The Mechanism of RecA Mediated DNA Patterning Interrogated by AFM

Abstract

Over recent years, advancements in bottom - up construction technologies are enabling the creation of heterogeneous and functional materials. These approaches offer the potential to surpass the physical limitations in traditional top - down micromachining. Of these, bionanotechnological approaches that harness the inherent molecular recognition and self-assembling properties of biological molecules - such as Deoxyribose nucleic acid (DNA) - are arguably the most promising. Over recent years, the field of DNA nanotechnology has advanced rapidly, enabling the creation of arbitrary structures in two and three dimensions. These substrates act as adapters enabling the arrangement of functional components at the nano-scale to be interfaced with the macro-scale world. One approach to spatially address DNA nano-architectures is to harness the sequence specific homologous recombination mechanism of the E.coli protein Recombinase A (RecA). This protein mediates the alignment of a supplied single stranded DNA (ssDNA) with a subject double stranded DNA (dsDNA) where homology is shared, making this method inherently programmable. Despite several successful demonstrations of the artificial application of RecA, the underlying mechanism which orchestrates this interaction remains widely debated. The lack of clear understanding surrounding this critical biological mechanism stems from the in-direct approaches taken to interrogate it, to date. In response to this, the work presented in this thesis, attempts to answer the open biological questions surrounding RecA. Here, recent advances in high speed atomic force microscopy (HSAFM) and high resolution Atomic force microscopy (AFM) - using rapid-force-curve imaging - are applied to directly interrogate the homology searching mechanism of RecA. When taken together, these structural and functional insights will inform the future development of RecA mediated patterning approaches within complex DNA topologies.

Metadata

Supervisors:	Wälti, Christoph and Hobbs, Jamie
Keywords:	High Speed AFM, RecA, DNA Nanotechnology
Awarding institution:	University of Leeds
Academic Units:	The University of Leeds > Faculty of Engineering (Leeds) > School of Electronic & Electrical Engineering (Leeds) > Institute of Microwaves and Photonics (Leeds)
Identification Number/EthosID:	uk.bl.ethos.692410
Depositing User:	Mr Andrew J Lee
Date Deposited:	22 Aug 2016 12:02
Last Modified:	25 Jul 2018 09:52
Open Archives Initiative ID (OAI ID):	oai:etheses.whiterose.ac.uk:13679

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The Mechanism of RecA Mediated DNA Patterning Interrogated by AFM

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