On the process of cellular division in Escherichia coli

Formation of duplex DNA from 4X174 single-stranded DNA by extracts of E. coli was previously shown to require the gene product of dna B. Using as an assay the stimulation of 4X174 DNA-dependent synthesis in inactivated extracts of dna B temperaturesensitive cells, we purified the dna B gene product from wild-type E. coli as well as from a dna B temperaturesensitive mutant. The dna B temperature-sensitive gene product is more thermolabile than its wild-type counterpart.Crude extracts of Escherichia coli catalyze the conversion of 4X174 single-stranded circular DNA to double-stranded structures (1). This reaction requires OX174 DNA, dNTPs, ATP, and MgCl2 (1, 2) and involves the products of the dna B, C (D), E, and G genes (2-5). The gene product of dna A and F, RNA polymerase, and DNA polymerases I and II appear to be unessential in this system (1, 2, 5). Using a transfection system, Taketo studied the ability of E. coli dna thermosensitive (ts) mutants to support the growth of OX174 (6). He found dna B, C, D, E, and G ts mutants are nonpermissive at 430 to single-stranded DNA, but not dna A or F mutants. Also, a mutant deficient in DNA polymerases I and II supports the growth of OX174 both in vivo (7) and in the transfection system (6).Stimulation of inactivated crude extracts prepared from ts dna B, C, D, E, and G cells has provided complementation assays for purification of the products of the dna genes of E. coli. Using this assay, we have isolated the dna C (D), E, and G gene products (4, 5). The purpose of the present communication is to report the isolation of dna B gene product from wild-type cells and a ts dna B mutant. MATERIALS AND METHODSMaterials and Reagents, unless otherwise indicated, were as previously described (2, 4). derived from a particular dna ts mutant that can be inactivated and used for measurement of the dna gene product. adjusted to 0.2 mg/ml of lysozymet-0.01 M dithiothreitol-0.5% Brij 58 and immediately distributed into centrifuge tubes. After 60 min in ice, the mixture was centrifuged for 30 min at 100,000 X g at 40 and the supernatant was frozen in 1-ml portions; crude extract, when thawed, was stored in ice and used the same day. The crude extract of dna B receptor (17 mg of protein per ml) has been stored frozen for at least 4 months without detectable change in its activity.(B) Preparation of dna B receptor ammonium sulfate fraction. E. coli BT1029 (280 g of cells), grown and frozen as above, was thawed and adjusted to 0.2 mg/ml of lysozyme; after it was mixed, the suspension was immediately distributed into centrifuge tubes and incubated for 60 min at 00 and then for 3 min at 200. The lysate was centrifuged for 30 min at 100,000 X g and the supernatant (340 ml, 10 mg of protein per ml) was frozen overnight. After the fraction was thawed at 200, it was adjusted to 60% with solid ammonium sulfate (36.1 g/100 ml), kept 10 min in ice, and centrifuged for 30 min at 100,000 X g. The pellet was resuspended successively in 100 ml of 40%, 50 ml of 30%, and 20 ml of 20% saturated ...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Studies on In Vitro DNA Synthesis Isolation of dna B Gene Product from Escherichia coli

Wright

Wickner

Hurwitz

1973

“…In B. subtilis, a newly synthesized membrane protein of 35,000 Mr is not found in the membrane when the initiation mutant dna-i is incubated at nonpermissive temperature (11). Other studies using E. coli also support a possible involvement of the membrane during initiation (12)(13)(14).To demonstrate a direct role of the origin-membrane complex during initiation, we have examined the effect of two initiation-defective mutations (dna-i and dnaBl9) on the membrane association of replication origin regions of the host chromosome and a plasmid, pSLL03, in B. subtilis. The origin-membrane association has been analyzed by preparing membrane-associated DNA in two ways.…”

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confidence: 97%

DNA-membrane association is necessary for initiation of chromosomal and plasmid replication in Bacillus subtilis.

Winston¹,

Sueoka²

1980

We examined the effect of the inhibition of initiation of DNA replication on the membrane association of the chromosomal origin of replication of Bacillus subtilis and the Staphylococcus aureus-Bacillus pumilus chimeric plasmid pSLl03, using temperature-sensitive mutants of B. subtifis that have specifically affected initiation. Inhibition of initiation of the chromosome and pSLlO3 in the initiation mutant dna-i results in a decrease in the membrane association of both a marker near the chromosomal origin, purA16, and the plasmid pSL103.The membrane association of both purA16 and pSLl03 can be recovered by allowing initiation to resume at the permissive temperature. In another initiation mutant, dnaBi9, only the initiation and membrane association of the host chromosome are affected at the nonpermissive temperature, whereas both initiation and membrane association are not affected in the plasmid pSL103. In experiments in.vitro, DNA containing the purAl6 marker and pSL103 DNA molecules are both selectively released during incubation of purified DNA-membrane complexes prepare from dna-i cells at the nonpermissive temperature. On the other hand, only purA16 DNA is released in vitro from the DNA-membrane complex prepared from dnaB19 cells. This consistent coupling between initiation and membrane association indicates that DNA-membrane association is critical for the initiation of the B. subtilis chromosome and the plasmid pSL103.The initiation of chromosome replication is the major regulatory process controlling the cell cycle. Very little is known about the mechanism and regulation of initiation of bacterial chromosomes, although some of the biochemical requirements for initiation have been elucidated (reviewed in refs. 1 and 2).A plethora of evidence exists which demonstrates that the replication origin is associated with the membrane fraction in both Bacillus subtilis (3-7) and Escherichia coli (8,9). The origin appears to remain associated with the membrane throughout the DNA replication cycle (3, 9, 10). In B. subtilis, a newly synthesized membrane protein of 35,000 Mr is not found in the membrane when the initiation mutant dna-i is incubated at nonpermissive temperature (11). Other studies using E. coli also support a possible involvement of the membrane during initiation (12)(13)(14).To demonstrate a direct role of the origin-membrane complex during initiation, we have examined the effect of two initiation-defective mutations (dna-i and dnaBl9) on the membrane association of replication origin regions of the host chromosome and a plasmid, pSLL03, in B. subtilis. The origin-membrane association has been analyzed by preparing membrane-associated DNA in two ways. In one method, the DNA-membrane fraction (M fraction) is isolated by centrifugation of cell lysates in CsCl/sucrose gradients (high salt condition) (7). In the second method, sucrose gradients (low salt condition) are used to isolate a DNA-membrane complex (M complex) and a soluble complex (S complex) (15). The M complex is similar in its enrich...

“…There is evidence that suggests that the defect in the dnaB mutant involves an alteration in the cell membrane (11)(12)(13). Furthermore, it is known that replication of M13 involves membrane-bound intermediates (3,9).…”

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confidence: 99%

Replication of Bacteriophage M13: Specificity of the Escherichia coli dna B Function for Replication of Double-Stranded M13 DNA

Olsen¹,

Staudenbauer²,

Hofschneider³

1972

Infection of the temperature-sensitive E. eli mutant HfrH 165/70 (dnaB) with the filamentous ..1-Stranded DNA phage M13 is abortive at the restrictive temperature. Upon infection at 410, singlestranded phage DNA penetrates the cell and is converted in a rifampicin-sensitive step to the double-stranded replicative form (RF). The parental RF attaches to the cell membrane, but subsequent replication of the RF is blocked. It is concluded that in M13 infection semiconservative RF replication of a double strand to a double strand, in contrast to single-stranded DNA synthesis, depends specifically on the dnaB function.In strains of E. coli containing a temperature-sensitive mutation in the dnaB region, infection by the single-stranded DNA phage M13 is inhibited at the restrictive temperature (1). However, synthesis of phage single-stranded (ss) DNA and liberation of mature phage particles continues after cells that are actively producing phage are shifted to the higher temperature (1)t. This result implies that there is an earlier step in the replication process of M13 that depends on the dnaB function. Since a precise localization of the block in M13 replication may contribute information both to the replicative process of single-stranded DNA phages and to the biochemical lesion in the dnaB mutant, abortive M13 infection at the restrictive temperature was studied in more detail. At the restrictive temperature, M13 ss DNA penetrates the cell and is readily converted to RF. In contrast, neither the parental nor progeny RF could replicate at the high temperature. The data indicate that the block in DNA replication in the dnaB mutant involves the inability to perform semiconservative replication of double-stranded RF DNA. For determination of membrane-bound DNA, cells were lysed by lysozyme treatment in the presence of 15% (w/w) sucrose at 00 for 30 min, followed by 3-4 cycles of freezethawing. The lysate was then analyzed by centrifugation in a 20-40% sucrose gradient over a shelf of 60% sucrose (3).Preparation of 82P-labeled M13 phage and 3H-labeled ss M13 DNA and radioactivity assays have been described (4). RESULTS Abortive infection at 410To detect abortive infection under the conditions used, E. coli HfrH 165/70 (dnaB) cells were infected with M13 both at the permissive (340) and restrictive (410)