Suppression of Tif-Mediated Induction of SOS Functions in Escherichia Coli

JOURNAL OF BACTERIOLOGY, Nov. 1979, p. 381-387 Vol. 140, No. 2 0021-9193/79/11-0381/07$02.00/0 Suppression of tif-Mediated Induction of SOS Functions in Escherichia coli by an Altered dnaB Protein RICHARD D'ARI,* JACQUELINE GEORGE,t AND OLIVIER HUISMAN Institut de Recherche en Biologie Moleculaire, Centre National de la Recherche Scientifique, 75221 Paris Cedex 05, France Received for publication 10 August 1979 The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in A lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tifexpression. Induction of A by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of X is known to proceed via repressor cleavage, presum- ably promoted by an activated (protease) form of the recA protein. Since A induction is normal after ultraviolet irradiation of the tif-1 dnaB252(X) strain, tif- mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation. In Escherichia coli, perturbations in DNA SOS response (6, 11, 19). There is evidence that replication induce a global cellular response, the this activation involves an effector generated so-called "SOS response" (24), including the ap- through DNA degradation at the replication pearance of repair and mutagenic activities, an fork (23). inhibition of septation, an inhibition of DNA The mutation tif-1, located in the recA gene degradation, the induction of the recA protein (4, 11, 19), causes, at high temperature, expres- ("protein X"), and the induction of certain pro- sion of all SOS functions without detectable phages such as A (see reference 32). A number of DNA damage or perturbations in DNA replica- DNA-damaging treatments are effective in this tion (2). The present work shows that the DNA induction: UV, X, or y irradiation, mitomycin C replication initiation mutation dnaB252 specifi- or nalidixic acid treatment, thymine starvation, cally suppresses tif-mediated SOS induction disruption of the replication fork by denatura- without affecting UV induction. This indicates tion ofthe dnaB, dnaG, orpolC (dnaE) protein, that, despite the absence of detectable altera- or introduction into the cell of certain UV-dam- tions in DNA replication during tif expression, aged plasmids. In all these cases, the induction the replication machinery is nevertheless in- process can be suppressed by mutations at the volved in this induction. The fact that UV in- recA locus (recA, zab, lexB) or at lexA. It has duction of A is not affected by the dnaB252 recently been shown that the purified recA prod- mutation suggests that tif expression is blocked uct can cleave purified A repressor in vitro (26). at the recA activation step and not in the pro- It has been hypothesized that the recA product, teolytic reaction, demonstrating the involve- in response to a signal from a perturbed repli- ment of the dnaB protein in this activation cation apparatus, may undergo a conformational process. change to an activated form thought to be a highly specific protease able to cleave the re- MATERIALS AND METHODS pressors of the various functions involved in the Bacterial and strains. The t After 5 years of heroic struggle against cancer, Jacqueline phage nonlysogenic George passed away on 14 August 1979. Despite weakened strains used in this study are listed in Table 1; all are health and debilitating therapy, she continued to stimulate derivatives of E. coli K-12. JM1 is the AB1157 strain and participate in the work of the microbial genetics group of Howard-Flanders and Theriot (14), reported to that she had created. carry in addition the markers xyl mtl ara thi tsx. All 381 382 D'ARI, GEORGE, AND HUISMAN J. BACTERIOL. TABLE 1. Bacterial strains' Strain Description Source (reference) JM1 thr leu pro his arg lac gal supE Strr (2) JM12 tifthr leu pro his arg lac gal supE Strr (2) GC2002 JM1 Arg+ malB Two-step transductant, of JM1, to Arg+ metA, then to Met' malB GC2004 JM12 Arg+ malB Two-step transductant of JM12, to Arg+ metA, then to Met' malB. GC2024 JM1 Arg+ dnaB252 Mal+ transductant of GC2002, donor strain 120/6 dnaB252 (1) GC2027 JM12 Arg+ dnaB252 Mal+ transductant of GC2004, donor strain 120/6 dnaB252 (1) GC2037 JM1 Arg+ dnaB70 Mal+ transductant of GC2002, donor strain Q1508 (5) GC2007 JM12 Arg+ dnaB70 Mal' tranaductant of GC2004, donor strain 91508 (5) GC2005 JM12 Arg+ dnaB6 Mal+ transductant of GC2004, donor strain Q1501 (5) GC2006 JM12 Arg+ dnaB59 Mal+ transductant of GC2004, donor strain Q1507 (5) GC429 dnaA46 ilv pro his lac gal supE Str' GC421 x JM1, Arg+Strr selection GC2021 tifdnaA46 ilv thr leu pro his lac gal GC421 x JM12(A), Arg+Strr selection, cured of A supE Str' GC2030 JM1 Thr+ dnaC325 Thr+ transductant of JM1, donor strain Q175 (5) GC2033 JM12 Thr+ dnaC325 Thr+ transductant of JM12, donor strain Q175 (5) JM620 thr leu pro his arg cysC reLA lac gal (4) supE Str' GC694 JM12 Leu+ sfiB Leu+ transductant of JM12, donor strain GC693 (7) HfrG6 malB his (PO 65 CW) (3) GC421 Hfr dnaA46 ilv thy (PO 7 CCW) (12) a See text. other strains in Table 1, except GC429, -GC2021, (2), and P1 phage were grown and titrated on LC JM620, HfrG6, and GC421, were derived from AB1157 plates (LB medium adjusted to pH 7.6 and supple- by a small number of mutational or Pl-transductional mented with 2.5 x lo-3 M CaCl2 and 1% agar [Difco steps and thus may carry any or all of these (untested) Laboratories]). alleles. The presence of the tif-I mutation in the tif-1 Chloramphenicol, when needed, was used at 25 ygl dnaB252 strain GC2027 was verified in two ways: first, ml; streptomycin sulfate was used at 200 ,ug/ml. it was transduced out of the strain into the cysC strain Transduction. Plvir was used by following a pro- JM620(X) (selection Cys+), where it conferred the nor- cedure described earlier (2). mal Tif phenotype, and second, when the dnaB252 Bacterial crosses. Bacterial crosses were done as mutation was eliminated from the tif- dnaB252 strain described earlier (3). by crossing with HfrG6 malB (selection Thr+ Leu+ Conditions for tifexpression. Exponential-phase SW), normal Tif recombinants were recovered. cultures growing in EMM at 30°C were transferred to Lysogens for A or Aind were formed by spotting the 40 or 420C, and 100 Ag of adenine per ml was added. phage on a bacterial lawn on a GT plate, incubating To monitor A induction, the optical density at 650 nm overnight at 300C, streaking out the bacteria growing was measured at regular intervals; after 120 min, chlo- inside the circle of lysis, purifying, and testing for roform was added, and the free phage were assayed. immunity; curing was by a similar process, except that To measure tif-induced lethality, viable bacteria were the phage spotted was Ab2imm21. Lysogens for assayed at regular intervals on LB plates incubated at PlCmbac-1 were selected by infecting exponential- 300C; incubation at 42°C was at cell densities greater phase cells in LB medium containing 2.5 x 10-' M than 108/ml to minimize the lethal effects of the CaCl2 and streaking on LB plates containing 25 jig of dhaB(Ts) mutations. chloramphenicol per ml. Detection of the recA protein. The polyacryl- The phage used were Apapa (16), Aind (15), aniide-sodium dodecyl sulfate gel technique was that Ab2imm21, Plvir' (29), and PlCmbac-1 (5), here re- used by Gudas (10). ferred to as Plbac. Media. Complete medium was LB medium (21), RESULTS which inhibits expression of the Tif phenotype in all Under certain conditions of perturbed DNA tif strains used. Minimal medium was M63 medium synthesis, the replication apparatus is thought (21) supplemented with thiamine (10 ,g/ml), glucose to generate a signal which activates the recA (0.4%), and required amino acids (100 Ag/ml). En- product, resulting in induction of the various riched minimal medium (EMM) contained, in addi- SOS functions. Thermal elevation of the tif-1 tion, Casamino Acids (0.4%); EMMA was EMM supplemented with adenine (100 pLg/ml) to enhance tif mutant also induces the SOS system. To test expression (2, 9). Guanosine and cytidine, when used whether tif-mediated SOS induction is coupled to inhibit tif expression (7), were added at 100 Lg/ml to DNA replication, tif expression was studied each. A phage were grown and titrated on GT plates in various tif-1 dna(Ts) double mutants. VOL. 140, 1979 SUPPRESSION OF tif BY AN ALTERED dnaB PROTEIN 383 The dnaB252 mutation is different from most (28). The complete prophage induction normally other dnaB mutations in that at 420C it blocks associated with tifexpression, however, is clearly specifically the initiation of new rounds of DNA suppressed by the dnaB252 mutation. In con- synthesis without affecting the progress of forks trast, UV irradiation induces complete lysis in already operative at the time ofthe temperature the tif-1 dnaB252(X) strain (Fig. 1), following shift (1, 33). Furthermore, phage A is able to the same kinetics as UV-irradiated wild-type or grow in dnaB252 bacteria at 420C (17). This is single-mutant lysogens (data not shown). in contrast to other known dnaB mutations, The massive induction of recA protein syn- which affect DNA chain elongation, either thesis normally associated with tif expression blocking both bacterial and A replication at non- (10) is not observed in the tif- dnaB252 double permissive temperatures [dnaB(Ts)] or specifi- mutant. As in the case of A induction, the cally preventing the replication of A DNA with- dnaB252 mutation causes a slight induction of out affecting that of chromosomal DNA (groP) recA, identical in the tif-1 and tir strains and (8). much less than that observed in the tif-1 dnaB+ The ability of tifto induce SOS functions was strain (data not shown). studied in a tif-1 dnaB252 double mutant. tif-1 In the tif-1 dnaB252 nonlysogen, the lethal dnaB252(A) bacteria are very poorly induced effect normally induced by tifis also suppressed under the usual coinditions for tifexpression (Fig. (Fig. 2); the fact that chloramphenicol prevents 1). The partial lysis observed also occurs in the cell death in the tif population confirms the tir dnaB252(A) culture and is due to a slight induced nature ofthis lethality, which is presum- induction of the prophage by the dnaB252 mu- ably due to the induced inhibition of septation tation, as evidenced (i) by the fact that the (7). corresponding Aind lysogens show no lysis (the The phage P1 is known to code for a function optical density rises to about 1.0 in 120 min ban analogous to dnaB. Plbac mutants, which under these experimental conditions; data not express this function constitutively in the pro- shown) and (ii) by a high level of free phage in phage state, suppress the temperature sensitiv- these cultures after 120 min at 420C. Induction ity of dnaB(Ts) strains (5, 22). The dnaB252 of A by dnaB252 has been reported previously mutation is no exception: dnaB252(Plbac) ly- , . WT ),x 2 W TA) WT 109

.ASl

0 s0 120 Time at 420 (min) tif FIG. 1. Suppression of tif-mediated induction of A by dnaB252. Cultures were grown to exponential phase in EMM at 30°C. At time zero, adenine was 0 60 120 added at 100 pg/ml, and the cultures were transferred Time at 42°(min) to 42°C and aerated vigorously. Samples were with- FIG. 2. Suppression of tif-induced lethality by drawnperiodically for optical density measurements. dnaB252. Experimental conditions were as described Strains were X-lysogenic derivatives of JM1 [WT(X), in the legend to Fig. 1. Viable bacteria were assayed xl JM12 [tif(AJ, 01 GC2024 [dnaB252(A), 1, GC2027 on LB plates at 30°C. Strains were JM1 (WIT, x), [tif dnaB252(V, A] and GC2027 LW irradiated (50 JM12 (tif-1, 0), GC2024 (dnaB252, *), GC2027 (tif J/m2) at time zero, then treated as the unirradiated dnaB252, A), and JM12, to which chloramphenicol cultures [tifdnaB252(;X) + UV, A]. (25 pg/ml) was added at time zero (tif + CM, 0). 384 D'ARI, GEORGE, AND HUISMAN J. BACTERIOL. sogens have 100% colony-forming ability at 420C broth, suggesting that they were not genetic (31a). In the tif-1 dnaB252 double mutant, the revertants. We shall assume that both pheno- presence of a Plbac prophage suppresses the typic characters are due to the dnaB252 muta- Dna(Ts) phenotype: tif-1 dnaB252(Plbac) ly- tion. sogens show no filamentation in EMMA at 42°C Since dnaB252 is a rather peculiar dnaB mu- and, indeed, form colonies on EMMA plates at tation, blocking only chromosome initiation but 420C with 100% efficiency; furthernore, in X- not affecting chain elongation or A replication, it lysogenic derivatives the prophage is not in- was possible that its suppression of tifis likewise duced under the usual conditions for tif expres- a special property. To test this, we examined a sion (Fig. 3), although induction and lysis are second double mutant, tif-1 dnaB70. The normal at 420C after UV irradiation (not shown). dnaB70 allele is an "immediate arrest" Similarly, tif-induced reversion of the his-4 dnaB(Ts) mutation, stopping chain elongation ochre mutation, observed in tif sfi strains at and preventing A replication at 420C. The lethal 410C in the presence of adenine (7), is com- effect induced by tif at 420C in the presence of pletely suppressed in the tif-1 dnaB252(Plbac) adenine is fully expressed and even enhanced in strain (data not shown). Finally, the presence of the double mutant (Fig. 4). A Plbac-lysogenic the Plbac prophage does not restore tif-pro- derivative has a DnaB+Tif phenotype: 100% col- moted induction of the recA protein. ony-forming ability at 420C on LB plates, where Control experiments showed that a Plbac pro- tif is not expressed, and 1 x 10-" to 2 x 1O-5 phage has no suppressive effect on tif-mediated survival on EMMA plates at 420C, due to tif- A induction in dnaB+ strains and that it is not induced lethality. Two other elongation muta- inducible by tif (Fig. 3 and unpublished data). tions, dnaB6 and dnaB59, were also transduced We tried to test whether thermosensitivity and suppression of the Tif phenotype could be I reverted simultaneously in the tif-1 dnaB252 "/X WT strain. Temperature-resistant colony formers 109 were isolated on LB plates at 420C at a fre- quency of 10-'. Among 100 tested, none had recovered the Tif phenotype. Three clones * dna B70 tested further, however, proved to be unstable for the temperature-resistant phenotype in LB ------0-- tif +CM

0I - tif dna B704CM

WT(Pibec)(A) 0 u OS. wx dna B 252 (PI bacXAI N .0 a R A* tif dna 8 252 (PlbecXM * 0 E * A 0 V 1 0 ~~~~tif

A.Atif dna B70 106 0 60 120 Time at 42'(min) 0 60 120 FIG. 4. Expression of tif-induced lethality in tif Time at 420(min) dnaB70. Experimental conditions were as described FIG. 3. Suppression of tif-mediated A induction in in the legend to Fig. 1. Viable bacteria were assayed the phenotypically Dna' strain tif-1 dnaB252 on LB plates at 30°C. Chloramphenicol (CM; 25 pg/ (PlCmbac)(A). Experimental conditions were as de- ml), when present (open symbols), was added at time scribed in the legend to Fig. 1. Strains were PlCmbac- zero. Strains were JM1 (WT, x), JM12 (tif, 0), 1, A double lysogens ofJM1 [WT(Plbac)(N, xl JM12 GC2037 (dnaB70, U), GC2007 (tif dnaB70, A), JM12 [tif(Plbac)(V, *01 GC2024 [dnaB252(P1bacX)(, 01 plus chloramphenicol (tif + CM, 0), GC2007 plus and GC2027 [tif dnaB252(P1bac)(A), A]. chloramphenicol (tifdnaB70 + CM, A). VOL. 140, 1979 SUPPRESSION OF tif BY AN ALTERED dnaB PROTEIN 385 into a tif-1 strain, and the resulting double mu- the culture remains normally inducible by mi- tants were lysogenized with Plbac. Like tif-1 tomycin C (data not shown); this partial inhibi- dnaB7O(Plbac), these two strains exhibited a tion of tifexpression is not observed in the tif-1 DnaB+Tif phenotype (temperature sensitivity dnaC325(A) strain under the same conditions. on EMMA plates, not on LB plates). Thus, the suppression of tifseems to be a special property DISCUSSION of the initiation mutation dnaB252 not general- izable to other dnaB(Ts) alleles. Inducing treatments (e.g., DNA damage) to A Two further mutations affecting the initiation lysogens result in proteolytic cleavage of the A of DNA replication, dnaA46 and dnaC325, were repressor (25); thermal elevation in the presence similarly crossed into a tif-1 strain. Since neither of adenine results in the same cleavage in tif- of these mutations prevents A growth, tifexpres- 1(X) lysogens (30). The recA product purified sion was monitored by the degree of cell lysis from recA+ or tif-1 strains has been shown to and phage production in A lysogens at 40°C in cleave purified A repressor in vitro (26). The tif- the presence of adenine. Neither mutation sup- promoted in vivo reaction takes place even if presses tif-mediated A induction under these chloramphenicol is added before the tempera- conditions (Fig. 5). If replication cycles are ter- ture shift (30); tif-induced synthesis of the recA minated before allowing tifexpression, however protein is known to be inhibited by chloram- (either by starving the culture for a required phenicol (18), suggesting that the tif protein amino acid for 150 min before the temperature acquires protease activity at high temperature shift or by adding guanosine and cytidine for the through modification of the preexisting recA first 90 to 120 min at 400C, then diluting into (tif) product. It is commonly hypothesized that EMMA), the degree of cell lysis is severely re- the wild-type recA protein similarly undergoes duced in the tif-1 dnaA46(X) lysogen, although a conformational change (activation) after inducing treatments (6, 11, 19). Recent evidence suggests that the effector responsible for stabi- lizing the protease configuration in recA+ strains dna C (A) may be generated by DNA at to degradation the dna A(A) replication fork when fork progression is abnor- mal (23). The spontaneous activation of the tif mutant protein at high temperature has not 0.5 * been explained. The fact that the dnaB252 mutation prevents tif-mediated induction of A, of lethality, of the 0.2 recA protein, and of mutagenesis strongly suggests that the replication apparatus is involved, O ~~~~~~~~~tifdna A(C directly or indirectly, in tif-mediated induction 0 - of the SOS functions, since the dnaB protein is thought to operate solely within the replication complex (20). The modifications of the replication machinery which interfere with tif-mediated SOS induction (dnaB252 and dnaA46) do not affect X induction under conditions of altered DNA replication (UV irradiation, mitomycin C treat- 0 60 120 ment, dnaB252 at 4200); when fork progression Time at 400(min) is blocked, the modified replication apparatus is FIG. 5. Expression of tif-mediated induction of A still able to generate the effector involved in the in tif dnaA46(A) and tif dnaC325(X). Overnight cul- activation of the recA protein, and activation tures were grown into exponential phase in EMM at and A induction take place normally. These ob- 30&C. At time zero, they were diluted to an optical servations suggest that under conditions of tif density (O.D.) at 650 nm of between 0.1 and 0.2, expression the dnaB252 and dnaA46 mutations adenine at 100 pg/mi was added, and the cultures do not prevent the proteolytic action ofthe recA were transferred to 400C and aerated vigorously. product, once activated; they may rather sup- Samples were withdrawnperiodically for optical den- induction sity measurements. Strains were A-lysogenic deriva- press A by preventing the activation of tives of GC429 [dnaA(A), 01 GC2021 ttif dnaA(X), the recA (tif) protein to its protease configura- 0], GC2030 [dnaC(,), Al and GC2033 [tif dnaC(X, tion. A]. The free-phage titers measured at 120 min were, Activation of the recA protein in the tif mu- respectively, 8 x 1i0, 2 x 109, 1.4 x 160, and 1.0 x 109. tant can be accounted for by different models. 386 D'ARI, GEORGE, AND HUISMAN J. BACTERIOL. (i) In the simplest one, tif activation is sponta- the dnaB70 mutation, which stops fork progres- neous at 400C, independent of effector. How- sion immediately on temperature shift, actually ever, the stimulatory effect of adenine and the enhances tif expression. inhibitory effect of guanosine plus cytidine have Specific suppression of tif expression without been more readily interpreted as suggestive of alteration of UV or mitomycin C induction can some effector requirement, the tif mutation de- also be achieved by the presence of guanosine fining the regulatory site of the recA protein for plus cytidine in the medium (unpublished data), effector interaction (4). (ii) The tif mutant pro- or by the presence of the plasmid R100.1 in the tein at 400C is activated by an analog of the strain (M. Bagdasarian, R. D'Ari, W. Filipowicz, normal effector, generated spontaneously by a and J. George, submitted for publication). The normal replication fork. (iii) The tif protein at presence of a lexA mutation likewise blocks tif- 400C is activated by the normal effector due ta mediated SOS induction completely, while al- an increased affinity for it or (iv) due to an lowing a slight but definite induction of A by UV abnormally high effector concentration. It is par- irradiation (3). It is conceivable that in these ticularly striking that the tif-1 mutation does not examples, as in the case of tif-1 dnaB252, it is sensitize bacteria to the rather poor induction of the recA (tif) activation step that is affected. X caused by the dnaB252 mutation at 420C (Fig. Recent results suggest the existence of different 1). Since UV induction of X is normal in these pathways leading to formation of the effector strains, the incomplete character of dnaB252- responsible for activation of the recA protein, mediated induction may be due to a limiting depending on the inducing agent used and dis- amount of effector. If this is the case, it suggests tinguishing, for example, UV irradiation or mi- that the tif-1 mutation does not affect the affin- tomycin C treatment from expression ofdna(Ts) ity of the recA protein for the effector. In agree- mutations or thymine starvation (31). Expres- ment with this suggestion, the last model implies sion of the tifmutation may be a novel pathway that the tif-1 mutation does not alter the site of of effector formation. the recA protein which recognizes the effector, In conclusion, we would like to point out var- but rather in some way affects the replication ious lines of evidence suggesting that the recA complex, causing it to generate effector gratui- protein may, under certain conditions, play an tously at high temperature. essential role in the DNA replication process. (i) Despite the speculative character of these "Stable DNA replication"-an SOS response models, in all cases the simplest hypothesis (Kogoma, Torrey, and Connaughton, in press) would be that the recA protein interacts directly conferring the ability to initiate new rounds of with replication proteins. A protein-protein in- replication in the absence of protein synthesis- teraction could: (i) inhibit spontaneous effector has been shown to be temperature sensitive in independent activation (model 1); (ii) inhibit the a recA(Ts) strain (17a). (ii) Sasakawa and Yosh- interaction of the recA (tif) protein with an ikawa (27) have described mutants of the plas- effector analog (model 2), or with the nornal mid R621a which restore temperature-resistant effector (model 3), although the latter seems DNA replication in a rec+ dnaG(Ts) strain but unlikely in view of the normal activation caused not in recA dnaG(Ts). (iii) The mutator locus by dnaB252 at 420C or after UV irradiation or dnaQ, because of the Dna(Ts) phenotype of the mitomycin C treatment; or (iii) inhibit effector dnaQ mutant, is thought to define a protein (or effector analog) production by the replication involved in DNA replication (13); the mut-8 complex (models 2 and 4). mutation, mapped at the same site, confers a Our results suggest that the dna(Ts) muta- recA-dependent mutator phenotype (12a). tions which interfere with tifexpression specifi- It is thus possible that the recA protein is a cally affect the initiation process. Nevertheless, nonessential component of the normal replica- tif-mediated SOS induction can take place nor- tion complex, perhaps an essential component of mally in the absence of initiation of new rounds the stable replication complex, and the tif-1 mu- of replication, as seen in the double mutants tant protein, through direct protein-protein in- tif-1 dnaA46(X) (without prealignment of the teraction with other replication proteins, may DNA molecules), tif-1 dnaC325(X), and tif-1 perturb the replication apparatus. dnaB70. If rounds of replication are allowed to terminate before the tif mutation is expressed, ACKNOWLEDGMENTS the dnaA46 mutation interferes considerably We thank Tokio Kogoma and Gerd Hombrecher for com- with tif-mediated induction of A. Nevertheless, municating results before publication. We are grateful to one cannot attribute this interference to the Antonia Kropfinger, Solange Dorsimont, Renee Marquise, Yvette Mallet, and Franfois Couessurel for services so cheer- absence of normal fork progression, since (i) the fully rendered. dnaC325 mutation under the same conditions O.H. was the recipient of a fellowship from the Ligue does not interfere with tif expression, and (ii) Nationale Franfaise contre le Cancer. This work was sup- VOL. 140, 1979 SUPPRESSION OF tif BY AN ALTERED dnaB PROTEIN 387 ported in part by grant A.T.P. no. 3058 from the Centre 17. Lanka, E., and H. Schuster. 1970. Replication of bac- National de la Recherche Scientifique. teriophages in Escherichia coli mutants thermosensitive in DNA synthesis. Mol. Gen. Genet. 106:274-285. 17a.Lark, K. G., and C. A. Lark. 1978. recA-dependent DNA LITERATURE CIED replication in the absence of protein synthesis: charac- teristics of a dominant lethal replication mutation, 1. Beyersmann, D., M. Schlicht, and H. Schuster. 1971. dnaT, and a requirement for recA+ function. Cold Temperature sensitive initiation of DNA replication in Spring Harbor Symp. Quant. Biol. 43:537-549. a mutant of Escherichia coli K12. Mol. Gen. Genet. 18. Maenhaut-Michel, G., A. Brandenburger, and S. Boi- 111:145-158. teux. 1978. Requirement of protein and RNA synthesis 2. Castellazzi, M., J. George, and G. Buttin. 1972. Pro- for A repressor inactivation by tif-1: effects of chloram- phage induction and cell division in Escherichia coli. I. phenicol, neomycin and rifamycin. Mol. Gen. Genet. Further characterization of the thermosensitive muta- 163:293-299. tion tif-l whose expression mimics the effect of UV 19. McEntee, K. 1977. Protein X is the product of the recA irradiation. Mol. Gen. Genet. 119:139-152. gene of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 3. Castellazzi, M., J. George, and G. Buttin. 1972. Pro- 74:5275-5279. phage induction and cell division in Escherichia coli. 20. McMacken, R., K. Ueda, and A. Kornberg. 1977. Mi- II. Linked (recA, zab) and unlinked (lex) suppressors of gration of Escherichia coli dnaB protein on the tem- tif-1 mediated induction and filamentation. Mol. Gen. plate DNA strand as a mechanism in initiating DNA Genet. 119:153-174. replication. Proc. Natl. Acad. Sci. U.S.A. 74:4190-4194. 4. Castellazzi, M., P. Morand, J. George, and G. Buttin. 21. Miller, J. H. 1972. Experiments in molecular genetics. 1977. Prophage induction and cell division in E. coli. V. Cold Spring Harbor Laboratory, Cold Spring Harbor, Dominance and complementation analysis in partial N.Y. diploids with pleiotropic mutations (tif, recA, zab and 22. Ogawa, T. 1975. Analysis of the dnaB function of Esch- lexB) at the recA locus. Mol. Gen. Genet. 153:297-310. erichia coli and the dnaB-like function of P1 prophage. 5. D'Ari, R., A. Jaffe-Brachet, D. Touati-Schwartz, and J. Mol. Biol. 94:327-340. M. Yarmolinsky. 1975. A dnaB analog specified by 23. Oishi, M., and C. L. Smith. 1978. Inactivation of phage bacteriophage P1. J. Mol. Biol. 94:341-366. repressor in a permeable cell system: role of recBC 6. Emmerson, P. T., and S. C. West. 1977. Identification DNase in induction. Proc. Natl. Acad. Sci. U.S.A. 75: of protein X of Escherichia coli as the recA+/tir gene 3569-3573. product. Mol. Gen. Genet. 155:77-85. 24. Radman, M. 1975. SOS repair hypothesis: phenomenol- 7. George, J., M. Castellazzi, and G. Buttin. 1975. Pro- ogy of an inducible DNA repair which is accompanied phage induction and cell division in Escherichia coli. by mutagenesis, p. 355-367. In P. C. Hanawalt and R. III. Mutations sfiA and sfiB restore division in tif and B. Setlow (ed.), Molecular mechanisms of repair of Ion strains and permit the expression of mutator prop- DNA. Plenum Publishing Corp., New York. erties of tif. Mol. Gen. Genet. 140:309-332. 25. Roberts, J. W., and C. W. Roberts. 1975. Proteolytic 8. Georgopoulos, C. P., and L. Herskowitz. 1971. Esche- cleavage of bacteriophage lambda repressor in induc- richia coli mutants blocked in lambda DNA synthesis, tion. Proc. Natl. Acad. Sci. U.S.A. 72:147-151. p. 553-564. In A. D. Hershey (ed.), The bacteriophage 26. Roberts, J. W., C. W. Roberts, and N. L Craig. 1978. lambda. Cold Spring Harbor Laboratory, Cold Spring Escherichia coli recA gene product inactivates phage Harbor, N.Y. A repressor. Proc. Natl. Acad. Sci. U.S.A. 75:4714-4718. 9. Goldthwait, D. A., and F. Jacob. 1964. Sur le meca- 27. Sasakawa, C., and M. Yoshikawa. 1978. Requirements nisme de l'induction du developpement du prophage for suppression of a dnaG mutation by an I-type plas- chez les bacteries lysogenes. C.R. Acad. Sci. 259:661- mid. J. Bacteriol. 133:485-491. 664. 28. Schuster, H., D. Beyersmann, M. Mikolajczyk, and 10. Gudas, L. J. 1976. The induction of protein X in DNA M. Schlicht. 1973. Prophage induction by high temper- repair and cell division mutants of Escherichia coli. J. ature in thermosensitive dna mutants lysogenic for Mol. Biol. 104:567-587. bacteriophage lambda. J. Virol. 11:879-885. 11. Gudas, L. J., and D. Mount. 1977. Identification of the 29. Scott, J. R. 1968. Genetic studies on bacteriophage P1. recA (tif) gene product of Escherichia coli. Proc. Natl. Virology 36:564-574. Acad. Sci. U.S.A. 74:5280-5284. 30. Shinagawa, H., K. Mizuuchi, and P. T. Emmerson. 12. Hirota, Y., J. Mordoh, and F. Jacob. 1970. On the 1977. Induction of prophage lambda by y-rays, mito- process of cellular division in Escherichia coli altered mycin C and tif repressor cleavage studied by immuno- in the process of DNA initiation. J. Mol. Biol. 53:369- precipitation. Mol. Gen. Genet. 155:87-91. 387. 31. Smith, C. L., and M. Oishi. 1978. Early events and 12a.Hombrecher, G., and W. Vielmetter. 1979. A recA mechanisms in the induction ofbacterial SOS functions: dependent mutator of Escherichia coli K-12: method analysis of the phage repressor inactivation process in of isolation and initial characterization. Mutat. Res. 62: vivo. Proc. Natl. Acad. Sci. U.S.A. 75:1657-1661. 7-17. 31a.Touati-Schwartz, D. 1979. A dnaB analog, ban, specified 13. Horiuchi, T., H. Maki, and M. Sekiguchi. 1978. A new by bacteriophage P1: genetic and physiological evidence conditional lethal mutator (dnaQ49) in Escherichia for functional analogy and interactions between the two coli. Mol. Gen. Genet. 163:277-283. products. Mol. Gen. Genet. 174:173-188. 14. Howard-Flanders, P., and L. Theriot. 1962. A method 32. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible for selecting radiation-sensitive mutants of Escherichia DNA repair in Escherichia coli. Bacteriol. Rev. 40: coli. Genetics 47:1219-1224. 869-907. 15. Jacob, F., and A. Campbell. 1959. Sur le systeme de 33. Zyskind, J. W., and D. W. Smith. 1977. A novel Esch- repression assurant l'immunite chez les bacteries lyso- erichia coli dnaB mutant: direct involvement of the genes. C.R. Acad. Sci. 248:3219-3221. dnaB252 gene product in the synthesis of an origin- 16. Kaiser, A. D. 1957. Mutations in a temperature bacterio- ribonucleic acid species during initiation of a round of phage affecting its ability to lysogenize E. coli. Virology deoxyribonucleic acid replication. J. Bacteriol. 129: 3:42-61. 1476-1486.