The replication of bacteriophage P4 DNA in vitro

Krevolin, Mark D.; Calendar, Richard

doi:10.1016/0022-2836(85)90237-2

Cited by 24 publications

(5 citation statements)

References 24 publications

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“…Some of our results on the in vitro P4 replication do not match results reported previously (7,5 (23). The plasmid's replicative helicase RepA unwinds DNA in 5'-3' direction with respect to the strand it has bound.…”

Section: Discussioncontrasting

confidence: 55%

“…In order to further characterize the P4 replication process in vitro, we have improved the P4 replication assay previously described (7). Here we present in vitro data on the origin and direction of replication, the products, and the requirement for several host proteins.…”

Section: Introductionmentioning

confidence: 99%

“…Such a system has been developed, based on extracts of P4 infected cells and partially purified gpa (5,7). In this system, P4 replication depended strictly on gpca, SSB, and DNA polymerase HI holoenzyme.…”

Section: Introductionmentioning

confidence: 99%

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Phage P4 DNA replicationin vitro

1994

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Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 a protein , J. Mol. Biol. 182, [507][508][509][510][511][512][513][514][515][516][517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in 0-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase Ill holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.

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Section: Discussioncontrasting

confidence: 55%

Section: Introductionmentioning

confidence: 99%

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Phage P4 DNA replicationin vitro

1994

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“…It binds to doublestranded DNA containing the ColE2 origin in a sequencespecific manner as revealed by a filter binding assay (Kido et al, 1991) and synthesizes a unique primer RNA at the origin. The gene a protein of bacteriophage P4 required for phage replication (Gibbs et al, 1973;Krevolin and Calendar, 1985) binds to double-stranded DNA containing the cis-acting regions required for replication in a sequence-specific manner (Ziegelin et al, 1993) and possesses primase and helicase activities (Krevolin and Calendar, 1985;Strack et al, 1992;Ziegelin et al, 1993). Although synthesis of oligoribonucleotides on singlestranded fd DNA or on synthetic oligodeoxyribonucleotides has been demonstrated (Ziegelin et al, 1993), primer RNA synthesized on double-stranded DNA containing the origin has not been examined.…”

Section: Discussionmentioning

confidence: 99%

Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

1995

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Initiation of in vitro ColE2 DNA replication requires the plasmid‐specified Rep protein and DNA polymerase I but not RNA polymerase and DnaG primase. The ColE2 Rep protein binds specifically to the origin where replication initiates. Leading‐strand synthesis initiates at a unique site in the origin and lagging‐strand DNA synthesis terminates at another unique site in the origin. Here we show that the primer RNA for leading‐strand synthesis at the origin has a unique structure of 5′‐ppApGpA. We reconstituted the initiation reaction of leading‐strand DNA synthesis by using purified proteins, the ColE2 Rep protein, Escherichia coli DNA polymerase I and SSB, and we showed that the ColE2 Rep protein is a priming enzyme, primase, which is specific for the ColE2 origin. The ColE2 Rep protein is unique among other primases in that it recognizes the origin region and synthesizes the primer RNA at a fixed site in the origin region. Specific requirement for ADP as a substrate and its direct incorporation into the 5′ end of the primer RNA are also unique properties of the ColE2 Rep protein.

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“…Characterization of a purified P4 α protein revealed that the 88-kDa protein contains RNA polymerase activity (38,39). To synthesize primers, P4 α protein requires a DNA template, NTPs, and Mg 2+ and K + ions for optimal activity (40)(41)(42). On ssDNA, the P4 α protein synthesizes two-to five-nucleotide-long primers that begin with pppAG (41).…”

Section: Bacteriophage P4 Primasementioning

confidence: 99%

DNA Primases

Frick

Richardson

2001

Annu. Rev. Biochem.

343

425

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DNA primases are enzymes whose continual activity is required at the DNA replication fork. They catalyze the synthesis of short RNA molecules used as primers for DNA polymerases. Primers are synthesized from ribonucleoside triphosphates and are four to fifteen nucleotides long. Most DNA primases can be divided into two classes. The first class contains bacterial and bacteriophage enzymes found associated with replicative DNA helicases. These prokaryotic primases contain three distinct domains: an amino terminal domain with a zinc ribbon motif involved in binding template DNA, a middle RNA polymerase domain, and a carboxyl-terminal region that either is itself a DNA helicase or interacts with a DNA helicase. The second major primase class comprises heterodimeric eukaryotic primases that form a complex with DNA polymerase alpha and its accessory B subunit. The small eukaryotic primase subunit contains the active site for RNA synthesis, and its activity correlates with DNA replication during the cell cycle.

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The replication of bacteriophage P4 DNA in vitro

Cited by 24 publications

References 24 publications

Phage P4 DNA replicationin vitro

Phage P4 DNA replicationin vitro

Primer RNA synthesis by plasmid-specified Rep protein for initiation of ColE2 DNA replication.

DNA Primases

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