Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.744851
Title: DNA double-strand break repair studied by atomic force microscopy
Author: Zabolotnaya, Ekaterina
ISNI:       0000 0004 7229 8790
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2018
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Abstract:
DNA double-strand breaks (DSBs), where both strands of the DNA duplex are simultaneously fractured, are considered the most lethal type of DNA damage. The conserved Mre11-Rad50 DNA repair complex enables the catalytic activities of the Mre11 nuclease and the Rad50 ATPase to function together to coordinate the recognition and processing of DSBs prior to the recruitment of long-range end-resection machinery required to trigger the DSB repair by the homologous recombination (HR) pathway. Fast-scan atomic force microscopy (AFM) in fluid conditions was primarily used to explore the architectural arrangement, DNA binding and processing machinery of the Mre11-Rad50 complex from the thermophilic crenarchaeote Sulfolobus acidocaldarius. The structural analysis identified four distinct architectural arrangements and demonstrates the key role of the Rad50 zinc hooks in the oligomerisation of the complex. AFM imaging showed a dynamic and Velcro-like interplay between Mre11-Rad50 protein complexes and the DNA double-helix using the Rad50 coiled-coils in a novel mode of DNA binding. The complex appears to use the Rad50 zinc hook region to bind to and track along dsDNA for broken DNA-terminals. Furthermore, the present study shows that this archaeal complex can drive extensive ATP-dependent unwinding of DNA templates. It is the first time that such unwinding has been observed in a single molecule study. These observations reveal novel activities leading to the proposal of a new model for Mre11-Rad50 action during DSB repair. AFM was also used to visualise the structure and activity of the HerA-NurA protein complex, which has been predicted to combine the activity of the NurA nuclease and hexameric HerA-translocase to generate long single-stranded DNA overhangs essential for DSB repair by HR in archaea. The present data verify and clarify the presumed biological role of this complex. Overall, the present study provides new insights into the initial steps of DNA DSB repair by the HR pathway and, most importantly, the detection of the broken ends.
Supervisor: Henderson, Robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.744851  DOI:
Keywords: DNA double-strand breaks ; DSB ; DNA ; AFM ; atomic force microscopy ; DNA unwinding ; DNA-protein interaction ; Mre11 ; Rad50 ; MR complex
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