In Vitro Studies on Escherichia coli DNA Replication Factors and on the Initiation of Phage λ DNA Replication

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

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The Escherichia coli Fis protein prevents initiation of DNA replication from oriC in vitro

Nucleic Acids Research ◽

10.1093/nar/24.18.3527 ◽

1996 ◽

Vol 24 (18) ◽

pp. 3527-3532 ◽

Cited By ~ 37

Author(s):

S Wold

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Initiation Of Dna Replication

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IN VITRO STUDIES ON THE INTERACTION BETWEEN MOUSE PERITONEAL MACROPHAGES AND STRAINS OF SALMONELLA AND ESCHERICHIA COLI

Journal of Experimental Medicine ◽

10.1084/jem.112.2.403 ◽

1960 ◽

Vol 112 (2) ◽

pp. 403-417 ◽

Cited By ~ 43

Author(s):

Charles Jenkin ◽

Baruj Benacerraf

Keyword(s):

Escherichia Coli ◽

Normal Mouse ◽

Peritoneal Macrophages ◽

In Vitro Studies ◽

Experimental Conditions ◽

Mouse Plasma ◽

Virulent Strains ◽

Mouse Macrophages ◽

Mouse Peritoneal Macrophages

Virulent strains of Salmonella opsonized with normal mouse plasma are never phagocytosed as well as avirulent strains. The virulent strains of Salmonella phagocytosed after opsonization with normal mouse plasma are able to multiply within normal mouse peritoneal macrophages, whereas under similar experimental conditions the avirulent strains are killed. When virulent strains of Salmonella are opsonized with specific antiserum or plasma from BCG-infected mice, they are treated by normal mouse macrophages as if they were avirulent. Virulent bacteria opsonized with BCG plasma are phagocytosed and killed better by peritoneal macrophages from BCG-infected mice, than peritoneal macrophages from normal mice.

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In vitro studies on the role of phage T7 gene 4 product in DNA replication

MGG Molecular & General Genetics ◽

10.1007/bf00433903 ◽

1974 ◽

Vol 135 (1) ◽

pp. 73-86 ◽

Cited By ~ 9

Author(s):

Eberhard Scherzinger ◽

Frank Litfin

Keyword(s):

Dna Replication ◽

In Vitro Studies ◽

Phage T7 ◽

Gene 4

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The Escherichia coli chaperones involved in DNA replicadon

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.1993.0025 ◽

1993 ◽

Vol 339 (1289) ◽

pp. 271-278 ◽

Cited By ~ 43

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Heat Shock ◽

Heat Shock Proteins ◽

Conformational Changes ◽

Heat Shock Genes ◽

Dnab Helicase ◽

Initiation Of Dna Replication ◽

Shock Proteins

Mutadons in the Escherichia coli heat shock genes, dnaK , dnaJ or grpE , alter host DNA and RNA synthesis, degradation of other proteins, cell division and expression of other heat shock genes. They also block the initiation of DNA replication of bacteriophages λ and P1, and the mini-F plasmid. An in vitro λDNA replication system, composed entirely of purified components, enabled us to describe the molecular mechanism of the dnaK , dnaJ and grpE gene products. DnaK , the bacterial hsp 70 homologue, releases λP protein from the preprimosomal complex in an ATP- and DnaJ-dependent reaction (GrpEindependent initiation of λDNA replication). In this paper, I show that, when GrpE is present, λP protein is not released from the preprimosomal complex, rather it is translocated within the complex in such a way that it does not inhibit DnaB helicase activity. Translocation of λP triggers the initiation event allowing DnaB helicase to unwind DNA near the ori λ sequence, leading to efficient λDNA replication. Chaperone activity of the DnaK -DnaJ-GrpE system is first manifested in the selective binding of these heat shock proteins to the preprimosomal complex, followed by its ATP-dependent rearrangement. I show that DnaJ not only tags the preprimosomal complex for recognition by DnaK, but also stabilizes the multi-protein structure. GrpE also participates in the binding of DnaK to the preprimosomal complex by increasing DnaK ’s affinity to those λP proteins which are already associated with DnaJ. After attracting DnaK to the preprimosomal complex, DnaJ and GrpE stimulate the ATPase activity of DnaK , triggering conformational changes in DnaK which are responsible for the rearrangement of proteins in the preprimosomal complex and recycling of these heat shock proteins. The role of DnaK , DnaJ and GrpE in λDNA replication is in sharp contrast to our understanding of their role in the oriC , P1, and probably mini-F DNA replication systems. In the cases of oriC and P1 DNA replication, these heat shock proteins activate initiation factors before they are in contact with DNA, and are not required during the subsequent steps leading to the initiation of DNA replication. The common feature of DnaK , DnaJ and GrpE action in these systems is their ATP-dependent disaggregation or rearrangement of protein complexes formed before or during initiation of DNA replication.

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RB Reversibly Inhibits DNA Replication via Two Temporally Distinct Mechanisms

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5404-5420.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5404-5420 ◽

Cited By ~ 25

Author(s):

Steven P. Angus ◽

Christopher N. Mayhew ◽

David A. Solomon ◽

Wesley A. Braden ◽

Michael P. Markey ◽

...

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Dna Synthesis ◽

Cellular Proliferation ◽

Negative Regulator ◽

Proliferating Cells ◽

Factor C ◽

Replication Factors ◽

Multiple Replication

ABSTRACT The retinoblastoma (RB) tumor suppressor is a critical negative regulator of cellular proliferation. Repression of E2F-dependent transcription has been implicated as the mechanism through which RB inhibits cell cycle progression. However, recent data have suggested that the direct interaction of RB with replication factors or sites of DNA synthesis may contribute to its ability to inhibit S phase. Here we show that RB does not exert a cis-acting effect on DNA replication. Furthermore, the localization of RB was distinct from replication foci in proliferating cells. While RB activation strongly attenuated the RNA levels of multiple replication factors, their protein expression was not diminished coincident with cell cycle arrest. During the first 24 h of RB activation, components of the prereplication complex, initiation factors, and the clamp loader complex (replication factor C) remained tethered to chromatin. In contrast, the association of PCNA and downstream components of the processive replication machinery was specifically disrupted. This signaling from RB occurred in a manner dependent on E2F-mediated transcriptional repression. Following long-term activation of RB, we observed the attenuation of multiple replication factors, the complete cessation of DNA synthesis, and impaired replicative capacity in vitro. Therefore, functional distinctions exist between the “chronic” RB-mediated arrest state and the “acute” arrest state. Strikingly, attenuation of RB activity reversed both acute and chronic replication blocks. Thus, continued RB action is required for the maintenance of two kinetically and functionally distinct modes of replication inhibition.

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