Suppression of E.coli dnaB Mutants by Prophage Plbac: A Biochemical Approach

Lanka, Erich; Schlicht, M.; Mikolajczyk, Maria; Geschke, B.; Edelbluth, Claus; Schuster, Hans‐Uwe

doi:10.1007/978-1-4684-0844-7_48

Cited by 13 publications

(49 citation statements)

References 7 publications

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“…The final preparation was about 90% pure as determined by NaDodSO4/polyacrylamide gel electrophoresis followed by staining with Coomassie blue (21) and was used in these experiments. The DnaA protein used was fraction III (19), the X 0 protein used was fraction III (22), the X P protein used was fraction III (22), and the DnaB protein used was fraction IV (23). DnaA, 0, and P proteins were about 90% pure, and DnaB protein was about 80% pure as determined by NaDodSO4/polyacrylamide gel electrophoresis (21 pSP102 and pCM960 plasmid DNAs were purified as described (2).…”

Section: Methodsmentioning

confidence: 99%

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

Wickner¹,

Chattoraj²

1987

Proc. Natl. Acad. Sci. U.S.A.

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Section: Methodsmentioning

confidence: 99%

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

Wickner¹,

Chattoraj²

1987

Proc. Natl. Acad. Sci. U.S.A.

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“…Unless otherwise indicated, these were as described (11)(12)(13). The following E. coli strains were used for preparation of receptor extracts: BT1071 dnaB (14), BC1304 dnaB dnaC (15), PC22 dnaC (16), BT1011 dnaG (14), AX727 dnaZ (17), and HMS83 dna+, polA, polB (18).…”

Section: Methodsmentioning

confidence: 99%

A DNA primase specified by I-like plasmids.

Lanka¹,

Scherzinger²,

Günther³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

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An enzyme has been isolated from Escberichia coli strains harboring the I-like plasmid R64drdll, which is capable of initiating DNA synthesis -on the circular, singlestranded DNA of phages 4X174, fd, and G4. In the conversion of these templates to duplex forms in vitro, the enzyme can substitute for the functions of E. coli dnaB-dnaC-dnaG proteins, E. coli RNA polymerase, and E. coli dnaG protein, respectively. The enzyme requires all four ribonucleoside triphosphates for optimal activity, although a combination of ATP, CTP, and GTP can almost completely satisfy the rNTP requirement. The enzyme appears to cooperate specifically witf DNA polymerase III because single-stranded DNA-dependent synthesis takes place in extracts deficient in DNA polymerases I and II but not in extracts from a dnaZ mutant. Highly purified enzyme preparations consist mostly of two major polypeptides, Mr 140,000 and 180,000, when analyzed by sodium dodecyl sulfate gel electrophoresis. These polypeptides cosediment with the enzyme activity through a glycerol gradient with a sedimentation coefficient of 3.6 S. DNA priming activity in extracts of E. coli strains harboring the mutant plasmids R64drdll or ColIdrdl, which are derepressed in functions of conjugational DNA transfer, is severalfold higher than the activity from strains carrying the corresponding wild-type plasmid. This correlation suggests that the enzyme may play a role in conjugational DNA synthesis. The I-like plasmids (R64, R144, and ColT) of the incompatibility group Ta are large, circular DNA molecules with molecular weights ranging from 62-72 X 106 (1, 2). Transfer properties specified by I-like plasmids are generally repressed, but derepressed (drd) mutants have been isolated (3, 4) and used to study the transfer of conjugative plasmids from donor to recipient cells of Escherichia coli (5-7). Only one of the strands of the plasmid is transferred during conjugational DNA synthesis (5). Conjugational DNA synthesis is independent of the E. coli dnaB and polA functions but requires the dnaE gene product in the recipient cell (6, 7). In addition, drd mutants of plasmids R64, R144, and ColT, but not those of F-like plasmids, partially suppress the phenotype of E. coli dnaG mutants (8).Many conjugative plasmids are able to suppress or enhance the temperature sensitivity of E. coli dnaB mutants (9), and five different R plasmids, including R64drdll and R144drd3, have been used to construct viable R-derivatives of the unsuppressed (sup+) E. coli dnaB266 amber mutant (10). This has been interpreted as evidence for the presence of dnaB analog (ban) genes being associated with these plasmids (10). During our studies in collaboration with V. N. Iyer (Ottawa) designed to identify a ban protein of plasmid R64drdll, we have discovered an enzyme capable of initiating DNA synthesis on circular single-stranded (ss) DNA templates. A similar enzyme activity has subsequently been detected in extracts from strains harboring the plasmids R144 and ColI. The enzyme level in extracts from st...

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“…Materials, reagents, and methods, unless otherwise indicated, were as previously described (14)(15)(16) Other Materials. DEAE-Sephacel, Octyl-Sepharose CL-4B, and Phenyl-Sepharose CL-4B were from Pharmacia, Phosphocellulose P11 was from Whatman, Brij 58 (polyoxyethylene monocetylether, n =20), Triton X-100 (polyoxyethylene octylphenylether, n = 10), and polyethylene glycol 20,000 were from Serva.…”

Section: Methodsmentioning

confidence: 99%

“…dnaB protein in 10 mM ATP (Fraction III) from strain Q1710 was used (15). ATP was removed, if necessary, by DEAE-Sephacel chromatography.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

ThednaCprotein ofEscherichia coli. Purification, physical properties and interaction withdnaBprotein

Lanka

Schuster

1983

Nucl Acids Res

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Suppression of E.coli dnaB Mutants by Prophage Plbac: A Biochemical Approach

Cited by 13 publications

References 7 publications

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

A DNA primase specified by I-like plasmids.

ThednaCprotein ofEscherichia coli. Purification, physical properties and interaction withdnaBprotein

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