TROTTER, ALEXANDER,JOHN (2015) DNA binding proteins of the phage λ ninR region. Masters thesis, Durham University.
|PDF (Alex Trotter MSc Thesis ) - Accepted Version|
Bacteriophages are known to disable host functions in order to impose their own enzymes to hijack the bacterium for maximal replication of the virus. One well-characterised example of this is the Red genetic recombination system of phage λ which blocks host RecBCD and replaces it with an exonuclease and synaptase to promote genomic rearrangements. Other substitute enzymes from λ include Orf, which can replace the RecA-loading activity of RecFOR, and Rap, which can substitute for the RuvC Holliday junction resolvase. This study has investigated the DNA binding activities of three gene products from the ninR region of phage λ that are linked to genetic recombination. These are Orf (NinB), Rap (NinG) and NinH, the latter an uncharacterised protein of 68 residues. DNA binding analysis with selected substitution mutants of the Orf protein established that the central channel and adjacent regions are critical for binding to single-stranded DNA. The predicted HNH catalytic site of the Rap DNA structure-specific endonuclease was also investigated by site-directed mutagenesis confirming that the HNH domain is essential for Holliday junction cleavage and may also participate in branched DNA binding. Finally, the NinH protein which resembles the bacterial nucleoid associated protein (NAP) Fis, was also analysed for its ability to bind double-stranded DNA. The in vitro data showed that NinH binds DNA and preferentially favours bent duplex substrates, consistent with its similarity to Fis. NinH could not compensate for the loss of various NAPs from E. coli in deletion strains, including a fis mutant, and generally conferred a negative effect on the growth of these strains. Thus NinH may play a role in perturbing normal bacterial replication to promote phage production. The results offer several new insights into the properties of these three DNA binding proteins and their possible roles in phage DNA replication and recombination.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Keywords:||DNA binding, phage λ, NinB, Orf, NinG, Rap, NinH, ninR|
|Faculty and Department:||Faculty of Science > Biological and Biomedical Sciences, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||07 Dec 2015 11:41|