The Characterisation of DNA Topoisomerase VI from Methanosarcina mazei Using Ensemble and Single-Molecule Methods

Mckie, Shannon (2020) The Characterisation of DNA Topoisomerase VI from Methanosarcina mazei Using Ensemble and Single-Molecule Methods. Doctoral thesis, University of East Anglia.

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DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase, found predominantly in archaea but with eukaryotic presence in various plant and algal species, and possibly plasmodia. In Arabidopsis thaliana, topo VI is an indispensable nuclear protein, essential for endoreduplication, despite the presence of other type IIA topoisomerases, capable of performing the same reactions. Whilst topo VI has been proposed to be a DNA decatenase since its discovery, robust evidence and a mechanism for its preferential decatenation activity was lacking. In this research, the activity of topo VI from the mesophilic archaeon Methanosarcina mazei (MmT6) was characterised using ensemble biochemistry, single-molecule magnetic tweezers and next-generation DNA sequencing (NGS). This demonstrated that MmT6 activity was significantly enhanced in the presence of catenated or braided DNA substrates, as opposed to supercoiled, through a strong preference for DNA crossings with angles close to 90°. In addition, MmT6 behaved as a true crossing sensor with dramatic increases in ATPase activity, DNA binding and the rate of strand-passage, with increasing DNA writhe. Taken together, these results strongly suggest that MmT6 has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes, simultaneously disfavouring the relaxation of supercoils, by sensing DNA crossings directly with geometries close to 90°. This provided an explanation for why topo VI homologues persist in higher eukaryotes during situations in which the genome undergoes rapid duplication, potentially as a dedicated DNA decatenase that cannot be substituted by other topoisomerases. It was also shown, using an NGS-based technique, that MmT6 cleaved DNA with the sequence preference 5ʹ-[G/C][G/C]^[A/C/T], generating 2-base overhangs. The in vitro plasmid-based NGS technique developed to attain this result was extended to begin exploring the in vitro DNA cleavage preferences of other type IIA topoisomerases, namely E. coli DNA topoisomerase IV and E. coli DNA gyrase.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 15 Feb 2021 11:47
Last Modified: 15 Feb 2021 11:47


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