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Title: The mechanism and consequences of recombination hotspot recognition by an AddAB-type helicase-nuclease
Author: Gilhooly , Neville S.
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2013
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Over billions of years cells have evolved numerous conserved repair pathways to deal with the continuous onslaught of DNA damage. One particularly toxic lesion is a double-stranded DNA break, which can be faithfully repaired by the ubiquitous homologous recombination pathway. In bacteria, the processing of double-stranded DNA breaks for recombinational repair is carried out by helicase-nuclease enzymes. These are stable multi-protein complexes that coordinate their helicase and nuclease activities to resect DNA ends. This activity generates the 3'-ssDNA overhangs that are required by the homologous pairing enzyme RecA/Rad51. In bacteria, the formation of this ssDNA tail is dependent upon recognition of the recombination hotspot sequence Chi; a specific single-stranded DNA sequence that acts to attenuate nuclease activity on the 3'-strand. Despite their extensive study, the mechanism by which helicase-nuclease enzymes recognise their cognate Chi sequence has remained elusive. This mechanism has been addressed in this thesis using the model helicase-nuclease AddAB, from Bacillus subtilis. Mutagenesis analysis reveals that Chi is recognised by the AddB subunit and interestingly, Chi binds to a domain that resembles a catalytically inactive SFl helicase. Further mutagenesis shows that an "ionic latch" structure regulates a conformational change that is linked to the recognition of Chi. This latch structure is also intimately linked to the specificity of the AddAB:Chi interaction. Using rapid reaction techniques it was also found, somewhat unexpectedly, that Chi attenuates the ATPdependent translocation activity of AddAB. Finally, preliminary data suggest that ATP binding to AddB stabilises the binding of Chi to AddB, but only after Chi has been recognised and AddAB has undergone a Chi-induced conformational change.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available