A study of DNA interactive proteins and their origin from thermophilic Siphoviridae phages

Sammanfattning: This thesis covers the results of studies on the amino acid sequence, three-dimensional structure, and activities of two types of DNA-interactive proteins: Holliday Junction resolving enzyme (represented by Hjc_15-6 isolated from phage Tth15-6) and DNA Polymerase I (represented by PolI_G20c, isolated from phage G20c), including studies on their homologous counterparts. As these proteins originate from two different, but related phages that infect Thermus thermophilus, the studies also include the evolutionary relationship between these and other related phages at genomic level. The novel phage Tth15-6 was isolated, its genome sequenced, and from the sequence data, a large number of genes were selected. The selected genes were cloned and expressed in Escherichia coli and the resulting production levels of soluble target protein were analysed. The DNA polymerase encoded in phage Tth15-6, PolI_15-6, was considered interesting, due to its relatively low sequence conservation compared to previously studied candidates from the same family of polymerases. It was, however, not possible to produce the protein in its soluble form in the selected E. coli production host. Therefore, a gene encoding a homologous protein, PolI_G20c, was identified, cloned and expressed, that resulted in soluble production of the target protein. After purification, PolI_G20c was crystallised, but its structure could not be solved by molecular replacement, using available structures in the Protein Data Bank, PDB. However, a fragment of a homologous protein, ExnV1, was also produced and crystallised. In this case, it was possible to solve the three-dimensional structure, which was subsequently used as a template, allowing the determination of the three-dimensional structure of PolI_G20c. The gene encoding the Holliday junction resolving enzyme, Hjc_15-6, was also cloned, and the enzyme was produced, purified and crystallised. In this case, production of a selenium derivatized variant was successful. Crystallisation of both variants, allowed determination of the three-dimensional structure. Studies were also made on how Hjc_15-6 interacts with branched DNA structures and single peptides in conjunction with polymerases and polymerase reactions. Analysis of the genome sequence of phage Tth15-6, showed that the identified open reading frames in many cases lacked homologues of known function. This sequence divergence makes it difficult to find related candidates of known function using traditional bioinformatics. However, the organisation of genes in the whole genome may be a valuable tool for this purpose. The evolutionary relationship between phage Tth15-6, phage G20c, and other related phages, were also reflected in the deduced amino acid sequences of the enzymes Hjc_15-6 and PolI_G20c. A three-part sequence signature motif of Hjc_15-6 differed from corresponding motif in earlier reported Hj resolving enzymes. It was thus proposed that this novel sequence signature is common among Hj resolving enzymes originating from the related phages. Furthermore, Hjc_15-6 is the first Hj resolving enzyme originating from a phage that could be classified as an archaeal type Hj resolving enzyme, regarding both its amino acid sequence, and its structure. This is the first time an archaeal type Hj resolving enzyme originating from a phage is reported. The structure of PolI_G20c and ExnV1 revealed a new structural motif that has not previously been reported among type 1 polymerases. However, based on the amino acid sequence analysis, this motif may also occur in enzymes from related phages. Finally, the studies show that Hjc_15-6 has unusual features not reported among Hj resolving enzymes elsewhere. Therefore, Hjc_15-6 was studied further, both regarding its capability to cleave DNA oligomers, and its function in conjunction with polymerases and polymerase reactions. It was revealed that Hjc_15-6 is capable of cleaving branched products from isothermal polymerase reactions based on strand replacement. It was also shown that it was possible to induce a polymerase reaction with neither DNA template nor primers present, when Hjc_15-6 was present. Moreover, Hjc_15-6 may form large networks of DNA, something that was studied in real-time under an electron microscope. Hence, Hjc_15-6 may be a valuable tool for DNA origami with a wide variety of applications. This thesis contains four research papers along with introducing chapters for the reader's orientation including brief presentations of the content in the Papers, followed by a chapter of Concluding remarks where the most important findings in the articles are presented, followed by a chapter of Future prospects, Acknowledgements and References.