Isolation and Characterization of dnaX and dnaY Temperature-Sensitive Mutants of ESCHERICHIA COLI

Abstract

Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage P1, dnaX and Y have been mapped at minutes 10.4–10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and Yts10 are recessive to wild-type alleles present on episomes. F13 carries both dnaX⁺ and Y⁺; the shorter F210 carries dnaY⁺, but not X⁺. Lambda transducing phages that carry dnaX⁺ or Y⁺ have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the Clarke and Carbon (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to 44°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially inhibited even at 30°.—When the dnaYts10 mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.—The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

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