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
De Dios R
(2025)
Saccharin disrupts bacterial cell envelope stability and interferes with DNA replication dynamics
in EMBO Molecular Medicine
Goodall DJ
(2023)
Interplay between chromosomal architecture and termination of DNA replication in bacteria.
in Frontiers in microbiology
| Description | It is known that DNA replication frequently suffers from blocks. These can be caused by small lesions to the DNA but also by protein complexes sitting tightly on the DNA template. One frequent obstacle is transcription. Both DNA replication and the generation of mRNA needed for protein synthesis use the same template, but operate with very different speeds, with DNA replication being much faster. This means that that conflicts will occur frequently and regularly. In older studies it was shown that proteins involved in homologous recombination are involved in the processing of replication forks stalled at sites of ongoing transcription. Especially the RecBCD exonuclease plays a role. However, RecBCD only processes double-stranded DNA ends, not structures that arise as replication forks arrest. This means that stalled forks must be processed by another protein to convert the structure of a stalled fork into a structure that has a dsDNA end which can be processed by RecBCD. Various proteins were implicated in this conversion, but so far it was not shown directly whether they are involved. By using a model block to DNA replication in E. coli we are now able to show via single molecule tracking that replication forks arrest upon meeting the block, and that some of the forks are disassembled. However, replication continues after a short period, which means that replisomes must be re-assembled at the site of arrest. In E. coli this is done by a class of proteins known as replication restart proteins, including PriA, PriB, PriC and DnaT. Previous genetic analysis suggests that at least three distinct pathways exist: a PriA/PriB pathway, a PriA/PriC pathway and a PriC-dependent pathway. Again, by using a model block we are able to show that replication restart at an obstacle such as a protein-DNA complex predominantly uses the PriA/PriB restart pathway. PriC is of less important, but becomes more important for more persistent blocks. In addition, PriA restart protein has two distinct functions: a replication restart activity and a helicase activity. The restart activity is important to re-recruit the replication machinery to a site of conflict. The role of the helicase is slightly less defined. It was thought that it unwinds the nascent lagging strand at a replication fork structure to generate a "landing pad" for re-recruiting a replisome. In our studies we show that cells lacking only PriA helicase activity (but not the restart activity) show significant problems if replication is blocked at a model block. In the presence of such a block cells show a growth defect and filament. However, this effect is temporary. After some filamentation these filamented cell break down and continue growth, and the impact on cellular viability is mild. Thus, blocks are overcome eventually in a different way. However, it was not shown so far that PriA helicase has a significant role in the processing of forks stalled at protein-DNA obstacles. This now opens the door for investigations looking into whether PriA helicase might be one of the factors that specifically remodel a stalled fork into a substrate that can be processed by the recombination proteins RecBCD. |
| Exploitation Route | Identifying proteins that directly remodel stalled replication forks is a tremendously important achievement. The finding now allows for systematic studies, both in vitro and in vivo, identifying what substrates are generated and what other processes are then involved to process the intermediates generated, both by our labs and labs of other researchers interested in the processing of stalled replication forks. |
| Sectors | Education Healthcare Pharmaceuticals and Medical Biotechnology |
| Description | Ed Bolt |
| Organisation | University of Nottingham |
| Department | School of Life Sciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | My lab is actively working on generating strains in which proteins of the Cas-CRISPR system can be localised in vivo. We are investigating the role of the CRISPR-Cas system in bacteria in general and in E. coli in particular. |
| Collaborator Contribution | Ed Bolt's lab is working on the in vitro reconstitution of the Cas-CRISPR system in Escherichia coli. The collaboration will allow us to combine both in vivo and in vitro approaches to characterise the system in E. coli and other bacterial organisms. |
| Impact | No outputs yet |
| Start Year | 2016 |
| 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 | Ronan McCarthy |
| Organisation | Brunel University London |
| Department | Division of Biosciences |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | My lab is providing a molecular biology of analysis of the effect of artificial sweeteners on cell growth and morphology, both E. coli and Acinetobacter baumannii |
| Collaborator Contribution | Dr McCarthy is the PI of this particular project. |
| Impact | Collaboration has led to a first high-profile publication in EMBO Molecular Medicine (https://doi.org/10.15252/emmm.202216397). |
| Start Year | 2022 |
| Description | Oral presentation at the DNA replication meeting in Cambridge |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Other audiences |
| Results and Impact | I presented my findings on DNA replication dynamics, termination of DNA replication and the role of CRISPR-Cas in DNA replication and repair at the DNA replication conference organised by the Biochemical Society, a national annual conference that was held in Cambridge in 2024. The meeting was attended by a large number of research groups working in the field of DNA replication in the UK. |
| Year(s) Of Engagement Activity | 2024 |
| URL | https://www.eventsforce.net/biochemsoc/frontend/reg/thome.csp?pageID=97695&eventID=188&traceRedir=2 |
| 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 |