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Initiation of asymmetric rolling-circle plasmid replication by RepD studied using magnetic tweezers

Toleikis, A; (2016) Initiation of asymmetric rolling-circle plasmid replication by RepD studied using magnetic tweezers. Doctoral thesis , UCL (University College London). Green open access

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Abstract

Bacterial drug resistance is often carried by circular DNA plasmids. As plasmids are copied separately from the bacterial genomic DNA, the copies can be passed to other bacteria in the colony spreading the antibiotic resistance. This PhD thesis describes studies of the replication machinery, used by the Staphylococcus aureus chloramphenicol-resistance plasmid pC221. The plasmid is duplicated by a process called asymmetric rolling circle replication, in which the two DNA strands are copied asynchronously. It is not fully understood how the replication process is regulated but the initiation requires a plasmid-encoded protein, in this case RepD. This initiator nicks the parent, supercoiled plasmid at the double-stranded origin of replication (here, oriD) and reveals a short length of single-stranded DNA, which allows a helicase and DNA polymerase to bind. These together unwind and copy the leading strand. Here, custom-built magnetic tweezers were used to control the extent of supercoiling and monitor the RepD nicking reaction of single molecules of linear DNA. I found that the nicking reaction is exquisitely sensitive to DNA supercoiling, which means replication cannot start if the plasmid is relaxed by damage or if replication is already in process. DNA supercoiling therefore acts as a mechanical signal to control initiation and as safety-catch to prevent re-initiation at the newly synthesized oriD site until the new, complete, circular daughter plasmid has been created and supercoiled. The role of DNA supercoiling therefore is not merely compaction but also an important regulator of protein-DNA interactions in DNA replication.

Type: Thesis (Doctoral)
Title: Initiation of asymmetric rolling-circle plasmid replication by RepD studied using magnetic tweezers
Event: University College London
Open access status: An open access version is available from UCL Discovery
Language: English
Keywords: DNA replication, antibiotic resistance, magnetic tweezers, single-molecule
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/1474862
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