Novel mutants of Escherichia coli that accumulate very small DNA replicative intermediates.

Abstract

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 ...

ThemutTDefect Does Not Elevate Chromosomal Fragmentation inEscherichia coliBecause of the Surprisingly Low Levels of MutM/MutY-Recognized DNA Modifications

“…It should be noted that the mutT1 recA mutants were reported to be viable (17); however, this is the first time that the viability of mutT recBC mutants was tested. For example, the rep and rnhA mutants are synthetically lethal with the recBC defect but viable with the recA defect (33,77 (23,39), apparently due to a rapid accumulation of suppressors (42). Finally, our study was the first one to employ a complete mutT deletion allele, which could have behaved differently from the mutT1 allele.…”

Characterization of a Mutant RecA Protein that Facilitates Homologous Genetic Recombination but not Recombinational DNA Repair: RecA423

“…Intrachromosomal recombination was assayed in a host carrying two copies of the lac operon, each with a non-overlapping deletion. Recombination between the two defective lac genes produces Lac + recombinants, which can be scored as red papillae on white Lac − colonies on MacConkey-lactose indicator plates (Konrad & Lehman, 1975). A mutant deficient in recombination, such as DrecA306, failed to produce any Lac + papillae out of 10 4 scored colonies, whereas recA + bacteria had an average of 24 papillae per colony (Table 1).…”

Discontinuous DNA Synthesis in Mammalian Cells

“…Recently a new mutant of E. coli has been obtained which makes fragments much smaller than normal-sized Okazaki fragments (Konrad & Lehman, 1975). One implication of this sof (small Okazaki fragments) mutation is that the first intermediate in E. coli DNA synthesis may be smaller (4-5S) than originally detected (lOS), and the mutation may cause some ligase deficiency that makes these pieces observable.…”

“…, 1973;Konrad & Lehman, 1975;Lark & Wechsler, 1975) that mayor may not be precursors to the 7-118 pieces.…”