Proc. NatL Acad. Sci. USA Vol. 78, No. 9, pp. 5764-5767, September 1981 Genetics

Functional cooperation of the dnaE and dnaN products in (DNA 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) , 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 (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 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). . 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). Revertants able dominant over its sueA+ allele, we proceeded to isolate a trans- to grow at 40°C were isolated from these Hfr strains, either ducing A phage that suppresses thermosensitivity of cells har- spontaneously or after mutagenesis with N-methyl-N'-nitro-N- boring the dnaE486 mutation. nitrosoguanidine treatment. They were then replica plated in Isolation of a Transducing AsueA Phage. Miki et aL (13) de- duplicate onto TY agar plates containing streptomycin and scribed a plaque-forming transducing phage, Aimm2idnaA-2, seeded with about 2 X 10' cells per plate ofNB70 (F- dnaE486 that has incorporated in its genome the tnaA-dnaA region ofthe dapD strA) as recipients. Conjugal transfer of the Hfr chro- E. coli chromosome. Although the attP attachment site was mosome took place on plates, and diaminopimelate-indepen- deleted from the phage, the phage could be integrated into the dent, streptomycin-resistant (Dap+ StrR) recombinants were E. coli chromosome by the tnaA-dnaA homology. NB99 tnaA selected. One plate was incubated at 300C, the other at 40°C overnight, and Hfr mutants unable to form Dap+ StrO recom- binants at higher temperature were identified. Because dapD is very closely linked to dnaE at 4 min on the E. coli chromo- some and is transferred proximally into recipient cells for both types of Hfr strains, we expected that suppressors distantly 81 82 83 84 linked to dnaE486 were transmitted to Dap+ StrR recombinants only rarely, and the majority ofthe recombinants would remain thermosensitive. Among approximately 2600 "revertants" ex- 84.0 amined by the replica plating, 9 thermoresistant Hfr mutants 32.5 (i.e., 6 from NB62 and 3 from NB66) bearing intergenic sup- 13.6 pressors were detected. These mutant Hfr strains were then 11.11.0 individually crossed with NB70 in nutrient broth and selected ! o.o again for Dap+ Str at 30"C, and alleles ofunselected markers 10.0: were scored. The results showed that, in two mutants obtained from NB66, about 1% ofDap+ StrRrecombinants grewat40°C. FiG. 1. Transductional mapping of the genes around sueA with Transmission of unselected proA, -metB, and Itl were phage P1. A selected marker is shown at the tail and an unselected 89%, marker at the head of an arrow. The number adjacent to an arrow rep- 18%, and 4%, respectively, suggesting that the suppressors resents the cotransduction frequency in percent. The scale, calibrated were located between netB and mtl on the E. coli chromosome. in min. shows a part of the 100-min standard map of E. coli K-12. Downloaded by guest on September 25, 2021 5766 Genetics: Kuwabara and Uchida Proc. Nad Acad.. Sci. USA 78 (1981)

sueA77 cells lysogenized with AdnaA-2 were induced by UV Table 2. Genetic and protein analysis of AsueA phages irradiation, and a plaque-forming transducing phage named Polypeptides AsueA-1 was isolated that could complement defects ofdnaE486 and dnaA46 (18) as well as recFl43 (11) mutations. The trans- AsuZA Suppression 50 49 58 53 43 ducing phage could not complement the tryptophanase-nega- phages tnaA dnaA dnaE recF kDal kDal kDal kDal kDal tive (Tna-) character of NB99 cells. AdnaA-2 + + - + + + + + + Determination ofbuoyant density in CsCl indicated that the AsueA-1 - + + + - + + + + AsueA-1 particle contained 104.6% A equivalent length ofDNA. del-2 - + + + - - - + + Electron microscopic examination of appropriate heterodu- del-33 - + + + - - - + + plexes showed that the lengths of chromosomal fragments in- del-48 - - + + - - -- + corporated into AdnaA-2 and AsueA-1 were 2.45% A and 26.8% del-19 - - - - - + - - - del-111 - - - - - + + - - A, respectively. Cleavage of AdnaA-2 DNA by HindIII endo- del-133 - + - - - + + + - nuclease a fragment of 6.1 kilobases (kb). However, yielded del-120 - + + - - + + + + HindIII new digestion of AsueA-1 DNA produced three frag- del-116 - + + - - + + + + ments, 4.8, 1.8, and 0.86 kb, respectively, in place of the 6.1- del-138 - + + + - + + + + kb fragment. Heteroduplexes formed by annealing strands of Agt-F101 - - + + - - - - + AdnaA-2 and AsueA-1 DNA showed unique eye-forms of un- equal single-stranded segments at the center ofthe phage DNA. Mutant cells were lysogenized with various transducing phages as These observations suggested that AsueA-1 sustained a tnaA listed, usingAimm2' ashelper. Thefollowingconditionswereemployed to test suppression of bacterial mutations. Tryptophanase: Activity of mutation produced by insertion ofa 1.3-kb sequence having two the tnaA gene product was tested by adding p-dimethylaminobenzal- HindIII sites into the tnaA gene and deletion of a 0.2-kb seg- dehyde solution to cultures of KH687 lysogens. Tryptophanase-posi- ment at the insertion point. The 1.3-kb sequence may be an IS3 tive cultures became pink, and tryptophanase-negative cultures re- element, because the latter is known to be about 1.4 kb in size mainedyellow. Thermosensitivity: Bacterial strainsusedwere KY8344 and has two HindIII sites (19). for dnaA, and NB93 and NB136 for dnaE. Nonpermissive tempera- Various deletion derivatives of AsueA-1 were isolated by (i) tures for their growth were 42, 40, and 42.50C, respectively. Growth in of lysogens at these temperatures is indicated by +, and no growth is vitro deletion of HindIII fragments (del-2); (ii) selecting vi- indicated by -; ± means slow growth. UV-sensitivity: recF function sually for phages forming large plaques when plated on C600 was tested by examining UV sensitivity of JC8111 lysogens. UV dose (del-19, del-33, and del-48); (iii) selecting for phages insensitive employedwas 14 erg/mm2. + means UVresistant, and - indicatesUV to P2 interference by plating on C600(P2) (20) (del-1l1, del-116, sensitive. The polypeptides are those encoded by the phages. del-120, del-133, and del-138). Structures of the transducing phages were determined by electron microscopic examination of heteroduplex DNA molecules formed by annealing various ±0.15 kb. AsueA del-138 complemented recF143, but AsueA combination of strands of the phage , and the re- del-116 did not. Therefore, dnaA, sueA, and recF genes are sults obtained are summarized in Fig. 2. distinct genes lying in this order on the right-hand half of the Suppressing activities of these transducing phages against transducing fragment of the AsueA-1 phage. various E. coli strains with dnaA46, dnaE486, and recF143 The dnaA region ofthe E. coli chromosome has attracted the mutations were measured, and they are summarized in Table interest ofseveral investigators, and information about detailed 2. From the complementation analysis and physical structures structure and function ofthe region has been accumulated rap- of the transducing phages, the sueA gene was allocated to a re- idly. In particular, a gene, dnaN, that codes for a protein in- gion of 2.3 kb between deletion ends of del-48 and del-116. volved in DNA synthesis was discovered recently (6), and the Similarly, the dnaA gene was allocated to an overlapping region gene has been mapped adjacent to the dnaA gene and within of 2.3 kb defined by ends of del-33 and del-133. The length of a 3.3-kb-long EcoRI fragment F of the AdnaA-2 DNA (21, 22). the region allocated to both dnaA and sueA was estimated to Because the location ofthe dnaN gene coincided almost exactly be 3.8 kb. Errors in determination ofrelevant ends were within with that of the sueA gene (Fig. 2), a DNA fragment corre- sponding to the fragment F ofAdnaA-2 was excised from AsueA- I DNA by EcoRl endonuclease treatment, and it was inserted -6 -4 -2 0 2 4 kb into Agt-AC and cloned to yield Agt-F101. The transducing phage complemented both dnaE486 and recF143 mutations (Table 2). AdnaA-2 Identification of the sueA Gene Product. The transducing v v VsYv v V = AsueA-1 phages were individually used to infect UV-irradiated uvrB- del-2 cells, and phage-coded proteins synthesized were labeled with del-33 "'C-labeled amino acids, separated electrophoretically, and vi- del-48 sualized by autoradiography. At least five proteins, with mo- 0 - del-19 lecular masses of approximately 58, 53, 50, 49, and 43 kDal, del-111 could be identified as products of the bacterial DNA fragment = del-133 present in the AdnaA-2 phage. AsueA-1 and its varous deletion del-120 derivatives failed to produce one or more ofthese proteins. An del-116 example ofthe autoradiograms is presented in Fig. 3, and bac- -_E del-138 terial proteins found to be encoded by each phage are listed in Agt-F1O1 Table 2. We may infer from the results that the 43-kDal protein was always associated with the suppressing activity ofthe sueA FIG. 2. Physical structures of transducing fragments of AsueA-1 gene, and this protein was the only protein identified as specific and related phages. The end points of deletions were determined by ofthe dnaN electron microscopic analysis of heteroduplexes. The sites of HindE to the EcoRI fragment F ofAgt-F10. The product as a (v) andEcoRI (v) were indicated. The scale shows distances in kb mea- gene has been identified by Yuasa and Sakaldbara 41-kDal sured from a HindIl site at the center. protein, and this was also the only protein detectable in cells Downloaded by guest on September 25, 2021 Genetics: Kuwabara and Uchida Proc. Natd Acad. Sci. USA 78 (1981) 5767

1 2 3 4 5 6 The sueA77 mutation was precisely mapped at 82 min on the _I 4t Ah E. coli chromosome by P1 transduction and by structural, func- tional, and product analyses of a AsueA77 transducing phage incorporating the suppressor mutation, and by analysis of var- ious deletion derivatives of the phage. The results showed clearly that the sueA gene is bracketed by dnaA and recF genes and is identical to the dnaN gene recently discovered as a gene involved in DNA synthesis ofE. coli (6, 21, 22). It is intriguing that a suppressor of dnaE is another dna gene, suggesting -1-omp- strongly an interaction of the two gene products as postulated by interpretation ii above. Moreover, the dnaN gene product kDal has been biochemically demonstrated to be identical to the /3 4_< 58 subunit of the DNA polymerase III holoenzyme by recent ex- 4_ 53 periments carried out in the laboratory of A. Kornberg (24). Therefore, we may deduce that our experiments give evidence alt _ a 49 for interaction ofthe a and / subunits ofholoenzyme function- ing in vivo in DNA synthesis ofE. coli as well as A phage (14).

The authors thank Drs. A. Kornberg and Y. Sakakibara for commu- _ Id nicating unpublished results. This work was supported in part by grants ~~~~~43 from the Ministry of Education, Science and Culture of the Japanese Government.

1. Kornberg, A. (1980) DNA Replication (Freeman, San Francisco), FIG. 3. Sodium dodecyl sulfate/polyacrylamide gel electrophore- pp. 174-178. sis of proteins produced by AsueA transducing phages. Bacterial pro- 2. Meyer, R., Shlomai, J., Kobori, J., Bates, D. L., Rowen, L., teins are marked by filled triangles (cf. Table 2). Lanes 1 and 6, Agt- McMacken, R., Ueda, K. & Kornberg, A. (1978) Cold Spring AC; lane 2, Agt-F101; lane 3, AsueA-1 del-2; lane 4, AsueA -1 del-48; lane Harbor Symp. Quant. BioL 43, 289-293. 5, AsueA-1 del-133. 3. Gefter, M., Hirota, Y., Kornberg, T., Wechsler, J. A. & Bar- noux, C. (1971) Proc. NatL Acad. Sci. USA 68, 3150-3153. infected with Agt-81 carrying fragment F of the AdnaA-2 DNA 4. McHenry, C. & Kornberg, A. (1977) J. Biol Chem. 252, 6478- (21). Considering possible errors inevitable in the determina- 6484. tion of the molecular weights by sodium dodecyl sulfate gel 5. Hfibscher, U. & Kornberg, A. (1979) Proc. Natl Acad. Sci. USA 76, 6284-6288. electrophoresis, it is very likely that our 43-kDal protein is iden- 6. Sakakibara, Y. & Mizutani, T. (1980) Mol Gen. Genet. 178, 541- tical to their 41-kDal protein, and therefore, to the dnaN gene 553. product. The AsueA-1 del-120 phage that showed slightly lower 7. Horiuchi, T., Maki, H. & Sekiguchi, M. (1978) Mol Gen. Genet. suppressing activity against dnaE486 (Table 2) produced 43- 163, 277-283. kDal protein almost indistinguishable from the other 43-kDal 8. Kimura, M., Miki, T., Hiraga, S., Nagata, T. & Yura, T. (1979) protein, delimiting further the extent of the DNA region allo- J. Bacteriol 140, 825-834. 9. Shizuya, H., Livingston, D. M. & Richardson, C. C. (1974) Proc. cated for the sueA gene. Regions assigned for the sueA and dnaN Natl Acad. Sci. USA 71, 2614-2617. genes overlap completely with each other (22). 10. Bachmann, B. J. (1972) Bacteriol Rev. 36, 525-557. Products of the tnaA and dnaA genes were identified as pro- 11. Horii, Z.-I. & Clark, A. J. (1973)J. Mol Biol 80, 317-344. teins of 50 and 53 kDal, respectively (Table 2). The product of 12. Ikeuchi, T., Yura, T. & Yamagishi, H. (1975) J. Bacteriol 122, the recF gene could not be detected (23). 1247-1256. 13. Miki, T., Kimura, M., Hiraga, S., Nagata, T. & Yura, T. (1979) DISCUSSION J. Bacteriol 140, 817-824. 14. Saito, H. & Uchida, H. (1977)J. Mol Biowl 113, 1-25. We have designed a simple system to isolate intergenic sup- 15. Saito, H., Nakamura, Y. & Uchida, H. (1978) Mol Gen. Genet. pressors of a thermosensitive dnaE mutation ofE. coli that are 165, 247-256. distantly linked to the primary mutation. The dnaE gene codes 16. Saito, H. & Uchida, H. (1978) Mol. Gen. Genet. 164, 1-8. for the a subunit of the DNA polymerase III holoenzyme (1, 17. Low, B. (1973) J. Bacteriol 113, 798-812. 3). One of the suppressors isolated presently was named 18. Hirota, Y., Mordoh, J. & Jacob, F. (1970)J. Mol Biol 53, 369- 387. sueA77, and the mutation was characterized further. 19. Sommer, H., Cullum, J. & Saedler, H. (1979) Mol Gen. Genet. Two interpretations may be considered to explain an inter- 177, 85-89. genic suppression of a thermosensitive mutation: (i) The sup- 20. Zissler, J., Signer, E. & Schaefer, F. (1971) The Bacteriophage pressor mutation may be in a gene whose mutationally altered Lambda, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, product substitutes directly for the defective function of the Cold Spring Harbor, NY), pp. 469-475. primary gene. (ii) Two gene products interact and a mutation 21. Yuasa, S. & Sakakibara, Y. (1980) Mol Gen. Genet. 180, 267-273. 22. Sakakibara, Y., Tsukano, H. & Sako, T. (1981) Gene 13, 47-55. in one gene alters the properties ofthe complex. Interpretation 23. Ream, L. W., Margossian, L. & Clark, A. J. (1980) Mol, Gen. (i) is difficult to explain in the present case, because the maximal Genet. 180, 115-121. permissive temperatures of dnaE486 and dnaEl026 mutations 24. Burgers, P. M. J., Kornberg, A. & Sakakibara, Y. (1981) Proc. suppressed by the same sueA77 are different. NatL Acad. Sci. USA 78, 5391-5395. Downloaded by guest on September 25, 2021