Subunits in Vivo in Salmonella Typhimurium

We show that temperature-sensitive mutations in dnaZ, the gene for the y subunit of DNA polymerase 111 holoenzyme, can be suppressed by mutations in the dnaN gene, which encodes the P subunit. These results support a direct physical interaction of these two subunits during polymerase assembly or function. The suppressor phenotype is also sensitive to modulation by the dnaA genotype. Since dnaA is organized in an operon with dnaN, and dnaA is a regulatory gene of this operon, we propose that the dnaA effect on suppression can best be explained by modulation of suppressor dnaN levels. N E. coli and Salmonella typhimurium, DNA replication is carried out priI marily by the DNA polymerase 111 (pol 111) holoenzyme complex in cooperation with other proteins that mediate initiation at the chromosomal origin, formation of RNA primers for Okazaki fragments and topological changes in the DNA (helix winding and supercoiling) (KORNBERG 1980; MCHENRY 1985). An enduring question is the mechanism for coordination of the various aspects of the replication process. One approach to this question relies on the analysis of genetic interactions manifested by certain replication mutants. These genetic interactions potentially are based on physical or functional interactions at the protein level and, thus, can reveal the existence and structure of a complex protein machine for DNA replication (HARTMAN and ROTH 19’73; BOTSTEIN and MAURER 1982; NOSSAL and ALBERTS 1983). Bacteria that escape the lethality of a temperature-sensitive mutation in a chosen DNA replication gene may carry suppressor mutations that are of interest for this type of analysis. Since pol I11 is likely to be at the heart of the replication complex, we have looked for genetic interactions involving genes for pol I11 subunits (MAURER, OSMOND and BOTSTEIN 1984). Here we report the characterization of suppressor mutations generated in Salmonella typhimurium using dnaZ, the gene encoding the subunit of pol I11 (HUBSCHER and KORNBERG 1980; KODAIRA, BISWAS and KORNBERG 1983; MULLIN et al. 1983) as the target gene. Several lines of investigation indicate that the y subunit (or, more likely, a complex of two subunits, 7 . 6 ) mediates the action of a key protein, the /3 subunit of pol 111, in the replication process. Genetics 113: 499-5 15 July, 1986. 500 J. ENGSTROM, A. WONG AND R. MAURER According to the early experiments of WICKNER (1976), the product of the 7mediated reaction is a stable complex of template-primer with @. This complex can then bind a polymerase moiety, presumably the pol I11 core consisting of three subunits (a, 6 and 6) (MCHENRY and CROW 1979) and rapidly initiate synthesis. Later work from the laboratories of KORNBERG and MCHENRY leads to the current, generally accepted view that 7, 6 and @ function as intrinsic subunit of the pol I11 holoenzyme. This picture was inferred from a comparison of the biochemical activity of pol I11 core, pol I11 holoenzyme and an intermediate assembly called pol 111” that is deficient in @. These forms of pol 111 differ in processivity (FAY et al. 1981, 1982; LADUCA et al. 1983) and ability to copy long single-stranded regions, such as single-stranded virus DNA (MCHENRY and KORNBERG 1977). In particular, holoenzyme, or pol 111” supplemented with @, can react with a primed viral template to form a stable “initiation complex” capable of rapid synthesis on addition of dNTPs. The importance of @ is shown by sensitivity of complex formation to inhibition by anti-@ antibody (JOHANSON and MCHENRY 1980, 1982) and to omission of @ (unsupplemented pol I11 * reactions). MCHENRY’S model does not address the specific role of y, but the y.6 complex and P are both required for polymerase activity on native templates (MCHENRY and KORNBERG 1977). Activity of @ requires ATP or dATP (WICKNER 1976; WICKNER and KORNBERG 1973) unless @ is present in excess (CRUTE et al. 1983). A possible alternative or additional role for y was proposed by WALKER on the basis of suppressor mutations occurring in the initiation gene, dnaA, directed against a temperature-sensitive mutation in dnaZ (WALKER, RAMSEY and HALDENWANG 1982). These findings were interpreted to mean that dnuA protein, in addition to its role in initiation, forms a structural matrix for the elongation complex containing pol 111. The genetic interaction of dnaA and dnaZ would thus be a manifestation of a direct physical interaction at the protein level. To date, this proposal has not been critically tested in biochemical experiments. The suppressor mutations we have derived also fall in the dnuA region; but in contrast to WALKER’S results, in our case the pertinent gene for suppression is clearly the adjacent gene, dnuN. Since dnaN codes for P (BURGERS, KORNBERG and SAKAKIBARA 1981), this finding supports a role for y in mediating the activity of P. Certain additional findings lead us to suggest that the level of dnaN protein may play a critical role in suppression. On this basis we propose a unifying explanation for WALKER’S results and our own that does not require a dnad-dnu2 protein interaction but, rather, depends on the ability of dnuA to influence the level of expression of dnaN. MATERIALS AND METHODS Strains: The following bacterial strains are described in detail in MAURER et al. (1984): DB4761, 4913, 4894, 4883, 4756 and 4884 E. coli replication mutants in dnaZ, dnaX, dnaN, dnaA, dnaB and dnaG, respectively; RB132, an E. coli strain used for transposition mutagenesis of phage X with TnIQA; RM42, an E. coli host for selective growth of A1059 recombinants; RM5 (=DB4673), RM6 (=DB4835) and RMlO (=DB9005) standard Salmonella strains in which temperature-sensitive mutations are POLYMERASE I11 SUBUNIT INTERACTIONS 50 1 derived and analyzed. In addition, we used RM34, an E. coli strain for transposition mutagenesis of A with TnlOAkanR. RM34 is identical to RB132, except that plasmid pNK289 (FOSTER et al. 1981) replaces pNK217. Salmonella dnaZ mutants are described below. Phage A clones are derivatives of A1059 (KARN et al. 1980), with the properties indicated in the tables and figures where they appear. Plasmids contain either a 7.3-kb BamHI fragment or a 3.7-kb EcoRI fragment inserted in the BamHI or EcoRI site, respectively, of pUC8 (VIEIRA and MESSING 1982). The orientation of the insert was determined by restriction enzyme mapping. Media and genetic procedures: Media and most genetic procedures were described previously (MAURER et al. 1984). Complementation and suppression mediated by phage A clones were assayed using a slight modification of the red plaque assay described by MAURER et al. (1984). The bacterial strain used in the overlay (the tester strain) was grown overnight in broth containing 30 g of tryptone, 5 g of yeast extract, 5 g of NaCl, 2 g of maltose, 1 mM MgSO,, and 1 ml of 1 M NaOH per liter. Such an overnight culture could be used directly in the assay without prior dilution and growth in fresh broth. Once the tester strain was added, the plates were prewarmed on a 60" slide warmer for 3 min and were incubated at 30" for 35 min to permit gene expression. Then plates were rewarmed for 3-4 min and were incubated at the nonpermissive temperature. For complementation assays involving strains DB476 1 and DB4894, the nonpermissive temperature used was 40"; for other strains, 42" was used. Suppression of dnaZ(Ts) mutations was assayed at 38". Complementation and suppression mediated by plasmid subclones were assessed by transforming appropriate mutants at 30" and then examining the growth of single transformant colonies restreaked at the nonpermissive temperature. In addition, in the case of the 3.7-kb EcoRI subclones, the relative efficiency of plating (e.0.p.) by the transformants +.e., colony formation at 38" relative to 30") was used to verify the qualitative assessment of suppression. In this assay, the suppressor derived from ARM560 produced an improvement of 30to 50-fold in relative e.0.p. of strain RM212 compared to derivatives harboring a wild-type 3.7-kb EcoRI subclone, or just pUC8. The effect of the same suppressor subclones on strain RM2 13, although detectable, was an order of magnitude weaker. DNA preparation and treatment: Bacterial DNA was prepared by the method of EBEL-TSIPIS, BOTSTEIN and FOX (1972) as modified in MAURER et al. (1984). Plasmid DNA was prepared by the rapid isolation procedure described in DAVIS, BOTSTEIN and ROTH (1980) or by the amplification protocol of MANIATIS, FRITSCH and SAMBROOK (1982). Phage A DNA was prepared as described in DAVIS, BOTSTEIN and ROTH (1980). Restriction digestions and ligations were performed under conditions recommended by the supplier or as described in DAVIS, BOTSTEIN and ROTH (1980). Gel electrophoresis was as described in MANIATIS, FRITSCH and SAMBROOK (1982). DNA fragments to be cloned were recovered from agarose gels by adsorption to glass beads (VOGELSTEIN and GILLESPIE 1979). The hybridization experiment shown in Figure 3 was conducted under "stringent" conditions, essentially as described in MANIATIS, FRITSCH and SAMBROOK (1982). Localization of dnaZ in cloned DNA fragments: Seven AdnaZ+ clones isolated from a wild-type A-Salmonella library have been reported previously (ARM 154-ARM 159 and ARM417; MAURER et al. 1984). All these clones complement dnaX as well as dnaZ mutants. Both dnaZ and dnaX were localized within the cloned fragments by restriction mapping to determine the limits of the DNA possessed in common by all seven phages. In addition, one phage (ARM4 17) was mutagenized with a transposon, TnlOAl6A17 (or more simply, TnlOA), and the sites of dnaZ::TnlOA and dnaX:TnlOA insertions were determined by restriction mapping. Isolation of Salmonella dnaZ(Ts) mutants: ARM4 17 was mutagenized with a hybrid kanamycin-resistance transposon, T n 10A16A17kanR (FOSTER et al. 1981) by lytic passage through E. coli strain RM34. The transposition system used is completely analogous to the system used previously for TnlOA (MAURER et al. 1984). The passaged A lysate 502 J. ENGSTROM, A. WONG AND R. MAURER was used to transduce Salmonella strain RM5 to kanamycin resistance. In such transductants, the drug-resistance determinant is chromosomally located, closely linked to dnaZ; thus, it can be used to direct local mutagenesis (HONG and AMES 1971). A P22 phage lysate grown on one such strain, RM70, was subjected to hydroxylamine mutagenesis in vitro (MAURER et al. 1984). The mutagenized lysate was then used to transduce Salmonella strain RM6, selecting kanamycin resistance at 30” after a growth interval of 2 hr to allow expression of the drug resistance. Six temperature-sensitive mutants were recovered among the transductants, at least three of which must be of independent origin because they derive from different aliquots of the mutagenized P22 lysate. The independence of the remaining isolates is in doubt because the mutants could have divided during the expression period. However, it is known that, under similar conditions, transduced Salmonella cells do not resume cell division for 2 hr following addition of phage (EREL-TSIPIS, FOX and BOTSTEIN 1972); therefore, the six mutations may well be independent. As expected, the six mutations are linked to kanamycin resistance in transductional crosses. Red-plaque complementation assays using the AdnaZ+ phage and the Tn lOA mutants thereof establish that all six mutants are temperature sensitive for dnaZ and apparently normal for dnaX (see Table 3). We designate these mutations dnaZ730, 731, 732, 735, 737 and 738, and the corresponding RM6 derivatives RM211, 212, 213, 239, 241 and 254. Construction of a “Z minus” genomic library: DNA isolated from the Salmonella dnaZ(Ts) strain RM262 (=RM10 dnaZ731, kanR) was partially digested with SauSAI, and 15to 20-kb fragments were gel purified. The fragments were cloned into BamHIdigested A1059 and were packaged in vitro (KARN et al., 1980; HOHN 1979). An aliquot of the packaged phage containing about 5000 distinct recombinants was amplified selectively by two successive passages in strain RM42.