JOURNAL OF BACTERIOLOGY, Feb. 1994, p. 815-821 Vol. 176, No. 3 0021-9193/94/$04.00+0 Copyright © 1994, American Society for Microbiology holE, the Gene Coding for the 0 Subunit of DNA Polymerase III of Escherichia coli: Characterization of a holE Mutant and Comparison with a dnaQ (6-Subunit) Mutant STEVEN C. SLATER,'t MIRIAM R. LIFSICS,1 MIKE O'DONNELL,2'3 AND RUSSELL MAURER'* Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4960,1 and Department of Microbiology, Cornell University Medical College,2 and Howard Hughes Medical Institute,3 New York, New York 10021 Downloaded from Received 4 October 1993/Accepted 2 December 1993 DNA polymerase III holoenzyme is a multiprotein complex responsible for the bulk of chromosomal replication in Escherichia coli and Salmonella typhimurium. The catalytic core of the holoenzyme is an arO heterotrimer that incorporates both a polymerase subunit ((; dnaE) and a proofreading subunit (£; dnaQ). The role of 0 is unknown. Here, we describe a null mutation ofholE, the gene for 0. A strain carrying this mutation was fully viable and displayed no mutant phenotype. In contrast, a dnaQ null mutant exhibited poor growth, chronic SOS induction, and an elevated spontaneous mutation rate, like dnaQ null mutants of S. typhimurium described previously. The poor growth was suppressible by a mutation affecting a which was identical to a http://jb.asm.org/ suppressor mutation identified in S. typhimurium. A double mutant null for both holE and dnaQ was indistinguishable from the dnaQ single mutant. These results show that the 0 subunit is dispensable in both dnaQ+ and mutant dnaQ backgrounds, and that the phenotype of e mutants cannot be explained on the basis of interference with 0 function. A goal of research on DNA polymerase III (Pol III) £, but not to a, and has no detectable effect on the polymerase holoenzyme is the elucidation of the precise role of each of its activity of a or an as complex. The only activity so far 10 or more distinct subunits. Research thus far has identified identified for 0 is a threefold stimulatory effect on the exonu- on August 7, 2015 by Rockefeller University the a subunit as the polymerase proper (22), the E subunit as clease activity of £ on a mispaired primer terminus (40). the proofreading exonuclease (34), and the 3 subunit as a Here, we report the construction of the first 0 (holE) doughnut-shaped clamp that topologically tethers the poly- mutation and describe its properties, both as a single mutation merase to the DNA and makes it highly processive (15, 29, 41). and in combination with a null mutation in dnaQ. In addition, the T subunit dimerizes the holoenzyme to allow coordinated leading- and lagging-strand synthesis by twin polymerases (27, 39). Most of the remaining subunits are MATERIALS AND METHODS involved in controlling the assembly of ,B with the template- primer, but the individual subunit roles in this function are not Bacterial strains and media. Bacterial strains and plasmids yet clear. are listed in Table 1. Phage Plvir was used for transductions. At least some of the subunits of the holoenzyme are Bacteria were generally maintained at 37°C on Luria-Bertani multifunctional. s, for example, affects the rate, processivity, (LB) medium (36). Sucrose plates were NaCl-free LB plus 5% and thermal stability of the enzyme, not just its fidelity (22, 23, sucrose (3). Antibiotics were used at the following concentra- 38). Null mutants affected in s, obtained in Salmonella typhi- tions (in micrograms per milliliter): ampicillin, 100; kanamycin, murium, exhibit both an elevated mutation rate and a severe 50; tetracycline, 25; chloramphenicol, 15; rifampin, 100; nali- growth defect (16). These phenotypes are experimentally dixate, 50. distinct because the growth defect can be specifically sup- Enzymes and reagents. Restriction enzymes were from New pressed by mutations affecting a (16, 17). Genetic and bio- England Biolabs, United States Biochemical, or Boehringer chemical analysis suggests that the s mutants suffer from Mannheim. Sequenase was from United States Biochemical. insufficient polymerization capacity and that the suppressors GeneScreen Plus hybridization membrane was from Dupont. increase this capacity (16, 19). Radiolabelled nucleotides were from Amersham. Western To date, the role of the 0 subunit has remained elusive. Early blots (immunoblots) were performed with a Tropix lumines- efforts to define its role biochemically were frustrated by the cent labelling kit. SeaKem agarose was from FMC. Constitu- scarcity of the protein and by the difficulty of preparing other ents of bacteriological media were from Difco. Other reagents core subunits free of 0. Conversely, the lack of 0 mutants has were from Sigma. prevented genetic analysis. Recently, however, the gene for 0 holE constructs. The holE disruption mutants were initially has been identified by reverse genetics (5, 40), making it constructed by using plasmid pUC18-0, which contains holE on possible to overproduce and purify 0 and begin its character- a 2.7-kb EcoRV fragment from Kohara X clone 336 (40). The ization. Initial biochemical analysis shows that 0 binds tightly to plasmid contains a unique EagI site 65 bp downstream of the holE initiation codon. It also contains three NruI sites, all within the insert. Site 1 is located approximately 1.7 kb * Corresponding author. Phone: 216-368-6021. Fax: 216-368-3055. upstream of holE, very near the insert-vector junction. Site 2 is Electronic mail address: [email protected]. within holE, 156 bp downstream of the holE initiation codon. t Present address: Department of Biochemistry and Molecular Site 3 is approximately 270 bp downstream of the holE Biology, Harvard University, Cambridge, MA 02138. termination codon. The antibiotic marker used for disruption 815 816 SLATER ET AL. J. BACrERIOL. TABLE 1. E. coli strains and plasmids Species and strain or plasmid Relevant characteristic(s) Source or reference E. colia RM990 A(lac proAB) thi rpsL supE endA sbcBIS hsdR4/F' traD36 25 proAB+ laclq A(lacZMS) RM1094 HfrH 25 RM1734 (=MG1655) F- wild type 1 RM2541 E. coli C-1 (susceptible to phage 4X174) E. coli Genetic Stock Center RM3979 RM1094 dnaQ903::tet Allele exchange (pFF596) RM3980 RM1734 dnaQ903::tet spq-2 P1 * RM3979 and spontaneous suppressor RM4006 RM1734 malE::TnS lexA3 (Ind-) This laboratory RM4021 RM1094 holE201::cat Allele exchange (pFF608) RM4029 (=CAG18580) RM1734 zae-3095::TnlOkan 35 Downloaded from RM4030 RM1734 holE201::cat P1 RM4021 RM4074 RM1094 holE202::cat Allele exchange (pFF622) RM4193 RM1734 holE202::cat Pl - RM4074 RM4194 RM1734 dnaQ903::tet spq-2 holE202::cat RM3980 + P1 RM4193 RM4196 RM1734 zae-3095::TnlOkan spq-2 RM3980 + P1 * RM4029 RM4221 (=W1485) F+ wild type 1 Plasmid pBIP3 Allele replacement vector 36 pFF441 mucA'B 37 http://jb.asm.org/ pFF498 mucA' 37 pFF499 mucB 37 pFF588 dnaQ This laboratory pFF589 cat This laboratory pFF590 tet This laboratory pFF596 pBIP3 derivative carrying dnaQ903::tet This work pFF608 pBIP3 derivative carrying holE201::cat This work pFF622 pBIP3 derivative carrying holE202::cat This work pUC18-0 holE+ 40 on August 7, 2015 by Rockefeller University a E. coli K-12 except as indicated. (cat, chloramphenicol resistance) was derived from pFF589, from one of the transformants. This plasmid contains the which is pBluescript IISK+ (Stratagene) carrying cat on a expected replacement of the dnaQ BssHII-MluI fragment with BamHI fragment of pJD12 (11). cat was excised from pFF589 tet. This deletion-insertion mutation, which we designated by digestion with EagI and SmaI. Plasmid pUC18-0 was dnaQ903, leaves the rnh promoter intact (28) and is very similar partially digested with NruI, and linearized plasmid was gel to the dnaQ200 allele previously constructed in S. typhimurium purified. This DNA was then digested to completion with EagI (16). The entire insert from pFF593 was cloned into the and ligated to the cat cassette. Escherichia coli RM990 was polylinker of pBIP3 (36), and the resulting plasmid (pFF596) transformed with the ligation products (6). As expected, three was used to replace the E. coli wild-type allele as described classes of recombinants were produced, each one deleting below. DNA from the EagI site to a different NruI site. In pFF605, cat Replacement ofchromosomal dnaQ and holE with disrupted replaced DNA from the EagI site through NruI site 3, while in aileles. Exchange of the disrupted alleles for the chromosomal pFF617 the replacement interval ended at NruI site 2. These wild-type alleles of strain RM1094 was performed by two-step alleles were designated hoIE201 and holE202, respectively. plasmid integration-excision using the method of Slater and Each disrupted allele was cloned into the polylinker of plasmid Maurer (36). The excision step was carried out in the presence pBIP3 (36), and the resulting plasmids (pFF608 carrying of tetracycline (for the dnaQ replacement) or chloramphenicol holE201 and pFF622 carrying holE202) were used to replace (for the holE replacement) to select for the excision product the E. coli wild-type allele as described below. preserving the disrupted allele. dnaQ constructs. Plasmid pFF588 is a derivative of pBlue- Mutation frequency analysis. The number of mutation script II SK+ that carries E. coli dnaQ on a 1.6-kb EcoRI events per culture was estimated from the number of mutants fragment subcloned from XRM406, -a dnaQ+ clone in our observed in at least 10 independent cultures of each strain, by collection. Plasmid pFF588 was digested to completion with the method of maximal likelihood as described previously (16, BssHII and MluI, deleting most of the 3' half of dnaQ. The 18). The standard deviation of this estimate was calculated vector fragment containing the remainder of dnaQ was end from Fig. 3 of Lea and Coulson (18). The estimate and its filled (31) and gel purified.