Characterization of REP− mutants and their interaction with P2 phage

Calendar, Richard; Lindqvist, Björn H.; Sironi, Gianpiero; Clark, Alvin J.

doi:10.1016/0042-6822(70)90380-6

Cited by 145 publications

(46 citation statements)

References 23 publications

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“…P2 does not grow on polAl strains, but does grow on the polB1 mutant, HMS85. Therefore, the rep-locus is probably not the gene for polymerase II (26). The malespecific phage fd gives a normal burst size when grown on HMS83 FTS114laC+, although the plaques are more turbid than those obtained on JG138 FTs114lac+.…”

Section: Mutagenesis Of E Coli Jg138 Polal Su7+mentioning

confidence: 98%

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Campbell

Soll

Richardson

1972

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

125

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A mutant of Escherichia coli deficient in DNA polymerase II has been isolated from E. coli polAl by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assay of polymerase activity in extracts of survivors. The polAl mutation was suppressed during mutagenesis by introduction of the suppressor, su7 +, into the parental strain. The mutant, HMS83 polAl polBi, contains less than 0.5% of the normal levels of DNA polymerase II. The only polymerase activity detected in the mutant is DNA polymerase III. E. coli HMS83 grows normally at both 25 and 420, and supports the growth of bacteriophages T4, T7, lambda, OX174, 186, P2, and fd. The poiB mutation does not affect sensitivity to ultraviolet irradiation or recombination frequencies.Three distinct DNA polymerases have been found in extracts of Escherichia coli. The major activity, polymerase I, has been purified to homogeneity and studied extensively (1). In order to determine the role of polymerase I in vivQ, DeLucia and Cairns (2) isolated a mutant of E. coli, E. coli polAl, that has greatly reduced levels of polymerase I activity in extracts. The mutant grows normally, but has definite physiological defects in repair mechanisms (2, 3).The residual polymerase activity detectable in extracts of E. coli polAl has been resolved into two enzymes: polymerase II (4-11) and polymerase III (5). In a survey of mutants temperature-sensitive for DNA synthesis, dnat8, Gefter et al. (12) found that strains with a mutation at the dnaE locus (13) contained a thermolabile polymerase III. Niisslein et al. (14) have partially purified the dnaE gene product using an in vitro complementation assay for replication, and have shown that the purified fraction contains an activity that has all of the properties of polymerase III. Therefore, it seems that polymerase III is essential for DNA replication.In contrast to polymerase III, polymerase II activity is normal in all the known classes of dnat8 mutants (7,12). Thus, in order to investigate the function of polymerase II in vivo, we decided to isolate a mutant lacking the enzyme. We tried to minimize the bias inherent in any selection by screening a highly mutagenized stock of E. coli for polymerase II in individual extracts. Because DNA polymerase I activity masks polymerase II activity in extracts, it was necessary to use E. coti polAl as the parental strain. This communication describes the isolation and partial characterization of a mutant lacking DNA polymerase II in extracts. MATERIALS AND METHODSBacterial and Phage Strains. The mutants were isolated from strains derived from E. coli W3110 thy-. The parental strain was JG138 thy-polAl rha-tacZam. Bacteriophage P1KC was grown on LS448F'14 and used for the transduction to su7+. (Calbiochem,10,000 units/mg) was added. The tubes were incubated for 5 min at 370, then chilled to 00 after the addition of 0.1 ml of 25% Triton X-100. The 2090 Abbreviations: NTG, N-methyl-N'-nitro-N-nitrosoguanidine; MMS, methylmethanesulfonate.

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Section: Mutagenesis Of E Coli Jg138 Polal Su7+mentioning

confidence: 98%

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Campbell

Soll

Richardson

1972

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

125

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“…There are several properties by which phage 186 resembles P2 and differs from X (2)(3)(4)(5)(6). In the present report we wish to determine if the similarities between phages P2 and 186 (and their differences from X) are also reflected in their respective modes of replication.…”

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confidence: 99%

Origin and Direction of Replication of Bacteriophage 186 DNA

Chattoraj

Inman

1973

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

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Intracellular bacteriophage 186 DNA replicates as a single-branched circle during the first round of replication. The free end of the branch is located at a unique position with respect to phage 186 DNA base sequence, and this point should, therefore, correspond to the origin of DNA replication. The position of the growing point has been mapped at various degrees of replication, and found to move unidirectionally from left to right with respect to the denaturation map of phage 186 DNA.A small proportion of the replicating molecules have two linear branches connected to the circle at two different branch points. These structures are consistent with two separate initiations from the same origin, again, with a unidirectional mode of replication. Branch points frequently have a single-stranded connection between the circle and the branch; significant numbers of branch points also possess an extra short single-stranded "whisker" protruding out of the branch point.The temperate bacteriophage 186 was isolated and the prophage site was localized on the Escherichia coli genetic map by Jacob and Wollman (1). There are several properties by which phage 186 resembles P2 and differs from X (2-6). In the present report we wish to determine if the similarities between phages P2 and 186 (and their differences from X) are also reflected in their respective modes of replication. Earlier investigations have already shown that both phages X and P2 possess unique starting points for replication and that replication is unidirectional for P2 and bidirectional for X (7,8).The strategy in these experiments is simply to isolate intracellular phage DNA and then to create a frame of reference on these molecules by partial denaturation. Examination of electron micrographs of partially denatured replicating molecules then allows the unambiguous location of the origin and direction of replication of each molecule. We will discuss the following experimental facts. (i) Intracellular replicative forms of phage 186 DNA frequently have a single-branched circular structure (a circular molecule possessing an extra linear segment attached to the circle at a branch point); more complex replicating structures are present at low frequency.(ii) Replication starts from a unique origin located 92.9% from the left end of the denaturation map of mature phage 186 DNA and always proceeds toward the right; replication in phage 186 DNA is, therefore, unidirectional. (iii) The molecular fine structure at branch points is complex. Frequently, single-stranded connections are observed at branch points and often there is an additional piece of single-stranded DNA protruding out of the branch point. STRUCTURE OF INTRACELLULARPHAGE 186 DNA When E. coli, growing in a heavy medium, is infected with light phage 186, it is possible to fractionate, by density-gradient centrifugation, various intracellular DNA species. During the first round of replication the phage DNA will have a density between light (LL) and hybrid (HL). In an electron microscope these fractions ...

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“…However, the P2 minichromosome still requires the E. coli Rep function, which is believed to be required to displace the parental strand to allow leading-strand synthesis during rolling-circle replication (11,13). Thus, during replication of the minichromosome, the P2 replicon seems to be able to recruit the DnaB-DnaC complex to the origin for lagging-strand synthesis, perhaps involving the A protein or some other origin-binding proteins.…”

Section: Discussionmentioning

confidence: 99%

“…P2 also utilizes several host components for its replication, like DNA polymerase III, primase, DnaB, and the Rep helicase (9,11), but encodes at least two proteins for its own replication. One is protein A, which initiates replication by introducing a sequence-specific, singlestranded cut at the origin of replication (ori) (33).…”

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confidence: 99%

The Interaction of Bacteriophage P2 B Protein with Escherichia coli DnaB Helicase

Odegrip

Schoen

Haggård‐Ljungquist

et al. 2000

J Virol

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Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where 35 S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.Many phages depend on the host DNA replication machinery, but they usually code for one or two proteins that direct the host machinery to their own genomes. For example, phage codes for O, an origin-binding protein, and P, a protein that interacts with O as well as with Escherichia coli DnaB helicase (36). The P protein can thus direct the helicase to the phage origin and is considered an analogue of E. coli DnaC protein, which loads the helicase onto the bacterial origin (51).Bacteriophage P2 is a temperate coliphage that replicates via a modified rolling-circle mechanism generating monomeric double-stranded circles (7). P2 also utilizes several host components for its replication, like DNA polymerase III, primase, DnaB, and the Rep helicase (9, 11), but encodes at least two proteins for its own replication. One is protein A, which initiates replication by introducing a sequence-specific, singlestranded cut at the origin of replication (ori) (33). The second P2 protein required for phage replication is the B protein (31,32). It is believed to be required for lagging-strand synthesis, since the displaced strand during rolling-circle replication remains single stranded in the absence of B (20). Since laggingstrand synthesis requires loading of the helicase (DnaB)-primase (DnaG) complex, a defect in helicase loading could account for the result. The requirement for the E. coli DnaB protein in P2 DNA replication is known (9). Moreover, a P2 rlb1 mutation, within the coding part of the B gene, has been shown to partially suppress a dnaB(Ts) mutation (49), suggesting that P2 B interacts directly with DnaB. These results led to the hypothesis that it is a DnaC analogue (23). However, the B protein was not required for replication of a P2 minichromosome containing only the P2 A gene...

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Characterization of REP− mutants and their interaction with P2 phage

Cited by 145 publications

References 23 publications

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Isolation and Partial Characterization of a Mutant of Escherichia coli Deficient in DNA Polymerase II

Origin and Direction of Replication of Bacteriophage 186 DNA

The Interaction of Bacteriophage P2 B Protein with Escherichia coli DnaB Helicase

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