Reed, Patricia (2006) Function of bacteriophage orf recombinasesin genetic exchange. Doctoral thesis, Durham University.
Recombination events in bacteriophages frequently occur by illegitimate exchange at short tracts of sequence homology, enabling these viruses to acquire novel genes and serve as vehicles for horizontal gene transfer. The emergence of new pathogenic organisms due to the acquisition of virulence determinants from bacterial viruses has stimulated considerable interest in the mechanisms of phage recombination. Bacteriophage λ encodes its own recombination system, consisting of Exo, β and γ proteins. An additional λ recombinase, Orf, participates in the early stages of exchange, supplying a function equivalent to the Escherichia coli RecFOR complex. The host enzyme complex promotes the loading of the RecA strand exchange protein onto SSB-coated ssDNA. This thesis describes the purification and biochemical analysis of the λ Orf protein, in parallel with two distantly related homologs from E. coli cryptic prophage DLP12 and Staphylococcus aureus phage ɸETA.X Orf was found to belong to a family of proteins originating from diverse lambdoid phage and prophage sources. Members of this family reside within a conserved genetic module located between phage replication and cell lysis functions. Orf exists as a homodimer, arranged as a toroid with a shallow cleft running perpendicular to the central cavity. K binds preferentially to DNA containing single- stranded regions, and associates with E. coli SSB protein in the presence of ssDNA. The Orf homolog from E. coli DLP12 displayed similar properties. This work suggests that members of the Orf family function as recombination mediator proteins, stimulating the assembly of strand exchange proteins onto ssDNA, and highlights the importance of overcoming the barrier presented by SSB proteins during lambdoid phage recombination.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||13 Sep 2012 15:58|