Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5764-5767, September 1981 Genetics Functional cooperation of the dnaE and dnaN gene products in Escherichia coli (DNA polymerase mII holoenzyme/subunits/intergenic suppressor/transducing A phages) NOBUHIKO KUWABARA AND HISAO UCHIDA Institute of Medical Science, University ofTokyo, P.O. Takanawa, Tokyo 108, Japan Communicated by Arthur Kornberg, May 26, 1981 ABSTRACT A system was designed to isolate second-site in- Table 1. Bacterial strainsa tergenic suppressors of a thermosensitive mutation of the dnaE gene of Escherichia coli. The dnaE gene codes for the a subunit Origin or ofDNA polymerase ff [McHenry, C. S. & Crow, W. (1979)J. BioL Strain Relevent characters ref. Chem. 254, 1748-1753]. One such suppressor, namedsueA77, was KH473 tonA dnaE486 T. Horiuchi (7) finely mapped and found to be located at 82 min on the E. coli KH687 F- tnaA phoS rbsK ilv trp T. Nagata (8) chromosome, between dnaA and recF, and within the dnaN gene lacZ tax rpsL [Sakakibara, Y. & Mizukami, T. (1980) MoL Gen. Genet 178, 541- AT982 Hfr KL16 dapD4 thi (9) 553]. The dnaN gene codes for the P3subunit of DNA polymerase CD4 HfrC metB proA metD CGSC 5096 (7) m holoenzyme [Burgers, P. M. J., Kornberg, A. & Sakaldbara, lac malA tsx red Y. (1981) Proc. NatL Acad. Sci. USA 78, 5391-53951. The sueA77 HC110 F- ilv pyrE met his rpaL Y. Sakakibara (6) mutation was trans-dominant over its wild-type allele, and it sup- BT1026 dnaE1026 Y. Hirota (3) pressed different thermosensitive mutations of dnaE with differ- KY8344 dnaA46 (8) ent maximal permissive temperature. These properties were in- AB1133 F- thr ara leu pro his argE (10) terpreted as providing genetic evidence forinteraction ofthe dnaE JC8111 recF143 (11) and dnaN gene products in E. coli. B582 HfrH A(gal attA chiA uvrB) thi (12) C600 It is now generally accepted. that Escherichia coli proteins in- NB62 HfrH tonA dnaE486 HfrH prototroph volved in DNA replication in vitro function as complexes (1). NB66 HfrC tonA dnaE486 CD4 Thus, by investigating in vitro conversion of phage 4X174 or NB70 F- dapD4 metB psL tonA AB1133 G4 single-stranded DNA to the double-stranded replicative dnaE486 form, the DNA polymerase III holoenzyme ofE. coli has been NB99 tnaA sueA77 tonA dnaE486 rpaL KH687 characterized as a complex multisubunit enzyme (2). Although NB93 dv tonA phoS rbsK rpaL tonA KH687 the holoenzyme has been shown to contain seven different sub- dnaE486 units [namely, a, E, 0, (3, y, 8, and T (2)], the holoenzyme struc- NB106 F- ilv met tonA rpoL dnaE486 HC110 ture has not yet been completely determined. Because repli- recA cation ofsmall DNA phages such as 4gX174 depends almost en- NB136 F- praA ilv tnaA phoS rbsK KH687 tirely on the cellular replication machinery, the multisubunit rpsL dnaEl026 complex characterized by employing DNA of such phages as probes may well be pertinent to replication ofthe hostcell chro- MATERIALS AND METHODS mosome. In fact, genetic loci of genes coding for a, y, and 8 subunits have been identified on the chromosome ofE. coli as Bacterial and Phage Strains. E. coli K-12 strains used are dnaE (3), dnaZ (4), and dnaX (5), respectively, indicating that listed in Table 1. Some ofthem were supplied by Y. Hirota, T. at least three subunits ofthe DNA polymerase holoenzyme are Horiuchi, T. Nagata, and Y. Sakaldbara. Others were either also required for the replication of the E. coli DNA. On the from our collection or prepared in this work by appropriate con- other hand, it is also possible that distinctive forms of the rep- jugal transfer and phage P1 transduction of genetic markers. A lication machinery are required for different chromosomal transducing phage, Aimm21dnaA-2 (13), was supplied by S. templates. Hiraga. Plvir, P2, AcI7l, Agt-AC, and Aimn2' were from our In order to characterize genetically the replication machinery laboratory collection. ofE. coli, we have exploited suppressor studies to detect func- Media and Buffers. The broth and agar media, as well as tional cooperation ofgene products. We have focused our anal- buffers used, were generally as described (14, 15). When nec- ysis on second-site suppression ofa thermosensitive dnaE mu- essary, media were supplemented with various compounds to tation because the a subunit coded by the dnaE gene is the the following concentrations: L amino acids, 20 pg/ml and D- largest subunit ofthe core polymerase III and has been consid- methionine, 5 ,g/ml; thymine, 30 ug/ml; adenine, 10 Ag/ml; ered to play a key role in DNA replication (1, 3). Evidence will thiamine, 1 ug/ml; a-DL-diaminopimelic acid, 40 ,Ag/ml; be presented that one such suppressor, isolated in this study, streptomycin sulfate, 100 .g/ml. is an altered dnaN gene (6). Enzymes. Restriction endonucleases HindIII and EcoRI, and phage T4 DNA ligase were purchased from Takara Shuzo The publication costs ofthisarticle were defrayed in partby page charge payment. This article must therefore be hereby marked "advertise- Abbreviations: kDal, kilodalton(s); kb, kilobase(s); Dap', diamninopi- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. melate-independent; StrR, streptomycin resistant. 5764 Downloaded by guest on September 25, 2021 Genetics: Kuwabara and Uchida Proc. NatL Acad. Sci. USA 78 (1981) 5765 (Kyoto, Japan). Digestion buffer used was 50 mM TrisHCl (pH The two mutants were named NB77 and NB78, and the sup- 7.4)/10 mM MgCl2/10 mM dithiothreitol/50 mM KCL. The pressor mutation present in NB77 was named sueA77 for sup- procedure for ligation and transfection of A DNA has been de- pressor ofdnaE. Existence oftheprimary mutation in NB77 was scribed (15). confirmed by P1 transduction of dnaE486, utilizing its close Electron Microscopic Analysis ofHeteroduplexes. The pro- linkage with dapD+. Mapping of sueA77 by P1 transduction cedure employed has been described (16). indicated that the suppressor was cotransducible with ilv or Analysis of Proteins Encoded by Transducing Phages. pyrE, but not with metE (Fig. 1). Therefore, sueA77was located Phage-coded proteins synthesized upon infection of UV-irra- at 82 min on the E. coli chromosome very near to tnaA and dnaA diated B582 cells were labeled with a '4C-labeled amino acid in the order pyrE-sueA-ilv-metE. mixture (NEC-445, New England Nuclear) by the procedure For revertant Hfr strains other than NB77, linkage of sup- described (15). Labeled proteins were precipitated with 10% pressors with ilv was tested by P1 transduction. For five out of trichloroacetic acid, washed with 5% trichloroacetic acid, and six mutants of NB62, as well as for NB78 derived from NB66, rinsed with acetone. Electrophoresis was carried out in the suppressors were shown to be linked to ilv on the other side of presence ofsodium dodecyl sulfate on slab gels (13 X 12 X 0.1 metE, suggesting that they were also mutations of the sueA cm) with polyacrylamide concentrations of 5% and a 10-20% gene. The remaining two suppressors were not linked to ilv. linear gradient for stacking and separation, respectively. After Some Properties of the sueA77 Suppressor of dnaE486. Ef- electrophoresis, gels were stained with Coomassie brilliant ficiencies ofcolony formation ofdnaE486 sueA+ cells incubated blue, dried, and exposed to Kodak x-ray films for autography. overnight on TY agar plates at 30, 40, and 42°C were 1.0, 10-6, The following proteins were used as size markers (kDal, kilo- and 10-6, respectively, whereas corresponding values for iso- daltons): E. coli RNA polymerase holoenzyme ((3', 160 kDal; genic dnaE486 sueA77 cells were 1.0, 1.0, and 2 X 10-3, re- (3, 150 kDal; a, 86 kDal; a, 40 kDal); bovine serum albumin, spectively. dnaE+ sueA77 cells formed the same number ofcol- 68 kDal; bovine pancreatic trypsin, 24 kDal; and egg white ly- onies at 30°C and at 42°C. Although sueA77 was originally sozyme, 14.4 kDal. isolated as a suppressor ofthe dnaE486 mutation, it suppressed thermosensitivity of another dnaE mutation with different ef- RESULTS ficiency. Thus, efficiencies ofcolony formation by NB136 cells harboring dnaE1026 (3) and isogenic sueA77 dnaE1026 double Isolation of Second-Site Suppressor Mutations of dnaE486. mutant cells were 1 x 10-7 and 1.0, respectively, at 42.50C. The a subunit of the DNA polymerase III holoenzyme of E. Replication ofphage A also depends on the function ofdnaE. coli K-12 is encoded by the dnaE gene (1, 3). At a frequency In fact, AcI71 did not multiply on dnaE486 sueA+ cells in broth of about 10-6, a culture ofE. coli cells having thermosensitive at 42°C. However, isogenic dnaE486 cells having the sueA77 mutation dnaE486 (3) contains "revertants" that are able to form suppressor supported multiplication of the phage at 42°C to an colonies at 40°C. Some ofthem may have acquired second-site extent similar to that of dnaE+ cells (data not shown). suppressors distantly linked to the primary mutation. This class F 111 factor (17) having the pyrE+-sueA+-metE+-malB+ frag- ofrevertants could be scrutinized by the following system: The ment of E. coli chromosome was transferred into NB106 F- dnaE486 mutation was introduced into streptomycin-sensitive sueA77 Al met recA dnaE486 to obtain NB106/F'111. The Hfr strains to yield NB62 (a Hayes-type Hfr, transferring its merozygote grew at 400C on minimal agar plates selective for chromosome clockwise with the point of origin at 98 min) and isoleucine-valine- and methionine-independent cells. Because NB66 (a Cavalli-type Hfr, transferring its chromosome coun- the experiment showed that the sueA77 suppressor is trans- terclockwise with the point oforigin at 14 min).