G4 DNA replication

Martin, Deijs; Godson, G. N.

doi:10.1016/0022-2836(77)90130-9

Cited by 18 publications

(7 citation statements)

References 22 publications

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“…This system is specific for OX DNA and does not operate on the SSs of phages G4 and M13 (26,27). Unlike phages G4 and M1S, which possess unique origins for complementary strand starts, both in vivo and in vitro studies indicate multiple starts on OX DNA (7,9,11,14). However, the strict specificity of the prepriming system for OX DNA suggests that a particular locus is recognized by one or more of the proteins of the prepriming system.…”

Section: Resultsmentioning

confidence: 99%

An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA.

Shlomai¹,

Kornberg²

1980

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

169

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Protein n', a prepriming DNA replication enzyme of Escherichia coli, is a 46X174 DNA-dependent ATPase DNA, by primase (4, 5). However, SSB-coated OX DNA, although primed by the same E. coli primase, must first be activated in a prepriming stage. In this prepriming reaction, the E. coli proteins n', n and n", i, dnaB, and dnaC form an activated complex with OX DNA (2, 6-8).The distinctions in primer synthesis on these three phage templates are probably due to structural differences in "promoter"-like sites recognized by the distinctive priming systems. Although the origin of complementary DNA strand replication is unique and well characterized for M13 (9) and G4 (10-14), it does not appear to be at a unique site for OX, as judged by in vivo and in vitro studies (7, 9, 11, 15).We will describe elsewhere the purification of protein n' to near homogeneity, its 4X DNA-dependent ATPase activity, and its capacity to destabilize an SSB-qX DNA complex. In this paper we report the recognition by protein n' of a specific sequence in qX DNA located at an intergenic region and suggest that it may be the signal that leads to the initiation of OX complementary DNA strand replication. MATERIALS AND METHODSNucleic Acids, Enzymes, Resins, and Nucleotides. OX, OX replicative form (RF)I, G4, and M13 DNAs were prepared as described (16) [a-32P]ATP and could also be applied to the production of 32p; from ['y-32P]ATP. Standard assays were carried out in 25-1ul reaction mixtures containing 10 mM KC1, 1 mM MgCl2, 1 mM labeled ATP or dATP, 120 pmol of XX DNA (as nucleotide), 50mM Tris-HCl (pH 7.5), 6% (wt/vol) sucrose, and bovine serum albumin at 0.2 mg/mi. Samples to be assayed were diluted in 50 mM imidazole.HCI, pH 6.8/25% glycerol/100 mM ammonium sulfate/bovine serum albumin at 0.2 mg per mI/i mM EDTA.When SSB was used, 1 Atg was added, unless otherwise noted.Reactions were carried out at 30'C unless otherwise noted.Aliquots of 2 Ml were applied to polyethyleneimine-cellulose strips (0.6 X 6 cm) together with unlabeled ATP, ADP, and AMP markers. The strips were developed with 1 M formic acid/0.5 M LiCI at room temperature, dried, and examined with UV light to locate and cut out the ATP and ADP spots. Radioactivity was determined in scintillation fluid without Abbreviations: OX, kX174; SS, single-stranded circular DNA; RF, double-stranded DNA of circular replicative form; SSB, singlestranded-DNA binding protein; DBM, diazobenzyloxymethyl.

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Section: Resultsmentioning

confidence: 99%

An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA.

Shlomai¹,

Kornberg²

1980

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

169

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“…In experiments with c$X using the isolation procedure described by Godson in his studies about the DNA replication of bacteriophage G4 (Godson, 1977a,b;Martin & Godson, 1977), we obtained a very low yield of rolling circles during +X RF DNA replication.…”

Section: Introductionmentioning

confidence: 81%

Bacteriophage φX174 and G4 RF DNA replicative intermediates

Baas

Teertstra

Ende

et al. 1980

Journal of Molecular Biology

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“…Nucleotide (2,32,38). The displaced strand is not used as a template until parental DNA unwinding has proceeded a significant way around the genome: in mammalian mitochondria, 2/3 of the genome (34,39,40); in phage G4, 1/2 of the genome (41); in Ff phages, completely around the genome (42). Thus, in both mitochondria and specific stages of viral strand synthesis of single-stranded phage, replication appears to occur primarily by continuous leading-strand DNA synthesis and therefore may require functionally similar DNA helicases.…”

Section: Resultsmentioning

confidence: 99%

DNA helicase from mammalian mitochondria.

Hehman

Hauswirth

1992

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

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In spite of the fact that a DNA helicase is clearly required for the predominately leading-strand synthesis occurring during mammalian mtDNA replication, no such activity has heretofore been Identified. We report the characterization ofa mammalian mitochondrial DNA helicase isolated from bovine brain tissue. The sucrose gradient-purified mitochondria in which the activity was detected had less than 1 part in 2500 nuclear contamination according to Western blot analysis using nuclear-and mitochondrial-specific probes.Mitochondrial protein fractionation by DEAE-Sephacel chromatography yielded a DNA helicase activity dependent upon hydrolysis of ATP or dATP but not other NTPs or dNTPs. The mitochondrial helicase unwound 15-and 20-base oligonucleotides but was unable to unwind 32-base or longer oligonucleotides, and the polarity of the unwinding is 3'-to-5' with respect to the single-stranded portion of the partial duplex DNA substrate. This direction of unwinding would place the bovine mitochondrial helicase on the template strand ahead of DNA polymerase yduring mtDNA replication, a situation analogous to that of the Rep helicase of Escherichia coli during leadingstrand DNA synthesis of certain bacteriophages.Mammalian mitochondria contain multiple copies of a 16.5-kilobase (kb) double-stranded DNA genome (mtDNA) (reviewed in refs. 1 and 2). Recent observations suggest that mtDNA copy number may be primarily responsible for regulation of mitochondrial gene expression in mammalian tissue (3,4). The proteins used for mitochondrial DNA replication, clearly important in controlling mtDNA copy number, are encoded by the nucleus and imported into the organelle. To date, much of the biochemistry of mtDNA metabolism has not been determined. Therefore, to understand that process, we are assembling and investigating components of the bovine mtDNA replication system. Utilization ofduplex DNA for DNA replication requires its unwinding to provide a single-stranded template for DNA polymerase (reviewed in refs. 5 and 6). Enzymes that catalyze this reaction, DNA helicases, act in an NTP/dNTPdependent manner to separate the two strands of a DNA helix. DNA helicases have been isolated from a number of organisms, including Escherichia coli, its bacteriophages, eukaryotic viruses, yeast, lily, Xenopus, mouse, cow, and human (5-11). The only presently identified DNA helicase found in mitochondria is involved in DNA repair and recombination in yeast (9, 12). To date no mitochondrial DNA helicase from mammals or any higher eukaryote has been identified.In this paper, we identify and partially purify a mitochondrial DNA helicase activity from purified bovine mitochondria. This activity unwinds partially duplex DNA in an ATPor dATP-dependent manner and shows a 3'-to-5' polarity of movement. Based on these properties, its potential role in the unidirectional synthesis of mtDNA is addressed. MATERIALS AND METHODSIsolation of Bovine Brain Mitochondria and Nuclei. All procedures were carried out at 40C or on ice. Mitochondria were iso...

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G4 DNA replication

Cited by 18 publications

References 22 publications

An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA.

An Escherichia coli replication protein that recognizes a unique sequence within a hairpin region in phi X174 DNA.

Bacteriophage φX174 and G4 RF DNA replicative intermediates

DNA helicase from mammalian mitochondria.

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