Volume 3

Gene expression / Benjamin Lewin.

  • Lewin, Benjamin.
Date:
1974-1977
Catalogue details

Licence: Attribution-NonCommercial 4.0 International (CC BY-NC 4.0)

Credit: Gene expression / Benjamin Lewin. Source: Wellcome Collection.

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    closed circular molecules of Col El DNA initiate and complete rounds of semiconservative replication in vitro; when examined just after replica tion has begun, linear molecules have a small loop of replicated DNA about 20% of the distance away from one end; this identifies the origin. Extensive replication takes place only in one direction, through the 80% of DNA on one side of the loop, since the left end of the loop remains fairly fixed in position while the right end (as usually aligned) moves. This suggests that replication is unidirectional, with a single replicating fork moving away from the origin. With replicating molecules extracted from minicells, Inselburg (1974a) also found that the position of one end of the loop was fairly constant while the other moved. If replication is unidirectional, the terminus should be adjacent to the origin. If the left end of the loop moves at all, an idea that is rendered unlikely but is not excluded by the results, the terminus should be a corresponding distance away from the origin, that is, should be closer to the left end (as usually aligned) of the molecule. Using the in vitro system, Sakakibara and Tomizawa (1974a) substituted NMN for the essential cofactor NAD; this inhibits the polynucleotide ligase of E.coli so that the open circles formed by replication cannot be closed. On subsequent incubation with appropriate components, the gap can be closed to gener ate a closed circle. Cleaving the open circular molecules with Eco RI nuclease showed that the gap lies 20% distant from the left end of the molecule; this is very close to the initiation site, measured in these experiments as 17% distant from the left end. Thus termination takes place at or very close to the origin; replication is therefore most probably unidirectional. The covalently closed circle of the Col El plasmid can be isolated from E.coli in the form of a DNA-protein complex. The purified closed and open circles of Col El DNA sediment at 23S and 17S respectively on sucrose gradients. Clewell and Helinski (1969) found that by using condi tions of gentle lysis, they could obtain DNA-protein complexes sediment- ing slightly faster, at 24S and 18S. Figure 3.18 compares the complexed and free forms of Col El DNA. The 24S form tends to decay to the 18S form; and agents that act on proteins, such as trypsin, pronase, SDS, convert the 24S complex to the 18S or 17S condition, although such treatment has no effect on free 23S supercoiled Col El DNA. The shift in buoyant density of the 24S complex from that of the free DNA suggests that about 240,000 daltons of protein are associated with the DNA (Clewell and Helinski, 1970c). The 24S DNA-protein complex contains covalently closed circles of DNA and the 18S DNA-protein complex contains open circles. Because the transition from 24S to 18S that is induced by attack on the protein