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Biomedical subjects

B E Funnell

Publications and source records attributed to B E Funnell.

25 records · Page 2Linked to original sources
PubMed1987

Interaction between the DNA polymerase and single-stranded DNA-binding protein (infected cell protein 8) of herpes simplex virus 1.

The herpes virus-encoded DNA replication protein, infected cell protein 8 (ICP8), binds specifically to single-stranded DNA with a stoichiometry of one ICP8 molecule/12 nucleotides. In the absence of single-stranded DNA, it assembles into long filamentous structures. Binding of ICP8 inhibits DNA synthesis by the herpes-induced DNA polymerase on singly primed single-stranded DNA circles. In contrast, ICP8 greatly stimulates replication of circular duplex DNA by the polymerase. Stimulation occurs only in the presence of a nuclear extract from herpes-infected cells. Appearance of the stimulatory activity in nuclear extracts coincides closely with the time of appearance of herpes-induced DNA replication proteins including ICP8 and DNA polymerase. A viral factor(s) may therefore be required to mediate ICP8 function in DNA replication.

Bacteriophages↗
PubMed1986

Complete enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome.

During enzymatic replication of plasmids containing the origin of the Escherichia coli chromosome, oriC, formation of an active initiation complex consisting of dnaA, dnaB, dnaC, and HU proteins, requires a supercoiled DNA template. Relaxed covalently closed plasmids are active only if supercoiled by gyrase prior to initiation; nicked and linear DNAs are inactive. Semi-conservative replication proceeds via delta structure as intermediates. Daughter molecules include nicked intermediates. Daughter molecules include nicked monomers and catenated pairs. Elongation is rapid, but late replicative intermediates accumulate because the final elongation and termination steps are slow. Production of covalently closed circular daughter DNA molecules requires removal of ribonucleotide residues (primers) by DNA polymerase I, assisted by ribonuclease H, gap filling, and ligation of nascent strands by ligase. Reconstitution of a complete cycle of oriC plasmid replication, beginning and ending with supercoiled molecules, has been achieved with purified proteins.

Centrifugation, Density Gradient↗
PubMed1986

Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome.

Early in the staged initiation of enzymatic replication of plasmids containing the unique origin of the E. coli chromosome (oriC), the plasmid is converted to a new topological form which is highly underwound, two to 15 times more than native supercoiled DNA. The underwinding reaction precedes priming of DNA synthesis and follows an initial complex formation, requiring ATP and proteins dnaA, dnaB, and dnaC; underwinding depends on the further addition of gyrase and SSB. DnaB protein as a helicase and gyrase as a topoisomerase drive the underwinding with the energy of ATP hydrolysis. The underwound template, extensively single-stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme.

Bacterial Proteins↗
PubMed1984

The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites.

Specific binding of dnaA protein to the E. coli origin of replication (oriC) is essential for initiation of chromosomal replication. Based on binding of plasmid DNA fragments, dnaA protein recognizes in addition to oriC a number of specific sites: within or near the replication origins of pSC101, pBR322, and ColE1; within the regulatory regions of the dnaA and "X-protein" genes; and in IRL-Tn5. All strong binding sites share a common 9 bp sequence, 5'-TTATCACACAA, repeated at four conserved positions within oriC. As shown by DNAase I footprinting, dnaA protein binding is highly cooperative, covering 250 bp at oriC and 100 bp at single 9 bp sequence sites. Consistent with filter-binding and nuclease-protection studies, complexes of 20 to 30 dnaA monomers are visualized at oriC and other sites by electron microscopy. The functions of dnaA complexes and 9 bp sequences at these sites are discussed.

Bacterial Proteins↗
PubMed1983

Bacteriophage P2 DNA replication. Characterization of the requirement of the gene B protein in vivo.

Replicative intermediates isolated from Escherichia coli cells infected with P2 gene B mutants were circular DNA molecules with single-stranded DNA tails, as opposed to the double-stranded DNA tails of wild-type replicative intermediates. The results show that the mutant replicative intermediates arose from aberrant DNA replication, aberrant due to a lack of lagging strand DNA synthesis, but with normal leading strand synthesis, so that only one circular duplex daughter DNA molecule was made from each duplex parent molecule. The single-stranded tails were shown to correspond to the nicked (and therefore displaced) parental DNA "l" strands. By partial denaturation mapping, the ends of the single-stranded tails tended to map close to the replication origin, but not all at a unique position, probably due to partial degradation or breakage in vivo, or during cell lysis or DNA isolation. By hybridization to separated strands of P2 DNA on nitrocellulose filters, DNA synthesis was shown to be asymmetric, and consistent with more leading strand than lagging strand synthesis having occurred. We concluded that the gene B protein is required for lagging strand DNA synthesis, but not for initiation, elongation or termination of the leading strand.

Centrifugation, Density Gradient↗