Observations of a Genome Carrying an Unreplicatable Region

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To block the progression of the DNA replication fork in E. coli cells, at least two factors are required ; one is the specific terminus (ter) sequence (-22bp) and the other is ter-binding protein. (1) To investigate behavior of an E. coli genome carrying the region unreplicated after DNA synthesis period and effects this genome have on host cells, we did the following experiments ; an E. coli strain, the genome of which carried a region flanked by two ter sequences, was constructed from a strain deficient in ter-binding protein. We expected that in the presence of ter-binding protein this strain would be lethal or would show poor growth due to the presence of an unreplicatable region on the genome. As expected, when the tau gene which codes for ter-binding protein was introduced into the E. coli strain, growth rate of the strain was greatly reduced, in comparison with that of the control strain. This suggested that blockage against' the DNA replication fork at the ter site is leaky. A greater inhibition for the replication fork at the ter site is needed to assess the fate of the genome with an unreplicatable region. (2) To determine whether or not E. coli ter system is functional on eucaryotic DNA replication, we investigated the DNA synthesis of SV40 DNA carrying the ter sequence, in both crude and purified enzyme in vitro, in the presence of ter-binding protein. As well as in the E. coli in vitro DNA replication system, blockage of the DNA replication fork at the ter site was evident under both conditions. The ter sequence-ter binding protein complex could impede the helicase action of SV40 large T antigen, in a polar fashion. A similar activity was observed previously when we used 3 types of E. coli helicases.