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The Escherichia Coli Polb Gene, Which Encodes DNA Polymerase II, Is Regulated by the SOS System HIROSHI IWASAKI,' ATSUO NAKATA,1 GRAHAM C

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The Escherichia Coli Polb Gene, Which Encodes DNA Polymerase II, Is Regulated by the SOS System HIROSHI IWASAKI,' ATSUO NAKATA,1 GRAHAM C JOURNAL OF BACTERIOLOGY, Nov. 1990, p. 6268-6273 Vol. 172, No. 11 0021-9193/90/116268-06$02.00/0 Copyright © 1990, American Society for Microbiology The Escherichia coli polB Gene, Which Encodes DNA Polymerase II, Is Regulated by the SOS System HIROSHI IWASAKI,' ATSUO NAKATA,1 GRAHAM C. WALKER,2 AND HIDEO SHINAGAWA1* Department ofExperimental Chemotherapy, Research Institute for Microbial Disease, Osaka University, Suita, Osaka 565, Japan,1 and Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 021392 Received 7 June 1990/Accepted 13 August 1990 The dinA (damage inducible) gene was previously identified as one of the SOS genes with no known function; it was mapped near the leuB gene, where the poiB gene encoding DNA polymerase H was also mapped. We cloned the chromosomal fragment carrying the dinA region from the ordered Escherichia coli genomic library and mapped the dinA promoter precisely on the physical map of the chromosome. The cells that harbored multicopy plasmids with the dimA region expressed very high levels of DNA polymerase activity, which was sensitive to N-ethylmaleimide, an inhibitor of DNA polymerase II. Expression of the polymerase activity encoded by the dinA locus was regulated by SOS system, and the dinA promoter was the promoter of the gene encoding the DNA polymerase. From these data we conclude that the polB gene is identical to the dinA gene and is regulated by the SOS system. The product of the polB (dinA) gene was identified as an 80-kDa protein by the maxicell method. In Escherichia coli, three DNA polymerases have been pSY343 (27), pSCH18 (10), and pRS528 (22) were used for identified (15). DNA polymerase III has been shown to be cloning. An E. coli gene library constructed with lambda essential for chromosomal DNA replication and to consist of vectors was kindly provided by Y. Kohara (14). LB medium multiple species of subunits. The nucleotide polymerizing was described by Miller (18). activity resides in the a subunit encoded by the dnaE (poIC) Materials. [a-32P]TTP, [35S]methionine, [35S]cysteine, and gene. DNA polymerase I, encoded by polA, has been shown 14C-labeled proteins for molecular weight standards were to be involved in DNA repair and to be required for DNA purchased from Amersham Japan (Tokyo, Japan). Mitomy- replication when the cell is growing fast on rich medium (11). cin C (MMC) and N-ethylmaleimide were from Kyowa However, no biological role has been assigned to DNA Hakko (Tokyo, Japan) and Sigma (St. Louis, Mo.), respec- polymerase II, encoded by polB. The polB mutants that have tively. The enzymes used for recombinant DNA techniques been isolated exhibit no defect in DNA replication and DNA were from Takara Shuzo (Kyoto, Japan). repair (5, 9). Assay of I0-galactosidase activity. P-Galactosidase activity We suspected that the existing polB mutants retained was assayed in cells harboring pSCH18 derivatives encoding substantial levels of DNA polymerase II activity in vivo and dinA'-'lacZ or other lacZ fusion genes grown in the presence that this might have prevented us from revealing the specific or absence of MMC (2 ,ug/ml) for 2 h. The procedures for the phenotype of the mutants so far. To elucidate the role of enzyme assay and calculation of the activity were as de- DNA polymerase II in DNA replication, repair, recombina- scribed by Miller (18). tion, and mutagenesis, we thought it necessary to construct Preparation of cell extracts. To measure the activity of apolB deletion mutant by using a cloned gene (25). Biochem- DNA polymerase II in the overproducing strains, cells of E. ical studies on DNA polymerase II have been hampered by coli HRS5200 harboring pSY343 derivatives carrying polB the relative scarcity of the enzyme in the cell. The overpro- were grown to an optical density at 600 nm of 0.1 in 10 ml of duction of the enzyme with the cloned gene would facilitate LB medium containing kanamycin (50 jxg/ml) at 28°C. After these studies. Therefore, we have been trying to clone the the incubation temperature was shifted to 37°C for 8 h, the polB gene. Bonnar et al. (3) presented evidence that synthe- cells were collected by centrifugation. sis of DNA polymerase X, presumably DNA polymerase II, To assay SOS-induced DNA polymerase II activity, the is regulated by LexA repressor. The dinA gene (13) was cells harboring pSCH18 derivatives were grown to an optical identified as an SOS-regulated gene with no known function; density at 600 nm of 0.3 in LB medium containing ampicillin it was mapped near leuB, where polB was also mapped (4, 9). (30 ,g/ml) at 37°C, and MMC was added to 2 ,ug/ml. The It occurred to us that dinA might be identical to polB. In this cells were cultured for 2 h and then collected by centrifuga- work, we examined this possibility and found that it is tion. The wet cell paste was washed with 5 ml of buffer indeed so. The polB gene was recently cloned by Chen et al. containing 50 mM Tris hydrochloride (pH 8.0)-25% sucrose (6). and then suspended in 50 fdl of the same buffer. After an equal volume of lysis buffer (50 mM Tris hydrochloride [pH MATERIALS AND METHODS 7.5], 25% sucrose, 60 mM spermidine hydrochloride, 20 mM Bacterial strains, plasmids, phages, and media. The E. coli NaCl, 12 mM dithiothreitol, 0.5 mg of lysozyme per ml) was strains used in this study are listed in Table 1. Strain added, the suspension was chilled at 4°C for 45 min. To the HRS5200 was constructed by P1 transduction of leu+ and suspension, 20 RI of 5% (wt/vol) Brij 58 was added, and the polB1OO of HMS83 (5) into ME6268. Plasmids pUC18 (26), mixture was incubated at 37°C for 5 min. After the lysate was chilled on ice, a clear supernatant was obtained by centrif- ugation (30,000 x g) for 30 min at 4°C. * Corresponding author. Assay of DNA polymerase activity. The standard reaction 6268 VOL. 172, 1990 CLONING AND REGULATION OF E. COLI polB GENE 6269 TABLE 1. E. coli strains used these operon fusions grown in the presence or the absence of MMC, an SOS-inducing agent (Table 2). Plasmid pTH1528, Strain Relevant markers Source or reference which carried the promoterless lacZ gene, was employed as ME6268 polAl leu A. Nishimura the negative control, and plasmid pTH1510, which carried HRS5200 polAl polB100 leu+ This study the dinA-lacZ fusion gene obtained from pGW510 (12), was DM2558 recA+ lexA+ (lac-pro)XIII 7 employed as the positive control for the induction of the DM844 lexA(Ind-) lacYl D. W. Mount SOS-regulated promoter. Strain DM2558 (recA+ lexA+) car- DM2570 lexA(Def) (lac-pro)XIII 7 either of or showed about DE241 recAl (lac-pro)XIII 7 rying plasmid pTH1116 pTH1118 DM2571 ArecA lexA(Def) (lac-pro)XIII 7 fivefold-higher P-galactosidase activity in the presence of CSR603 recAl uvrA6 phr-1 21 MMC than in its absence. Strains DM2570 [lexA(Def)] and DM2571 [ArecA lexA(Def)] carrying pTH1116 or pTH1118 showed constitutive expression of ,-galactosidase; the lev- els of activity observed were similar to that in MMC-treated mixture (40 p,l) contained 30 mM HEPES (N-2-hydroxyeth- DM2558 carrying pTH1116 or pTH1118. Strains DM844 ylpiperazine-N'-2-ethanesulfonic acid)-KOH (pH 7.6), 8 [lexA(Ind-)] and DE241 (recAl) carrying these plasmids mM magnesium acetate, 100 mM potassium glutamate, 5 showed low levels of expression that were similar to those of mM dithiothreitol, 33 ,uM (each) dATP, dGTP, and dCTP, uninduced DM2558 carrying the same plasmids. These re- 3.3 ,uM [a-32P]TTP (ca. 5,000 to 10,000 cpm/pmol), 25 mM sults suggest that promoter carried on pTH1116 and activated salmon DNA (1) for the template, and 5 ,ul of the pTH1118 is repressed by LexA repressor and regulated by cell lysate (usually the original lysates described above were the SOS system. Any strain carrying pTH1115 expressed diluted 25-fold with buffer containing 30 mM HEPES-KOH little 3-galactosidase. Therefore, we conclude that the 1.2-kb [pH 7.6] and 10% glycerol). After incubation at 37°C for 30 BamHI-ClaI-1 fragment does not contain an SOS-inducible min, the reaction was stopped by adding EDTA to 20 mM. promoter or any other strong promoter oriented from the left DNA synthesis was assayed by the radioactivity adsorbed to to the right of the restriction maps in Fig. lA. All strains DE81 filter disks (Whatman), which was measured by liquid carrying pTH1100, pTH1102, pTH1104, or pTH1117 showed scintillation counting, and the activity was expressed in high levels of P-galactosidase activity, and always the levels picomoles of dTTP incorporated into DNA during a 30-min of expression observed were higher in the MMC-induced reaction (20). The specific activity of the extract was calcu- cells than in the uninduced cells. However, the induction lated by dividing the activity by the quantity of protein was independent of the recA and lexA gene functions. measured with a protein assay kit (Bio-Rad). Therefore, another promoter that is not under SOS control Other methods. We used standard recombinant DNA should be located between the ClaI-2 and MluI-2 sites. From techniques (17). The proteins encoded by plasmids were these results, we concluded that the SOS-regulated dinA analyzed after the cells were labeled with [35S]methionine promoter is located in the ClaI-1-AccI region in Fig. 1C. and [35S]cysteine by the maxicell procedure of Sancar et al. Some new information obtained by the present work was (21).
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