PhD student profiles

     
     
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Algirdas Toleikis
Wellcome Trust 4-year Interdisciplinary PhD Programme, beginning in Autumn 2011

Project title
Initiation of plasmid replication studied using magnetic tweezers

Principle investigators: Justin Molloy and Martin Webb, NIMR, ISMB

 
Background

I started my “journey to science” during my undergraduate degree in Biochemistry in the group of Prof Virginijus Siksnys at the Institute of Biotechnology of Vilnius University, Lithuania. There I conducted a 2-year project on protein-DNA interactions during CRISPR/Cas mechanisms of bacterial immunity for viruses. After being awarded an interdisciplinary Wellcome Trust PhD scholarship I had an exciting opportunity to expend my viewing angle of protein-DNA interactions to different angles.

 
Rotation projects

Rotation 1
John Offer: Chemical peptide synthesis

Rotation 2
Justin Molloy: DNA unwinding by PcrA helicase studied with magnetic tweezers

Rotation 3
Willie Taylor: RNA secondary structure prediction
 
PhD Project

Bacterial drug resistance is often carried by small pieces of circular DNA called plasmids. Plasmids are copied separately from the bacterial genomic DNA by the coordinated action of several proteins which act to produce two identical daughter plasmids which are passed to other bacteria in the colony. During my PhD I have studied the replication machinery used by the Staphylococcal aureus plasmid, pC221. 

The plasmid is duplicated by a process called rolling circle plasmid replication in which the two DNA strands are copied one after the other. It is not fully understood how the replication process is regulated but we know that the first step, termed initiation, requires a plasmid-encoded protein called RepD which nicks at a specific site, oriD, and reveals a short length of single-stranded DNA which allows a helicase and DNA polymerase to bind and together unwind and copy the first, or leading, strand.

As part of my PhD I constructed a custom-built magnetic tweezers apparatus which I used to apply controlled amounts of twist to individual pieces of plasmid DNA so that we could monitor the RepD nicking reaction at the single molecule level. We found that the nicking reaction is exquisitely sensitive to DNA supercoiling which means replication cannot start if the plasmid is damaged, because single-strand nicks would need to be repaired for the plasmid to be supercoiled. Furthermore, once replication has commenced and the oriD site recreated, another round of initiation cannot occur until the entire plasmid has been copied and the new circular duplex religated and supercoiled again. Negative DNA supercoiling therefore acts as a mechanical signal to control initiation and as safety-catch to prevent re-initiation at the new oriD site until the new, intact, circular daughter plasmid has been created.

 
 
 
Magnetic tweezers is a simple yet very exciting technique not only allowing to manipulate single DNA molecules but also is a unique tool to twist and supercoil DNA and investigate protein-DNA interactions. This video is a lecture by Dr Justin Molloy given at MRC National Institute for Medical Research (NIMR, London) in 2014.

 

 

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