RecA and repressor: Working together to save phage
Pawlowski, David Ryan
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The CI repressor protein of bacteriophage 434 is a transcriptional regulator of the life cycle of bacteriophage 434. 434 repressor directs transcription of its own gene while repressing transcription of genes that are required for lytic growth. In order for a prophage to enter the lytic pathway of its life cycle, 434 repressor's function must be altered. Through an interaction with the co-protease, active RecA (RecA*), 434 repressor is stimulated to auto-cleave. Auto-cleavage results in a switch of the phage life cycle from lysogenic growth to lytic growth due to the loss of repressor's ability to bind DNA. The preferred form of repressor for auto-proteolysis was identified in order to further understand the mechanism of auto-cleavage. This was done by examining the kinetics of repressor auto-cleavage as a function of repressor concentration and the sequence of added DNA. The results show that repressor bound to specific DNA is the preferred substrate for RecA* mediated auto-cleavage. These results also indicate that the preference for the repressor/OR1 complex is due to an increase in affinity for RecA*, not a change in the chemical mechanism of cleavage. The discovery that 434 repressor is preferentially cleaved as a dimer led to the question of whether each monomer in a dimer cuts itself (intra-molecular) or whether they cut each other (inter-molecular). Using specific repressor mutants we show that 434 repressor likely utilizes an intra-molecular mechanism of cleavage. The data can be fit to a model in which the operator bound repressor molecules assume a cleavage competent conformation favoring intramolecular autoproteolysis. Finally, the rate of RecA* mediated homologous strand exchange was measured as a function of repressor concentration. This experiment was performed in response to data that suggested that the operator DNA bound to repressor was not released into solution upon repressor cleavage rather it remained bound to RecA*. Surprisingly, the rate of exchange increased at low concentrations of repressor suggesting that there is an overall increase in RecA*'s affinity for DNA, leading to increased strand exchange. The results described throughout this thesis offer new insight into the way RecA*/repressor interactions are viewed.