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Structural Gene for Rnase H of Escherichia Coli by Cloning of The Proc. NatL Acad. Sci. USA Vol. 78, No. 6, pp. 3770-3774, June 1981 Genetics Identification of the dnaQ gene product and location of the structural gene for RNase H of Escherichia coli by cloning of the genes (mutator/maxicell/insertion mutant) TAKASHI HORIUCHI, HISAJI MAKI, MASAO MARUYAMA, AND MUTSUO SEKIGUCHI Department of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, Japan Communicated by Motoo Kimura, March 11, 1981 ABSTRACT By in vitro recombination we have constructed MATERIALS AND METHODS hybrid plasmids capable of complementing a conditional lethal mutator mutation, dnaQ49, in Escherichia coli K-12. The dnaQ+ Bacteria, Plasmids, and Phages. All bacterial strains used in plasmids consist of a full-length pBR322 DNA and a 1.5-kilobase these experiments are derivatives of E. coli K-12. Strains DNA fragment derived from the E. coli chromosome. Specific la- KH1171 (F-, recAl dnaQ49 strA) and KH1188 (Hfr, dnaQ49) beling of plasmid-encoded proteins by the maxicell method re- were isolated in this laboratory. Strain CSR603 (F-, uvrA6 vealed that the 1.5-kilobase insert codes for two proteins, one recAl phr-1) is a gift of D. Rupp and was used in maxicell ex- whose molecular weight is 25,000 [the 25-kilodalton (kDal) protein] periments (6). Strain KP370 (F+, recAl), provided by T. Miki, and the other whose molecular weight is 21,000 (the 21-kDal pro- was used in y8 insertion experiments (7). The vector plasmid tein). Because insertion of y8 sequence into the dnaQ gene of the pBR322 (8) and the vector phage Agt-AC (9) were furnished by plasmid resulted in disappearance of the 25-kDal protein, it was Y. Sakaki and Y. Nakamura, respectively. concluded that the 25-kDal protein is the dnaQ gene product. The Construction of Hybrid Plasmids. F+ strains harboring the 21-kDal protein was identified as RNase H on the basis of the fol- ColEl hybrid plasmids in the Clarke and Carbon colony bank lowing evidence. (i) Cells harboring the dnaQ+ plasmids, with or (10), provided by K. Ueda, were screened by conjugation. Cells without the y8insertion in the dnaQ gene, had a 5- to 7-fold higher harboring pLC28-22 and pLC34-20 were found to complement level of RNase H activity than cells harboring pBR322. (ii) After the dnaQ and dnaE defective mutants, respectively. The plas- induction of cells that are lysogenized with dnaQ+-transducing A mids and their derivatives were isolated (10) and digested with phages, RNase H activity increased considerably. A similar high EcoRI. The DNA fragments were inserted into the EcoRI site level of RNase H activity was observed with transducing phages of pBR322. Details of the construction and properties of the whose dnaQ function was inactivated by insertion ofa transposon, hybrid plasmids will be published elsewhere. Transformation Tn3, into the gene. (iii) The plasmid-encoded RNase H, labeled Mandel and with [3S]methionine, was purified in a manner essentially similar and transfection were performed as described by to that ofthe chromosome-encoded enzyme. These results suggest Higa (11). that the dnaQ gene and the structural gene for RNase H, termed Transposition of the y6 Sequence to Plasmids. The y8 se- gene rnh, are closely linked and located at 5 min on the linkage quence was inserted into plasmids according to the method of map. Guyer (7). Strain KP370 (F+, recAl) was transformed with the dnaQ+ plasmids, into which the yc was to be inserted, and the Mutators are a special class ofmutations that render many other transformants were subsequently conjugated with strain KH1171 genes unstable (1). In Escherichia coli, many types ofmutators (F-, recAl dnaQ49 strA). Because only the plasmids that re- have been isolated and characterized, mostly by genetic means ceived the y8 from F factor can be transferred to the recipient (24). Although speculative interpretations for mechanisms cells (7), the recombinants showing ampicillin- and tetracycline- leading to increment mutation frequency by the mutator mu- resistance characters were selected and their plasmids were tation have been proposed, little is known about the nature of analyzed. The existence ofthe y8 sequence (5.7 kb) in the plas- products encoded by mutator genes and their functions. mids was confirmed by electrophoresis ofthe DNA after EcoRI Recently, we discovered a strong mutator mutation that also digestion. Some ofthe plasmids with the y8 sequence exhibited caused temperature-sensitive growth and defective DNA syn- dnaQ- character, probably due to insertion ofthe y8 inside the thesis in cells carrying the mutation (5). It was found that the dnaQ gene. mutation was in a new gene, designated dnaQ, which was lo- Construction of Hybrid Phages. The dnaQ+-transducing A cated at 5 min on the E. coli genetic map. phage, designated AdnaQ+, was constructed by recloning the By using in vitro recombination techniques we have con- 1.5-kb DNA fragment derived from pMM5, which carries the structed plasmids carrying the dnaQ gene. The hybrid plasmids dnaQ gene (see Table 1), into a vector phage, Agt-AC (9). To contain a 1.5-kilobase (kb) insert of E. coli DNA that codes for transpose Tn3 to the AdnaQ+, the phage was propagated in cells two proteins whose molecular weights are 25,000 and 21,000. that harbor pKY2283 (ColEl: :Tn3). Progeny phages that had By biochemical and genetic analyses, the 25-kilodalton (kDal) acquired ampicillin resistance, conferred by Tn3 (12), were protein was identified as the dnaQ gene product and the 21- isolated and their DNAs were analyzed. kDal protein, as RNase H. It was suggested that the dnaQ gene Labeling of Plasmid-Encoded Proteins. The maxicell method and the structural gene for RNase H are adjacent on the E. coli of Sancar et al. (6) with a minor modification (13) was used. chromosome. Bacteria were grown at 37°C in M9 medium supplemented with 1% Casamino acids and 0.2% glucose. When OD at 660 nm x was irra- The publication costs ofthis article were defrayed in part by page charge reached about 0.4 (3 108 cells per ml), the culture payment. This article must therefore be hereby marked "advertise- nent" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviations: kb, kilobase pair(s); kDal, kilodalton(s). 3770 Genetics: Horiuchi et al. Proc. Natl. Acad. Sci. USA 78 (1981) 3771 diated with ultraviolet light (60 J/m2). After the culture was Table 1. Properties of hybrid plasmids shaken for 1 hr, D-cycloserine was added to give a final con- centration of 100 ,gg/ml and the culture was shaken overnight Complemen- Size of DNA, kb at 370C. One milliliter of the culture was centrifuged: the cells tation E. coli Y3 were washed and resuspended in 1 ml ofM9 supplemented with Plasmid dnaQ dnaE pBR322 chromosome insertion 25 ug each of 18 amino acids other than methionine and cys- pMM1 - + 4.4 9 - teine. [3S]Methionine (1000 Ci/mmol; 1 Ci = 3.7 X 1010 pMM4 + - 4.4 4.5 - becquerels) was added to give 82 ,uCi/ml, and the proteins syn- pMM5 + - 4.4 1.5 - thesized under these conditions were labeled for 1 hr at 370C. The labeled cells were centrifuged, washed, resuspended in 0.1 pMM5::y8(dnaQ+) + NT 4.4 1.5 5.7 ml of a buffer, and heated for 2 min. The sample (25 pl) was pMM5: :y(dnaQ-) - NT 4.4 1.5 5.7 applied to 12.5% NaDodSO4polyacrylamide gel. NT, not tested. Assay of RNase H Activity. The procedure for preparation ofcell extract was essentially the same as described by Wickner et al. (14). RNase H activity was assayed according to Berkower hand, pMML, which complements the dnaE but not the dnaQ et al. (15). The reaction mixture contained 3 ,uM poly(A) (483 defect of recipient cells, had a 9-kb EcoRI fragment. cpm/pumol: 40-140 nucleotide residues) hybridized with 10 ttM Identification of the dnaQ Gene Product. To identify the poly(dT), 40 mM Tris HCl (pH 7.7), 4 mM MgCl2, 1 mM di- dnaQ gene product, proteins encoded by the dnaQ+ plasmids thioerythritol, bovine serum albumin at 30 Ag/ml, 4% (vol/ were specifically labeled by the maxicell method developed by vol) glycerol, and an extract. The reaction ran for20 min at 30°C, Sancar etal. (6) and analyzed by NaDodSOjpolyacrylamide gel and then the radioactivity converted into acid-soluble form was electrophoresis. Besides two known pBR322-encoded proteins determined. One unit of activity was defined as the amount of (28 and 37 kDal), pMM5 produced two specific proteins, a 25- enzyme producing 1 nmol ofacid-soluble material in 20 min at kDal protein that comigrated with 25-kDal chymotrypsinogen 300C. A and a 21-kDal protein that ran slightly faster than 21.5-kDal Analysis of RNase H. E. coli CSR603 harboring pMM5 or soybean trypsin inhibitor (Fig. 1). The corresponding bands pBR322 (60 ml each) was labeled with [35S]methionine (1000 Ci/ were found also in pMM4 (data not shown). mmol; 40 ,Ci/ml) by the maxicell method. The procedure and To determine which protein is specified by the dnaQ gene, the conditions were as described in a preceding section except we introduced the yO sequence into pMM5. Two types ofplas- that the dose ofultraviolet irradiation was 30 J/m2. The labeled mids with yv insertion were selected: pMM5:: y6(dnaQ+), cells were collected by centrifugation, washed, and frozen in which still complements the dnaQ mutation, and liquid nitrogen. The cells were thawed, resuspended in 2 ml pMM5:: y8(dnaQ7), which does not complement the mutation of2.5 M NaCVl mM EDTA/1 mM 2-mercaptoethanoV20 mM (Table 1).
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