Abstract
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.
Highlights
During enzymatic replicatioonf plasmids containing termined which enzymes are required to form supercoiled the origin of the Escherichia coli chromosome, oriC, daughter molecules
Plasmid replication can be separatedinto stages of: (i) formation of a prepriming complex, (ii) priming, (iii) elonga
No detectable supercoiled species were measured inthe absence of allthree enzymes;,low, but measurable levels were seen with ligase only or RNase H plus ligase (Table IV). This may be due to pol I contaminating some of the replication enzyme preparations, or to DNA polymerase I11 repair synthesis of gapped form I1 molecules during the pulse. pol I and ligase were required for significant levels of termination, and were stimulated by RNase H
Summary
During enzymatic replicatioonf plasmids containing termined which enzymes are required to form supercoiled the origin of the Escherichia coli chromosome, oriC, daughter molecules. Preparation of template is described under "Experimental Procedures." "Nicked ligase" denotes Nicked pCM959 (1 pg) incubated with 0.5 pg of T4 DNA ligase in 66 mM Tris-HC1 (pH 7.6), 5 mM MgC12,5 mM dithiothreitol, 1mM ATP (volume = 5 pl) for 4 hat 4 "C and heat-treated (55 "C for 15 min) to inactivate ligase, prior to theaddition of replication proteins; agarose gel electrophoresis confirmed that the DNA was converted to the covalentlv closed form.
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Published Version
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