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Resolution of phage N15 telomeres by its protelomerase enzyme in mammalian cells
thesisposted on 27.02.2017, 23:59 by Chen, Qingwen
N15 is a unique coliphage that replicates as a linear plasmid with covalently-closed hairpin telomeres during its prophage stage. Linearity of the N15 plasmid is conferred solely by two elements, which are the phage-encoded telomerase enzyme TelN and its recognition sequence telomerase occupancy site (tos). Due to the functional independence of TelN and tos from other host or phage accessory proteins, they have been used to linearise a wide range of non-N15 DNA both in vitro and in vivo of Escherichia coli. Furthermore, linear vectors generated by TelN and tos can shuttle mammalian transgenes into eukaryotic cells to facilitate their correct expression in vivo. Based on these findings, it is hypothesised that the TelN-tos system of phage N15 will be functional when expressed in eukaryotic hosts. Hence this study aimed to test this hypothesis by evaluating the properties of the TelN-tos system, and in particular examine the linearisation of DNA containing a tos site by TelN in mammalian cells. By transiently delivering circular tos-containing DNA into TelN-expressing HeLa cells, resolution of the substrate DNA into its linear form was observed in vivo. When the same tos-DNA was incubated with total proteins extracted from TelN-expressing HeLa cells in vitro, linearisation of the tos-DNA was also detected. Hence the hypothesis that TelN-tos will be functional when expressed in mammalian cells is supported, as indicated by TelN’s cleavage activity on tos site in mammalian environment. These results demonstrate the functionality of TelN-tos system in eukaryotic cells for the first time, and can potentially drive the development of a novel DNA linearisation tool for mammalian research, with applications ranging from generation of linear vector systems to artificially inducing chromosomal breakpoints.