DNA synthesis in an Escherichia coli dnaB dnaC mutant

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

doi:10.1007/bf00446907

Cited by 20 publications

(12 citation statements)

References 19 publications

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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). E. coli dnaB groPA15 (19) was used as a source for DNA polymerase III.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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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Section: Methodsmentioning

confidence: 99%

“…Electrophoresis was for 1 hr at 2.2 V/cm, followed by a 13-hr run at 4.5 V/cm (3.3 mA per tube) at 40C. The gel was cut into 2.6-mm slices, which were incubated for 15 hr at 4°C in 50 Ml of buffer B to elute the protein. Aliquots were assayed for DNA primase activity as described in the legend to Table 2.…”

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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“…Other chemicals were as described (10,11). Anti-body to E. coli dnaB (13.5 mg of gamma globulin protein per ml) was a gift of A. Kornberg and R. McMacken.…”

Section: Methodsmentioning

confidence: 99%

Escherichia coli dnaB mutant defective in DNA initiation: isolation and properties of the dnaB protein.

Lanka¹,

Geschke²,

Schuster³

1978

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

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Extracts of the DNA initiation-defective mutant Escherichia coli dnaB252 are inactive in a dnaB complementation assay but yield a ribonucleoside triphosphatase activity of native molecular weight of about 270,000 (60,000-dalton polypeptide as subunit) that can be inactivated by antibody to dnaB. On the other hand, extracts of a dnaB252(PI bac) lysogen, in which the dnaB mutation is suppressed in vivo by the constitutive expression of the P1 dnaB analog (ban protein), are active in dnaB complementation and the activity is also sensitive to dnaB antibody. Upon further purification two proteins (with polypeptide molecular weights of 60,000 and 56,000, respectively) are found associated with each other (native molecular weight about 270,000). The larger and the smaller protein are tentatively identified as the dnaB and P1 ban protein. It is suggested that suppression of the dnaB mutation by prophage PI bac is accomplished by a stabilization of dnaB252 by P1 ban subunit molecules in a heteromultimer. Escherichia coli dna ts mutants are usually divided into two classes, DNA initation-and DNA elongation-defective mutants.

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“…Besides its role in conjunction with dnaB in the prepriming reaction of 0X174 complementary strand synthesis (9) it was found that dnaC protein protects dnaB protein in vitro from inactivation by the P protein of phage X (24). Results obtained with an E.coli dnaB-dnaC double mutant suggest that inactivation of dnaB protein in vivo also affects the dnaC function (19). Moreover, suppression of the dnaB252 initiation mutant by elevated dnaC gene dosage (25) as well as suppression of dnaC alleles by the dnaB analog (ban) protein of phage P1 (26) convincingly point to an interaction of dnaB (ban) and dnaC protein in vivo.…”

Section: Discussionmentioning

confidence: 97%

“…dnaB and dnaC activities were determined by a complementation assay (5,14) using 90 to 120 pg of Fraction I ( 43 % saturated ammonium sulfate) of BT1071 dnaBts (16) and PC22 dnaCts (18), respectively. The activity of the dnaB-dnaC complex was determined in some experiments by complementation of Fraction I from the double mutant E.coli BC1304 dnaBts-dnaCts (19).…”

Section: Methodsmentioning

confidence: 99%