How bacteria replicate their DNA in spite of barriers, one molecule at a time
Lead Research Organisation:
Brunel University London
Department Name: Life Sciences
Abstract
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Technical Summary
Activities of DNA polymerases in replication result in collisions which, if remaining unresolved, can be lethal and therefore must be resolved efficiently. Extensive knowledge exists from genetics and biochemistry about the enzymes involved, but we know little of how this is achieved at the level of single molecules. Here we integrate interdisciplinary expertise, using E. coli as a model to study replication collisions and subsequent crucial repair, modifying cells to label key proteins used in replication used in replication and repair. We will use artificial blocks to controllably mimic collisions both in vitro and in living cells and native blocks involving RNAP from transcription that will allow us to study collisions in defined areas of the chromosome in any orientation. Some replisomes remain following collisions while others disassemble. By using labelled replisomes and blocks probed with super-resolved single-molecule microscopy, microfluidics and novel AI biocomputation tools, we will be able to define the conditions when forks disassemble under physiological conditions. We will then visualise which repair proteins are recruited and, finally, be able to characterise how restart proteins can re-recruit active replisomes to continue synthesis. We will also search for hitherto undiscovered factors that assist in vital replication collision resolution.
Our analyses will address fundamental questions concerning the resolution of collisions between replication molecular machinery and nucleoprotein blocks. DNA replication and repair offer key antibiotic targets, and we anticipate our findings will have longer term societal benefit in addressing how poisons targeting these processes can be tolerated by cells and lead to antibacterial resistance, aiding development of new antibiotics in addition to substantive development of new microscopy instrumentation, bioinformatics and high-precision analytical software tools of wide benefit to the biosciences.
Our analyses will address fundamental questions concerning the resolution of collisions between replication molecular machinery and nucleoprotein blocks. DNA replication and repair offer key antibiotic targets, and we anticipate our findings will have longer term societal benefit in addressing how poisons targeting these processes can be tolerated by cells and lead to antibacterial resistance, aiding development of new antibiotics in addition to substantive development of new microscopy instrumentation, bioinformatics and high-precision analytical software tools of wide benefit to the biosciences.
People |
ORCID iD |
Christian Rudolph (Principal Investigator) |
Publications
Goodall DJ
(2023)
Interplay between chromosomal architecture and termination of DNA replication in bacteria.
in Frontiers in microbiology
Description | Mark Leake |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | My lab provides cell biology expertise and research materials. |
Collaborator Contribution | Mark Leake provides single molecule imaging expertise, access to instrumentation and research materials. |
Impact | No specific outputs yet. |
Start Year | 2019 |
Description | Michelle Hawkins (York) |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We are investigating the issues arising from the termination of DNA replication from two separate angles. My lab focuses on the events associated with the fusion of two replication forks in vivo and the consequences that arise if certain protein activities are not present in cells. |
Collaborator Contribution | Michelle Hawkins is a very good biochemist who was trained in the lab of my former collaborator Peter McGlynn. She has taken over the investigations of the events associated with the fusion of two forks in an in vitro system where DNA replication is reconstituted from purified components. |
Impact | Research papers: PMID 30869136 |
Start Year | 2018 |
Description | Seminar Okinawa Institute of Science and Technology |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to present the latest research of my group as a Research Seminar at the Okinawa Institute of Science and Technology, Graduate University, Onna, Okinawa, Japan. I was personally invited by Prof. Simone Pigolotti to give this research seminar. This meeting was attended both in person in via online connections by a large number of researchers from all over Japan and other locations. My talk sparked questions and discussions afterwards. |
Year(s) Of Engagement Activity | 2022 |
Description | Seminar Tokyo Metropolitan |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | I was invited to present the latest research of my group as a Research Seminar at the Institute of Medical Science, Tokyo Metropolitan University. I was personally invited by the Director of the Institute of Medical Science, Prof. Hisao Masai, to give this research seminar. This meeting was attended both in person in via online connections by a large number of researchers from all over Japan and other locations. My talk sparked questions and discussions afterwards. |
Year(s) Of Engagement Activity | 2022 |