ATP-dependent DNA supercoiling catalyzed by Escherichia coli DNA gyrase was inhibited by oxolinic acid, a compound similar to but more potent than nalidixic acid and a known inhibitor of DNA replication in E. coil. The supercoiling activity of DNA gyrase purified from nalidixic acidresistant mutant (naL4U) bacteria was resistant to oxolinic acid. Thus, the nalA locus is responsible for a second component needed for DNA gyrase activity in addition to the component determined by the previously described locus for resistance to novobiocin and coumermycin (cou). Supercoiling of X DNA in E. coli cells was likewise inhibited by oxolinic acid, but was resistant in the nalAR mutant. The inhibition by oxolinic acid of colicin El plasmid DNA synthesis in a cell-free system was largely relieved by adding resistant DNA gyrase.In the absence of ATP, DNA gyrase preparations relaxed supercoiled DNA; this activity was also inhibited by oxolinic acid, but not by novobiocin. It appears that the oxolinic acid-sensitive component of DNA gyrase is involved in the nicking-closing activity required in the supercoiling reaction. In the presence of oxolinic acid, DNA gyrase forms a complex with DNA, which can be activated by later treatment with sodium dodecyl sulfate and a protease to produce double-strand breaks in the DNA. This process has some similarities to the known properties of relaxation complexes. Previous work (1-3) has described an enzyme activity, DNA gyrase, that is responsible for the supercoiling of DNA in Escherichia colh. As isolated from extracts of E. colh, the enzyme introduces negative superhelical turns into covalently closed circular DNA in an ATP-dependent reaction; the hydrolysis of ATP presumably provides the free energy needed to accumulate mechanical strain energy in the DNA.One genetic locus (cou), which determines resistance to coumermycin A1 and novobiocin, has been identified as controlling the activity of DNA gyrase (2). The enzyme isolated from wild-type cells is inhibited by both these antibiotics, while DNA gyrase from a coumermycin-resistant mutant strain is unaffected. Intracellular DNA supercoiling is similarly blocked by coumermycin.In this paper we report the involvement of a second genetic locus (nalA), which determines resistance to nalidixic acid and oxolinic acid (4, 5), in controlling DNA gyrase activity. These two drugs are inhibitors of DNA replication in E. coli (4, 5). They also inhibit replication in cell-free systems of colicin El plasmid (ColEl) DNA (6, 7) and of phage qX174 replicative form DNA (8), but they do not inhibit the synthesis of the complementary strand of /X174 single-stranded DNA (8). These properties are parallel to those described for coumermycin A1 and novobiocin.Nalidixic acid-resistant mutants of two classes have been identified and mapped (9). Mutations at one locus (naIB, 57 min on the standard E. coli map) are responsible for low-level resistance and have been characterized as interfering with the permeability of the cells to nalidixic acid. Mutations ...

Novobiocin and coumermycin are known to inhibit the replication of DNA in Escherichia coli. We show that these drugs inhibit the supercoiling of DNA catalyzed by E. coli DNA gyrase, a recently discovered enzyme that introduces negative superhelical turns into covalently circular DNA. The activity of DNA gyrase purified from a coumermycin-resistant mutant strain is resistant to both drugs. The inhibition by novobiocin of colicin El plasmid DNA replication in a cell-free system is partially relieved by adding resistant DNA gyrase. Both in the case of colicin El DNA in E. coli extracts and of phage X DNA in whole cells, DNA molecules which are converted to the covalently circular form in the presence of coumermycin remain relaxed, instead of achieving their normal supercoiled conformation. We conclude that DNA gyrase controls the supercoiling of DNA in E. coli.Novobiocin and the related drug coumermycin are preferential inhibitors of DNA replication in intact Escherichia coli cells (1, 2). In toluenized cells of E. coli, these drugs inhibit replicative DNA synthesis but not repair synthesis (3, 4). They are also effective inhibitors in cell-free systems for the replication of colicin El (Col El) DNA (5) and of phage OX174 replicative form DNA (6), but they do not inhibit the synthesis of the complementary strand of OX174 single-stranded DNA (6).Coumermycin-resistant mutants of E. coli have been isolated, and the mutation has been mapped near the dnaA locus (2).An enzyme, DNA gyrase, that introduces negative superhelical turns into double-stranded closed circular DNA, has recently been purified from E. coli (7). In this paper, we show that DNA gyrase activity in vitro as well as in vivo is specifically inhibited by coumermycin and novobiocin. Among several spontaneous coumermycin-resistant mutants of NI708 which were tested, all gave resistant extracts for the in vitro Col El DNA replication system. One of these mutants, N1741, was chosen for further work because of its good growth. This strain apparently has a partial reversion of the permeability mutation of N1708. The couR mutation of NI741 was transferred, by phage P1 cotransduction with dnaA+, to strain CRT46 dnaA (10). The resulting strain, N1748, was used as a source for purification of drug-resistant DNA gyrase. DNA gyrase sensitive to both novobiocin and coumermycin was isolated from N99 recB21 (7). Strain N1071(Xind-) (11) was used for experiments of superinfection by phage X, and strain YSl (8) for testing supercoiling of Col El DNA in extracts. MATERIALSOf these strains, the coumermycin-resistant isolates NI741 and N1748 are able to grow in liquid culture containing 60 gg/ml of coumermycin; growth of the other strains is blocked by 15 gg/ml of the drug.Chemicals. Novobiocin was obtained from Sigma Chemical Co. Samples of coumermycin A1 (referred to as coumermycin throughout this paper) were gifts from W. F. Minor (Bristol Laboratories) and J. Davies (University of Wisconsin). Sources of other materials have been described previously (7,8,12).Meth...

Meiotic recombination of S. cerevisiae contains two temporally coupled processes, formation and processing of double-strand breaks (DSBs). Mre11 forms a complex with Rad50 and Xrs2, acting as the binding core, and participates in DSB processing. Although these proteins are also involved in DSB formation, Mre11 is not necessarily holding them. The C-terminal region of Mre11 is required only for DSB formation and binds to some meiotic proteins. The N-terminal half specifies nuclease activities that are collectively required for DSB processing. Mre11 has a DNA-binding site for DSB formation and another site for DSB processing. It has two regions to bind to Rad50. Mre11 repairs methyl methanesulfonate-induced DSBs by reactions that require the nuclease activities and those that do not.

A plasmid that consists of an 812-base-pair segment containing the replication origin of lasmid ColEl and of a 1240-base-pair segment containing a' lactamase gene has been constructed. The plasmid DNA ias three principal sites where transcription is initiated in vitro. One is located in the CoIEl segment 555 nucleotides upstream from the origin. Most transcription from this site extends past the origin; some of the transcripts form hybrids spontaneously with the template at their 3' portions. Cleavage of these transcripts by RNase H generates 3' termini at the origin region. When DNA polymerase I is included in the reaction along with RNA polymerase and RNase H, dAMP or dCMP is added directly onto the cleaved RNA molecules, most of which retain the intact 5' terminus. The addition of a deoxyribonucleotide to the cleaved RNA can be regarded as the first step of ColEl DNA synthesis. Once it has served as a primer, the RNA is eliminated from the product by RNase H.Closed circular DNA of plasmid ColEl of Escherichia coil can be replicated in vitro by a soluble enzyme system (1). Replication starts most frequently at any of the three consecutive nucleotides (dA, dA, and dG) located at a unique position on the DNA (2). No plasmid-encoded protein is required for this replication (3). Initiation of replication can be demonstrated with a mixture of three highly purified E. coil enzymes: RNA polymerase, DNA polymerase I, and RNase H (4), an enzyme that creates a 3'-OH end by endonucleolytic cleavage of RNA that is hybridized to DNA (5). Previously we proposed (4) that RNA polymerase synthesizes a transcript that is processed by RNase H and then used as a primer by DNA polymerase L. Here we report the identification of such a transcript and demonstrate that it is processed by RNase H to form the primer. The replication of ColEl has been recently reviewed (6-8).MATERIALS AND METHODS Plasmid DNA. Plasmid pNT7 which contains the replication origin of ColEl and the f3-lactamase gene of the Tn3 transposon was derived from pNT5 (9) and pBR322 (10) as described below. Supercoiled molecules of plasmid DNA were purified as described (4).Enzymes. E. coli RNA polymerase (600 units/mg) was the holoenzyme (4, 11). DNA polymerase 1 (15,000 units/mg) (12) was a gift of A. Kornberg. RNase H (470,000 units/mg) was prepared as described (13) with the following modifications: The first DEAE-cellulose chromatography was performed at pH 8.9 instead of pH 7.5. An additional DEAE-cellulose chromatography was performed at pH 7.5 without a salt gradient. Sephadex G-50 chromatography replaced glycerol gradient centrifugation. NaDodSO4/polyacrylamide gel electrophoresis (14) A linear gradient gel of 2-20% polyacrylamide was also used. The 25% gels were 0.4 mm thick, whereas the others were 1.5 mm thick. The length of the slab gels was 38 cm and their width was 16 or 33 cm. Samples dissolved in 1 mM EDTA/5 M urea were heated at 80'C for 3 min immediately before loading. To recover nucleic acids from the gel, the gel was crushed and shaken f...