PUB - Publikationen an der Universität Bielefeld

Some aspects of the involvment of the terminal reduntant regions of T7 DNA on phage production have been studied by transfection experiments with T7 DNA after treatment of the molecules with [lambda] exonuclease or [lambda] exonuclease plus exonuclease I. It was found that terminal 5prime gaps between 0.08 and 6.4% of the total length did not decrease the infectivity of the molecules although such gaps cannot be filled directly by DNA polymerases. Rather, compared to fully native DNA the infectivity of gapped DNA increased up to 20 fold in rec + spheroplasts and up to 4 fold in recB spheroplasts. This indicates a protective function of the single-stranded termini against the recBC enzyme in rec + and possibly another unidentified exonuclease present also in recB. The possibility that spontaneous circularization of the gapped molecules in vivo provides protection against exonucleolytic degradation was tested by transfection with T7 DNA circularization in vitro by thermal annealing. Such molecules were separated from linear molecules by neutral sucrose gradient centrifugation. They displayed a 3 to 6 fold higher infectivity in rec + and recB compared to linear gapped molecules, which shows that T7 phage production may effectively start from circular DNA. When the 3prime single-stranded ends from gapped molecules were degraded by treatment with exonuclease I the infectivity of the molecules was largely abolished in rec + and recB as soon as 40 to 80 base pairs had been removed per end. It is concluded that the terminal regions of T7 DNA molecules are essential for phage production and that the redundancy comprises probably considerably less than 260 base pairs. The results are discussed with respect to the mode of T7 DNA replication.