Replication of the Escherichia coli Chromosome with a Soluble Enzyme System

Abstract

Semi-conservative DNA synthesis is observed when the isolated, folded E. coli chromosome is supplemented with a DNA-free, soluble enzyme fraction, the four deoxynucleoside 5'-triphosphates, ATP, and Mg++. The DNA synthesized in vitro remains associated with the folded chromosome during sedimentation through neutral sucrose, but is released as small DNA fragments in alkali. Sealing of these replicative intermediates to the chromosome requires the presence of both E. coli DNA polymerase I (EC 2.7.7.7) and DNA ligase (EC 6.5.1.2).Study of the replication of Escherichia coli DNA has been complicated both by the need for coordinated interplay of many enzymes and by the fragile nature of the template. For these reasons E. coli DNA replication has not been successfully reconstructed in solution. DNA synthesis observed in cell-free extracts (1-7) represents catalytic actions of the DNA polymerases (1, 5, 7-10), but lacks many of the important features of replication. Moses and Richardson (11) showed that E. coli cells rendered permeable by toluene to low-molecular-weight precursors are able to replicate their DNA; however, the cells remained impermeable to proteins and thus to fractionation and dissection of the enzymatic steps of replication. Schaller et al. (12) demonstrated that replication could proceed in cells lysed on cellophane discs; however, this system appears to require the tight complex of macromolecular components of the bacterial cell, rendering its fractionation extremely difficult.We were encouraged to attempt the replication of DNA in a soluble system by two recent advances. First, a special highspeed supernatant from gently lysed cells (13,14) has been shown to contain the active gene products (14, 15) defined genetically to be essential for E. coli DNA synthesis (the dnaA, B, C, E, and G proteins) (16) as well as other replicationproteins not yet defined by genetic lesions (17). Second, a method has been developed whereby E. coli spheroplasts yield a nonviscous preparation of intact, folded, and supercoiled chromosomes (18,19).These isolated folded chromosomes behave like compact particles, and sediment either as free, folded chromosomes at 1300-2200 S or at 3000-4000 S in a membrane-attached form (20,21). The physical properties of these extracted particles vary with the cell's physiological state: replicating chromosomes isolated at 10°-15°in 1 M NaCl remain associated with the bacterial envelope, whereas nonreplicating chromosomes are released membrane-free (20). The size distribution of the chromosomes also reflects their growth during replication (19).The exploitation of these techniques has allowed us to demonstrate and characterize the unique DNA synthesis that occurs when the folded chromosomes are combined with a DNA-free, soluble enzyme system. As reported below, this DNA synthetic reaction contains many of the features embodied in in vivo chromosome replication. Henry; DNA polymerase I (DNA nucleotidyl transferase, EC 2.7.7.7, 3 mg/ml, 18,000 units/mg) a gift from Dr. D. Brutlag;...

Growth phase dependent changes in the structure and protein composition of nucleoid in Escherichia coli

“…After examining several parameters including nucleoid buffer composition (different concentrations of salt, lysozyme, detergent, spermidine), reaction conditions for preparation of cell lysates, different centrifugation speeds and times, we developed a procedure for the isolation of nucleoids in a single visible fraction from the sucrose density gradient centrifugation (Figure 1). Our established method was mainly based on nucleoid isolation procedures described previously by Kornberg et al [31], and modified by Murphy and Zimmerman [32].…”

“…Preparation of nucleoids and their sucrose density gradient centrifugation run were performed according to the protocol devised by Kornberg et al [31] and modified by Murphy Figure 1 Schematic procedure for preparation and isolation of E. coli nucleoids. E. coli cells were cultured from exponential phase to early staitionary phases, harvested, prepared cell lysates and their nucleoids were separated from the whole cell extracts by sucrose density gradient centrifugation (see Materials and methods for detail).…”

Unfolding of the bacterial nucleoid both in vivo and in vitro as a result of exposure to camphor

“…If the procedure is carried out at 25ЊC, then membrane-free nucleoids (type I) are predominant, whereas if the procedure is carried out at 10ЊC, membrane- attached nucleoids (type II) are predominant (17). Nucleoids can also be isolated by using 10 mM spermidine to stabilize the nucleoids instead of 100 mM NaCl (type III) (9). This is reported to result in somewhat more physiologically relevant nucleoids that are more stable than type I or type II nucleoids (4).…”

Isolation, Purification and Characterization of Nucleoids from <i>Synechococcus elongatus</i> PCC 7942

“…Our established method was mainly based on nucleoid isolation procedures described previously by Kornberg et al [27] and modified by Murphy and Zimmerman [28]. In this method, cells were treated with lysozyme and then lysed with detergent NP-40 in low salt media containing spermidine.…”

“…Collected cell were chilled in an ice-cold water bath and harvested by centrifugation (12,000 ×g, 4˚C, 10 min). Preparation of nucleoids and their sucrose density gradient centrifugation run were performed essentially according to the protocol devised by Kornberg et al [27] and modified by Murphy and Zimmerman [28] and this study (for details see Figure 2). Linear gradients (12% -60% sucrose in 10 mM TrisHCl (pH 7.8 at 0˚C) containing 10 mM KCl, 1 mM EDTA, 0.2 mM DTT (dithiothreitol), 1 mM spermidine HCl) were formed in a cold room in 5 ml Ultraclear plastic centrifuge tubes (13 × 51 mm, Beckman) and were run at 10,000 rpm at 4˚C for 120 min in a RPS50-2-517 rotor (the centrifugal force was reduced to 7000 g).…”