The migration behaviour of DNA replicative intermediates containing an internal bubble analyzed by two-dimensional agarose gel electrophoresis.

Initiation of DNA replication in higher eukaryotes is still a matter of controversy. Some evidence suggests it occurs at specific sites. Data obtained using two-dimensional (2D) agarose gel electrophoresis, however, led to the notion that it may occur at random in broad zones. This hypothesis is primarily based on the observation that several contiguous DNA fragments generate a mixture of the so-called 'bubble' and 'simple Y' patterns in Neutral/neutral 2D gels. The interpretation that this mixture of hybridisation patterns is indicative for random initiation of DNA synthesis relies on the assumption that replicative intermediates (RIs) containing an internal bubble where initiation occurred at different relative positions, generate comigrating signals. The latter, however, is still to be proven. We investigated this problem by analysing together, in the same 2D gel, populations of pBR322 RIs that were digested with different restriction endonucleases that cut the monomer only once at different locations. DNA synthesis begins at a specific site in pBR322 and progresses in a uni-directional manner. Thus, the main difference between these sets of RIs was the relative position of the origin. The results obtained clearly showed that populations of RIs containing an internal bubble where initiation occurred at different relative positions do not generate signals that co-migrate all-the-way in 2D gels. Despite this observation, however, our results support the notion that random initiation is indeed responsible for the peculiar 'bubble' signal observed in the case of several metazoan eukaryotes.