which occurs aa a direct effect of the absence of normal ret gene products. However, secondary kinds of damage may also occur in these cells which are non-viable and non-dividing. First, we have found that non-dividing cells of all three Ret- strains are unable to synthesize normal amounts of DNA. We consider it likely that recB- recC- and recA- recB- recC- non-dividing cells are unable to synthesize DNA at all and that the incorporated [3H]thymidine found in the non-dividing cell band (Fig. 1 (b) and (d)) reflects a trailing of dividing cells (see Capaldo & Barbour, 1973, Fig. 4 (b)). This may also be true of the WA- culture (Capaldo & Barbour, 19’73, Fig. 4(c)). Because of the small size of the non-dividing cell fraction, we cannot draw this conclusion with the same degree of certainty for the recA- strain as for the other two Retstrains. We cannot clearly distinguish at this time whether the defect in DNA synthesis is the primary cause of the non-viability or merely a secondary effect of the primary damage. However, two observations suggest that in the recB- recC- and recA- recB recC- strains DNA synthesis occurs at near normal rates in non-viable cells until the cells can no longer divide, First, non-viable cells of these two mutants undergo an average of two to fiveresidual generations (Capaldo et al., 1974), producing non-dividing cells which contain normal or even slightly higher than normal amounts of DNA per particle. Thus it is unlikely that the residual divisions reflect the segregation of chromosomes which were synthesized in viable cells. Rather, DNA was being synthesized in cells undergoing residual divisions. Secondly, the rate of incorporation of [3H]thymidine into trichloroacetic acid-insoluble material in unfractionated Retcultures is reduced relative to the Ret+ culture, but this reduction is concomitant with the reduction in growth rate (F. Capaldo, unpublished data). This is consistent with the non-dividing cells not synthesizing DNA at all, but all dividing cells (viable and non-viable) synthesizing DNA at the Ret + rate. Thus, we believe that the inability of recB- recC- and recA- recB- recC- non-dividing cells to synthesize DNA is not,

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itself, the primary effect of the absence of normal ret gene products, but is rather a secondary effect resulting from a defect in DNA repair. This would lead to accumulation of damage in the chromosome and failure to replicate. Alternatively, the DNA synthesized in the residually dividing cells may be abnormal for reasons other than a defect in DNA repair, but would have the same effect of blocking DNA replication. The second important linding we have reported is that recA- non-dividing cells contain little or no DNA. As with the DNA synthesis experiment, we believe that the incorporated [3H]thymidine found in the non-dividing cell band (Fig. 2(c)) reflects a trailing of dividing cells. Although this contamination prevents us from stating categorically that recA- non-dividing cells totally lack DNA, it appears certain that recA - cultures have reduced amounts of DNA relative to ret+ cultures (Hout & Schuite, 1973; our results) and that the deficiency is most pronounced in the nondividing cells. The absence of, or deficiency in, DNA in the recA- non-dividing cells could result degradation, and experiments from either faulty DNA segregation or from chromosome are currently in progress to test these alternatives. However, in this regard, it is significant that recA- recB- recC- non-dividing cells contain normal amounts of DNA, and further that in the triply mutant strain, old DNA is noticeably altered compared with newly synthesized DNA. That old DNA in the recB- recC- strain a,ppears normal suggests that the DNA damage in the recA- recB- recC- strain is not a result of non-specific DNA degradation caused by non-viability. Rather, we suggest that the accumulation of single-&and breaks is a direct consequence of the absence of the recA gene product. We propose that in a recA single mutant (recB+ red+) these breaks are immediately attacked by exonuclease V, the product of the recB and recC genes (Tomizawa & Ogawa, 1972), and completely degraded. This would account for the absence of intermediate size pieces of DNA in our recA alkaline sucrose gradients. This is consistent with the known mode of attack of this enzyme in vitro (Goldmark & Linn, 1970,1972). Probably the excessive DNA breakdown observed in recA- strains, even under normal growth conditions (Clark et al., 1966; Howard-Flanders & Theriot, 1966; Willetts & Clark, 1969), reflects the complete degradation of chromosomes in the non-dividing cells rather than a low level of degradation in all cells of the population. The reduced level of diphenylamine-reactive material in the unfractionated recA- culture indicates that the breakdown products are released from the cells. The introduction of a recB- or a recC- mutation (resulting in a loss of exonuclease V activity) into a recA- strain blocks the excessive DNA breakdown (Willetts & Clark, 1969). We believe that as a consequence of blocking the degradation of damaged chromosomes, the initial damage, in the form of singlestrand nicks or gaps, is stabilized and perhaps even accumulates. Thus we observe this DNA damage in alkaline sucrose gradients prepared from the recA - recB- red?strain (Fig. 4(d), solid triangles). We have further analyzed this data in the following mamer. When the recA - recBrecG’- strain is labeled with [3H]thymidine for one generation and then chased with unlabeled thymidine for six generations, 65% of the trichloroacetic acid-precipitable 3H radioactivity is found in non-dividing cells; the remainder is found in dividing cells. Thus, the alkaline sucrose sedimentation profile of DNA from cells labeled in this manner is actually a composite of the dividing cell DNA profile and the nondividing cell DNA profile. We know the proportionate contributions of each component and we know the sedimentation profile of dividing cell DNA (Fig. 4(d), open triangles).

64

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BARBOUR

We assume that in the recA - recB- recC- culture, dividing cell DNA has the same single-strand molecular weight distribution whether it is newly synthesized or is six generations old (as is true for ret + DNA). On this basis we have determined the sediment(ation profile of non-dividing cell DNA by subtracting the contribution of dividing cell DNA from the total. This analysis is shown in Figure 5. Non-dividing cell DNA sediments in a very broad band. The molecular weight of the DNA in several of the fractions has been calculated using the equations developed by Abelson & Thomas (1966) and Studier (1965) and are shown in Figure 5. Much of the non-dividing cell DNA is one-half to one-seventh as large as the largest DNA isolated from dividing cells; however, we do not see appreciable amounts of DNA more degraded than this.

I I-- r---7-3-5

3.7 x IO’ I

Bottom

Fraction

-7

no

FIQ. 5. Analysis of the sedimentetion profile of DNA isolated from pulse-chased recA - recBrecC- cells. -- 0 -- O--, Dividing cell DNA; -A--A---, non-dividing cell DNA. The moleculer weight of fractions 12, 21 and 33 is indicated. The analysis is described in the Discussion.

Although Kapp & Smith (1970) looked at the single-strand molecular weight profile of u&radiated recA- recB- DNA and saw no increase in smaller molecular weight pieces, this would be expected since their labeling protocol would result in their looking at predominantly dividing cell DNA. Monk and co-workers have observed that when the polA recA double mutant is shifted to the non-permissive temperature it immediately begins to degrade its DNA (Monk $ Kinross, 1972) but that there is no change in the single-strand molecular weight distribution (Monk et al., 1973). Furthermore, they found that introduction of the recB- allele into the polA- recA- strain blocked the DNA breakdown but did not improve the viability of the strain at the non-permissive temperature These results are consistent with our own observations. A plausible function for the recA gene product would be to provide or help maintain a particular conformation of the DNA which would at once facilitate the repair of certain single-strand breaks or gaps and limit or eliminate the exonucleolytic attack by exonuclease V at the site of the breaks. A similar function was suggested by Tomizawa & Ogawa (1968) on the basis of the different sensitivities of ret + and recA strains and phages grown on these strains to DNA breakage caused by the radioactive decay of incorporated aaP.

DNA

IN

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E. COLI

66

If breaks occur normally in the course of cell metabolism, then, because the recA strain retains considerable viability, it must possess some mechanism for repairing these breaks. Since the products of the recA, recB and red? genes on the one hand, and DNA polymerase I on the other hand, comprise the only two independent systems currently known for the repair of X-ray-induced single-strand breaks in the DNA (Town et al., 1971) it is likely that in ret- strains polymerase I provides the alternative mechanism for the repair of single-strand breaks produced during cell growth. This would explain why all polA ret double mutants are inviable (Gross et al., 1971; Monk & Kinross, 1972; Smirnov et al., 1973). We still do not know why strains carrying mutations in the recB and recC genes have low viabilities. Since these mutants are also defective in the repair of X-rayinduced single-strand breaks, and since the alkaline sucrose sedimentation technique is not sufficiently sensitive to detect the introduction of only a few additional breaks, it remains possible, and we think likely, that exonuclease V functions in the repair of single-strand breaks or gaps produced during normal cell growth. ExonucIease V may be absolutely required to repair a minority class of breaks in the chromosome, having perhaps a particular structure or location. Thus, we postulate that the primary lethal event in all three Ret- strains is the failure to repair adequately single-strand breaks or gaps in the chromosome produced during normal cell growt,h. These breaks may be required to allow replication (Cairns, 1963; Ogawa et al., 1968) and transcription (Pauling & Hanawalt, 1965) or may be produced as a result of discontinuous DNA replication (Sugino et al., 1972). This investigation was supported by research grant GM17329 of the United States Public Health Service, Public Health Service training grant GM00171 from the National Institute of General Medical Sciences, and Public Health Service research career development award GM38140 from the National Institute of General Medical Sciences. We thank David Shlaes for helpful discussion and criticism of this work. This work is taken from a thesis submitted by F. Capaldo in partial fulfillment of the requirements for the Ph.D. degree, Case Western Reserve University, 1973.

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19,442-454. Demerec, M., Adelberg, E. A., Clark, A. J. & Hartman, P. E. (1966). Genetics, 54, 61-76. Goldmark, P. J. 85 Linn, S. (1970). Proo. Nat. Aoad. Sci., U.S.A. 67, 434-441. Goldmark, P. J. & Linn, S. (1972). J. BioZ. Chem. 247, 1849-1860. Gottesman, M. M., Hicks, M. L. & Gellert, M. (1973). J. Mol. BioZ. 77, 531-547. Gross, J., Grunstein, J. & Witkin, E. M. (1971). J. Mol. BioZ. 58, 631-634. Haefner, K. (1968). J. Bactetiol. 96, 652-659. Hertman, I. M. (1969). Genet. Rm. 14, 291-307. Hout, A. & Schuite, A. (1973). Biochim. Biophgs. Actu, 308, 366-371. Howard-Flanders, P. & Theriot, L. (1966). Genetics, 53, 1137-1150. Kapp, D. S. & Smith, K. C. (1970). J. Bacterial. 103, 49-53.

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Lowry, 0. H., Rosebrough, N. J., Fsrr, A. L. & Randall, R. J. ( 1951). J. Biol. Chem. 193, 265-275. Marinus, M. G. & Morris, N. R. (1974). J. Mol. Bid. 85, 309-322. Monk, M. & Kinross, J. (1972). J. Bacterial. 109, 971-978. Monk, M., Kinross, J. & Town, C. (1973). J. Ba.&rioZ. 114, 1014-1017. Ogaws, T., Tomizawe, J.-I. & Fuke, M. (1968). Proc. Nat. Ad. Sci., U.S.A. 60, 861-865. Oishi, M. (1969). Proc. Nat. Acud. Sci., U.S.A. 64, 1292-1299. Pauling, C. & Hanswalt, P. (1965). Proc. Nut Acad. Sci., U.S.A. 54, 17281735. Smirnov, G. B., Filkovs, E. V., Skavronskaya, A. G., Saenko, A. S. & Sinzinis, B. I. (1973). Mol. Gen. Genet. 121, 139-150. Stscey, K. A. & Simson, E. (1965). J. Bacterial. 90, 554555. Studier, F. W. (1965). J. Mol. BioZ. 11, 373-390. Sugino, A., Hirose, S. & Okazaki, R. (1972). Proc. Nat. Ad. Sci., U.S.A. 69, 1863-1867. Tomizawa, J.-I. & Ogawa, H. (1968). Cold Spring Harbor Symp. Quant. BioZ. 33, 243-251. Tomizawa, J.-I. & Ogrtwa, H. (1972). Nature New BioZ. 239, 14-16. Town, C. D., Smith, K. C. & Kaplan, H. S. (1970). J. Bueteriol. 105, 127-135. Town, C., Smith, K. C. & Kaplan, H. S. (1971). Science, 172, 851-854. Willetts, N. S. & Clark, A. J. (1969). J. Bacterial. 100, 231-239.