Slaloms on the double helix. Workshop on Regulatory Mechanisms of DNA Replication, sponsored by the Human Frontier Science Program, Les Arcs, France, March 17-23, 1991.
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Animals↗
Biomedical subjects
M Kohiyama
Publications and source records attributed to M Kohiyama.
The E. coli cell surface specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates.
A particular outer membrane fraction previously defined as possessing specific affinity for the hemimethylated form of the origin of replication of the E. coli chromosome (oriC) is shown to inhibit the initiation of DNA synthesis at this site on hemimethylated DNA templates in vitro. The replication of fully methylated or unmethylated DNA templates is not affected. Also, no inhibition is observed if initiation takes place at random sites on the hemimethylated template. The key inactivation step appears to be membrane inhibition of DnaA initiator protein binding to oriC. Remethylation of the membrane-bound hemimethylated DNA results in reactivation. Our results demonstrate direct involvement of the membrane in the control of DNA replication. We propose that association/dissociation of the origin from the cell membrane is one of the control elements governing interinitiation times in E. coli.
Bacterial Proteins↗
Circulating antibodies against c-myc oncogene product in sera of colorectal cancer patients.
We developed a Western blot assay using purified human c-myc protein expressed in E. coli in order to look for circulating anti-c-myc antibodies in human sera. The presence of IgG antibodies against c-myc was observed in 25 out of 44 sera from patients with colorectal cancer diagnosed and treated at the Hôpital Curie in Paris, compared to the sera of 46 normal donors of which only 8 samples were positive (p = 0.001).
Antibodies, Monoclonal↗
dam methylation and the initiation of DNA replication on oriC plasmids.
Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. Here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oriC region.
Bacterial Proteins↗
Dam methylated and hemimethylated oriC plasmids are replicated symmetrically; a novel and general test of replication symmetry.
Deoxyadenosine methylation (dam) of the numerous GATC sequences present in the Escherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleases MboI, MboII and DpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylated oriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part of MboII restriction sites.
Base Sequence↗
Purification and characterization of a low-molecular-weight membrane protein with affinity for the Escherichia coli origin of replication.
A purification procedure was devised for a low-molecular-mass (about 10-kilodalton) membrane protein from Escherichia coli that was shown to bind specifically to the chromosomal replication origin region (oriC). Nitrocellulose membrane retention assays showed the binding site to be adjacent to the right boundary of the oriC minimal sequence. We determined the amino acid sequence of the N-terminal and C-terminal regions as well as the global amino acid composition of this membrane protein. Specific antibodies against the protein were produced and used to confirm the cell membrane location of the protein. These results demonstrate that this is a new membrane protein, different from the previously described B' protein, with specific binding activity for the oriC region. We propose that this protein be called membrane oriC-binding protein 2 (MOB2 protein).
Amino Acid Sequence↗
A novel role for cAMP in the control of the activity of the E. coli chromosome replication initiator protein, DnaA.
DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.
Amino Acid Sequence↗
DNA primase activity found in an alpha-like DNA polymerase obtained from Halobacterium halobium.
An aphidicolin-sensitive DNA polymerase was purified from extracts of Halobacterium halobium. The analysis of this alpha-like DNA polymerase on polyacrylamide gels under denaturing conditions revealed two peptides with molecular masses of 70 kDa and 60 kDa in equal amounts. Like the DNA polymerase alpha isolated from eukaryotes, the alpha-like DNA polymerase possesses primase activity using UTP and polydeoxyadenylate as template. The primase activity was sensitive to aphidicolin and inhibited by an antiserum against the alpha-like DNA polymerase of H. halobium. The primase activity was dependent on the presence of high salt concentrations.
Antibodies↗
A protein of Halobacterium halobium immunologically related to the v-myc gene product.
A 70 kDa protein of Halobacterium halobium cross-reacts with an antiserum directed against the v-myc gene product of the avian myelocytomatosis virus (MC29). This cross-reaction is in agreement with hybridization studies which indicate that H. halobium possesses DNA and RNA sequences homologous to the v-myc gene.
Bacterial Proteins↗
Stimulation of an alpha like DNA polymerase by v-myc related protein of Halobacterium halobium.
Partial DNA sequencing of a genomic clone of the archaebacterium Halobacterium halobium, which hybridized with an avian v-myc probe, showed especially the presence, in the organism of one of the conserved regions through myb, myc and adenovirus E1a oncogenes. The archaebacterial deduced amino acid sequence displayed significant homology with the v-myc gene product. In accordance with the partial DNA sequencing which assured a sufficient homology to have similar epitopes, a protein having a molecular weight of 70,000 and possessing high antigenicity with a polyclonal antiserum against avian v-myc protein was isolated and purified from H. halobium extracts. The purified v-myc like protein stimulated in vitro DNA synthesis carried out by the alpha like DNA polymerase of H. halobium.
Amino Acid Sequence↗
Detection of circulating antibodies against c-myc protein in cancer patient sera.
We have partially purified an archaebacterial protein of 84 kD which shares common epitopes with the human c-myc protein as shown by its cross-reactivity with a commercialized anti-human c-myc antiserum. An antiserum raised against the 84 kD protein recognizes a 60 kD protein from HL-60 nuclei. This protein is also recognized by the anti-human c-myc antiserum. Using this archaebacterial protein as antigen for Western blot analysis, we found that the human c-myc oncogene product could be immunogenic and that it is possible, in some spontaneously occurring human tumours, to detect antibodies against the c-myc gene product in the serum of cancer patients.
Antibodies, Neoplasm↗
An alpha-like DNA polymerase from Halobacterium halobium.
Two DNA polymerases have been isolated from extracts of Halobacterium halobium, one having a sedimentation coefficient of 11 S, designated as alpha-like polymerase and possessing the following characteristics. It is sensitive to both aphidicolin and N-ethylmaleimide but indifferent to the presence of a dideoxynucleoside triphosphate. Therefore this polymerase is very similar to the alpha DNA polymerase of eukaryotes. The enzyme requires 5 M NaCl for maximum activity. The other polymerase has a sedimentation coefficient of 4.4 S and is resistant to both aphidicolin and N-ethylmaleimide. However, it is inhibited by a dideoxynucleoside triphosphate.
Aphidicolin↗
[Existence of sequences homologous to the V-MYB oncogene in the genome of archaebacteria].
The presence of DNA sequences homologous to the v-myb oncogene in the genome of both halophilic and methanogenic archaebacteria was revealed after hybridization of restriction fragments with cloned probes. No myb-related sequences were detected in the DNA from S. acidocaldarius.
Archaea↗
In vitro replication of a dam methylated and non-methylated ori-C plasmid.
We have examined the replication of a dam methylated and non-methylated ori-C plasmid in an in vitro ori-C dependent replication system. The results show that the non-methylated plasmid is 50% to 80% less efficient in the initiation of DNA synthesis; that the methylation state of the plasmid does not change the site of initiation at ori-C, and that in both cases initiation at this region requires the presence of exogenously furnished dnaA protein.
Bacterial Proteins↗
Studies on the role of dam methylation at the Escherichia coli chromosome replication origin (oriC).
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Base Sequence↗
Aphidicolin inhibits growth and DNA synthesis in halophilic arachaebacteria.
Aphidicolin, a specific inhibitor of eucaryotic alpha DNA polymerase, inhibits the growth of halophilic arachaebacteria. In Halobacterium halobium, aphidicolin prevents cell division and DNA synthesis. These results suggest that arachaebacterial replicases are of the eucaryotic type.
Antiviral Agents↗
Purification and characterisation of a 12 K dalton peptide with an affinity for oriC containing DNA fragments from Escherichia coli K12 membrane fractions.
A DNA binding protein capable of interacting specifically with oriC containing DNA was purified to near homogeneity from E. coli membrane fractions. It has a molecular weight of 12 KD in denaturing conditions. The specific binding to oriC DNA is more resistant to the presence of salt than the non specific binding. Neither the DNA binding activity nor the specificity for oriC DNA was destroyed by heating at 100 degrees C for 2 min.
Cell Membrane↗
Gamma-ray induction of deoxyribonucleic acid synthesis in temperature-sensitive DNA initiation mutants of Escherichia coli.
DNA synthesis was followed after gamma-ray irradiation of several different temperature-sensitive mutants with defects in the initiation process. The results indicate that only dnaA and dnaI mutants show induction of supplementary DNA synthesis after gamma-ray irradiation. The induction of DNA synthesis by gamma-ray irradiation was also shown to be recA+ dependent in the dna5 mutant.
DNA, Bacterial↗