A mutant strain of E. coli, initially identified by an abnormally high frequency of recombination, has been found to be defective in the 5' -. 3' exonuclease associated with DNA polymerase I, but not in the polymerase activity. This defect is tolerated at 300, but is lethal at 43°. Like other polymerase I mutants, the strain is unusually sensitive to methyl methanesulfonate and to ultraviolet irradiation; it is also unable to support the growth of phage X defective in general recombination, and shows a reduced rate of joining of 10S "Okazaki fragments." These results demonstrate that a functional DNA polymerase I is essential for normal growth and viability in E. coli K12.Escherichia coli K12 strains defective in the structural gene for DNA polymerase I (polA) have been extensively described (1-5). These mutants are abnormally sensitive to ultraviolet irradiation and to the alkylating agent methyl methanesulfonate, and show retarded joining of the 10S "Okazaki fragments" (6)(7)(8). A recent analysis of partially purified fractions derived from..several of the polA mutants has demonstrated that although they contain much reduced levels of DNA polymerase I activity the 5' --3' exonuclease associated with the polymerase I is present in nearly normal amounts (9).We have found that polA mutants are also characterized by an increased frequency of recombination at least under some conditions (7). By screening mutagenized cells for this phenotype, we have isolated a novel polA mutant (polAexl) that we describe in this paper. This mutant has a normal level of polymerase I activity but a greatly reduced level of 5' -_ 3' exonuclease. Unlike other polA mutants, it is conditionally lethal. MATERIALS AND METHODSBacterial and Phage Strains. All bacterial strains are derived from E. coli K12. Their origin and genotypes are given in Table 1. Phage X defective in general recombination (A red 3) was provided by D. Freifelder.Microbiological Procedures. Pivir phage stocks were prepared and used for transduction, and Hfr crosses were performed as described by Miller (10). Sensitivity to ultraviolet irradiation was determined as follows. Cells grown at 30°in H broth (11) to A595 = 0.5 were chilled, harvested, and resuspended in an equal volume of chilled M63 medium (10); 10 ml of a 5-fold dilution of this suspension in a glass petri plate was irradiated (25 ergs per mm2 per sec) with a General Electric germicidal lamp; and samples were removed at various times, diluted in chilled H broth, and plated on yeast extract-tryptone plates (10) that were incubated at 300 in the dark. Sensitivity to methyl methanesulfonate was determined on fresh yeast extract-tryptone plates supplemented with 0.04% methyl methanesulfonate. Cultures were treated with ethyl methanesulfonate as described by Miller (10). Lactosetetrazolium indicator plates were prepared according to Miller (10). Ability to form plaques of phage A was determined on tryptone plates as described by Miller (10). Unless otherwise noted, E. coli polAexl strains were grown at ...

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A method is described for the isolation of Escherichia coli mutants that show increased recombination between a pair of chromosomal duplications. These "hyper-rec" mutants display a variety of secondary phenotypes. I have isolated a large number of hyper-rec mutants and found them useful in screening for mutants that accumulate labeled DNA fragments after short pulses with [3H]thymidine. The mutants so recovered include ones that are defective in deoxyribonucleic acid ligase, deoxyribonucleic acid polymerase I and its associated 5' yields 3' exonuclease, and a group of mutants, dnaS, that accumulate abnormally short Okazaki fragments. Evidence is presented that suggests that the lac-att80 segment of the chromosome cannot be inverted.

A new group of mutants has been isolated which, during short pulses, incorporate [3Hjthymidine into DNA fragments that are substantially smaller than Okazaki fragments. These small fragments can be chased into DNA of high-molecular-weight, and thus may be precursors in DNA replication. During longer pulses, label also appears in DNA of higher molecular weight, although at an abnormally slow rate. The mutations map at a previously undescribed locus (dnaS) at 72 min on the E. coli chromosome.DNA replication in Escherichia coli appears to be a discontinuous process whereby daughter strands are synthesized as short fragments that are joined by DNA ligase to form covalently continuous strands (1-5). Intervening steps might include removal of an RNA primer on the 5' end of each fragment (6), perhaps by the 5' --3' exonuclease of DNA polymerase I (7) and filling of gaps between fragments also by DNA polymerase I (8, 9). Among the evidence in support of this model are kinetic studies which show that during short pulses, [8H]thymidine is incorporated predominantly into fragments about 1000 nucleotides long (Okazaki fragments) and later appears in DNA of high-molecular-weight (1, 2). The life-time of these normally short-lived fragments is greatly lengthened by deficiencies in DNA polymerase I, its associated 5' -* 3' exonuclease, or in DNA ligase (3-5, 7-9). This paper describes a novel group of mutants in which short pulses of [3H]thymidine are incorporated into fragments substantially smaller than the Okazaki fragments found in wild-type strains. These mutations are tentatively assigned to a new locus, dnaS, which is linked to pyrE at 72 minutes on the E. coli map. MATERIALS AND METHODSPhage and Bacterial Strains. All bacterial strains are derived from E. coli K12. Genetic nomenclature is from Taylor and Trotter (10).Strains KS391 (Hfr Hayes thi-lacMS286480dIIlacBK1) and KS418 (F-metB-ara-thi-lacMS2864dIIlacBK1) were used in isolating dnaS mutations; both of these are diploid for lac. c80dIIlac is a defective prophage inserted at att8O, that carries the lac operon but not 080imm (11), and lac-MS286 is a deletion including lacY and lacZ, furnished by M. Malamy. IacBK1 is a deletion of part of lacZ that does not overlap lacMS286. Strains RS5087, RS5083, RS5091, and KS474 were constructed by transducing strain KS468 (FmetB-thi-pyrE-lacMS286,8OdIIlacBKlstr') to pyrE+ and dnaS1, dnaS2, dnaS3, and dnaS+, respectively (see below). Phage Plvir was provided by J. Shapiro, X and X redS by Dr.Freifelder, and the recA 1 mutation by A. J. Clark.Scoring the "Hyper-rec" Phenotype. Strains carrying lacMS286480dIIlacBK1 were tested for the "hyper-rec" character by streaking to single colonies on lactose tetrazolium indicator plates. These strains are lactose negative, and initially form red colonies. On prolonged incubation, white lactose positive papillae, each representing a clone of recombinant (lac-080-dIIlac+) cells, appear on the surface of colonies. Colonies showing conspicuously more papillae than wild type were scored ...

F-prime heterogenotes of dam-3 bacteria segregate F-prime homogenotes at a frequency 30-200 times higher than the isogenic dam+ strain. A hyperrecombination mutant which shows increased recombination between chromosomal duplications was characterized as a dam mutant. The dam-3 allele causes a reduction in linkage of proximal unselected markers in transconjugants and increases the recombination frequency between a pair of closely linked markers. It is concluded that dam mutations confer a hyperrecombination phenotype to the cell.