MBI PhD Oral Defence
Time: 10AM
Date: Wednesday, 15 April 2020
Venue: Webcast only
Supervisor: Prof. Yan Jie
DEVELOPING LABEL-FREE SINGLE-MOLECULE ASSAYS TO ENABLE PRECISE QUANTIFICATION OF DYNAMICS IN DNA-PROCESSING PROTEINS
by LU Chen, Yan Jie Group
Cells rely on a series of complex functions to copy, utilize, protect and correct DNA information. Among these processes, dynamic transitions between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) are frequently regarded as the core components. A class of protein called helicases play the pivotal roles behind these transitions, for their active engagement in DNA unwinding, rewinding, and translocation functions. Studying helicases is therefore not only beneficial for understanding any of the particular functions mentioned, but also helpful in decoding the complex DNA processes as a whole. Despite the great importance, the molecular mechanisms of helicase activities remain largely elusive, and one of the major obstacles is the lack of accurate assays to probe these activities. That is especially apparent for the study of less-elucidated helicase-dependent DNA translocation function. To cater to this problem, two label-free single-molecule translocation assays were designed, constructed and validated in parallel based on the our ultra-stable magnetic tweezers. Using these assays, precise quantification of translocation, unwinding and re-zipping rates for Saccharomyces cerevisiae Pif1 (ScPif1) and its ΔN mutant were achieved. The comparison of these rates suggested that ScPif1 is a rather active helicase, while the N-terminal domain may play a crucial role in maintaining that property. In addition, our assays also helped identify two translocation modes of ScPif1, one dubbed as mobile translocation and the other involving an unexpected loop-generation activity. It was found that the switch between the dual translocation modes was heavily dependent on the helicase’s concentration. Besides that, our studies also revealed the same dual translocation modes being evident for another helicase UvrD. Together with the rare rewinding activity discovered on both helicases, our observations suggested that the properties more common than expected may be shared among helicases, implying a general molecular mechanism behind. Finally, experimental designs utilizing the novel translocation assays implemented in this thesis were provided to further test the mechanism, and hopefully to decipher all the helicase-dependent functions in the near future.
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