There is a lot of interest in the study of protelomerase enzymes, due to their unique functionality and potential value in the biotechnology industry. Linear DNA is vulnerable to exonuclease degradation and suffers from genetic loss due to the end
replication problem. Eukaryotes commonly have open-ended DNA and encode telomerases: enzymes responsible for protecting the chromosome termini from shortening during replication. Most
prokaryotes harbour their genetic material in circular plasmids and chromosomes. However, some bacteria and viruses do have linear DNA molecules. These linear genomes are created via recruitment
of protelomerase enzymes. The temperate Escherichia coli phage N15 is a well characterised example, maintaining its prophage
extra chromosomally as a linear replicon with covalently closed telomere ends. Related phage with linear genomes includes phiKO2, PY54, VP882 and phiHAP1. The Lyme disease causing bacteria, Borrelia, and the plant pathogen, Agrobacterium tumefaciens C58, also have linear genomes. Research to date has largely focused on characterizing protelomerase recognition sequences, solving 3-dimensional structures, and exploring the effects of protein mutations on activity. This thesis explores the structural, biophysical dynamics and biochemical actions of the phage protelomerase, TelN. Work is also presented as steps to biophysical characterisation of the bacterial protelomerase, ResT. Further research was conducted computationally to identify uncharacterised proteins with regions of structural homology to known protelomerases. New protocols for the heterologous expression and purification of these proteins, including variants,
have been developed, and the results from activity assays are presented. Cryo-EM analysis of TelN in complex with a mutated DNA substrate reveals a dynamic assembly and represents the first known structure of this protein.